My Reference Manual

Biochemistry &
Molecular Biology
ATP Synthase
Introduction to Biochemistry & Molecular Biology
BMB Moodle Site

We provide a website for the Biochemistry & Molecular Biology (BMB) course using the
University’s Virtual Learning Environment (VLE), Moodle, accessed from www.vle.cam.ac.uk.
You will be registered for the BMB site once we have collected your User IDs at the
practical registration. There is a lot of important information on Moodle:
• Look out for course announcements
• Information about the course is provided on Moodle
• Moodle provides on-line access to course materials and resources:
E.g. lecture handouts, films of lectures (via Panopto – see below), pre-practical
quizzes, interactive practical materials, exam quizzes, background information (E.g.
techniques posters and films), information for the practical classes, tutorial films, etc.
• Additional material to support the BMB course will be provided on Moodle
throughout the academic year – do keep an eye out for new additions or
developments.
Do take the time to explore the BMB Moodle site, as there are many resources that will
support your learning, both for lectures and practical classes.
Please note that there are resources on Moodle that are absolutely essential for
your course work. In addition, there are also a large number of additional resources that
have been provided to support your learning (E.g. quizzes, interactive practical materials,
extra experimental design exercises, information about biochemical techniques etc.), and it
is not essential that you complete or engage with all of the material provided.
The virtual learning environment should support the way YOU learn best, so
feel free to pick and choose from the resources provided to use those best
suited to your individual learning style.
A few practical details about using Moodle for BMB
The format of the BMB Moodle site may be arranged slightly different to what you’ve used
previously. Here are a few helpful tips to get you started:
• Helpful information can be found in the blocks on the right-hand side of Moodle
• Click on each coloured box on the front page to access the various course materials
or information
• Lecture materials etc. in each block are generally organised into ‘Moodle Books’,
where there are separate Moodle Books for each lecture set.
E.g. Within the block ‘BMB Lectures – Michaelmas Term’:
• To access the contents of Moodle Books, click on the book and the chapters will
appear in the top right-hand side of the screen, and the contents of each chapter will
be in the middle of the screen:
E.g. Chapter 1: Lecture handout 2017
• All of the materials on the BMB Moodle site can be accessed by

o Navigating from the front page (click on the top image (collage) at any time to
take you back to the BMB front page)
o Using the drop-down menu in each section ‘Jump to…’
o Using the navigation arrows/text at the sides of each section
E.g.
Some course information will also be available via the Biochemistry Department at
http://www.bioc.cam.ac.uk/teaching/second-year/bmb
Lectures
Lecture handouts are provided in advance on Moodle and in hard copy at the lectures. To
aid discussion in supervisions, lecturers will provide some questions related to the lecture
material, if you and your supervisors wish to use them. Your supervisors will be provided
with guides to answering these questions.
Various resources to support the lectures are included on Moodle. These are as follows:
• Lecture handouts
• Lecture slides
• Questions based on the lectures as provided by lecturers
• Literature sources, reading lists etc.
• Lecture recordings – via Panopto software (see below)
Lecture Recordings – Panopto

The majority of lectures in BMB will be recorded using ‘Panopto’ and will be accessible via
the BMB Moodle site. Here are a few tips for using Panopto for BMB.
• Lecture recordings for BMB can be accessed in two ways:
o via the Panopto block (top right-hand side of BMB Moodle page) where the
recordings will be listed by date
o via links in the Moodle book for each set of lectures

• You can search for key words in recordings – these will be identified by voice
recognition and from text on slides:
E.g. if you type ‘translation’ in search box (top left-hand side of Panopto screen), you
will get a list of places where that word occurs, and at what time. You can then skip
forward to the part you are looking for.
• You can also use bookmarks to mark bits that you might want to go back to later:
Practical Classes
Aim of the practicals
Progress in science is achieved through observation and experiment. Biochemistry (and its
close cousin, molecular biology) is an experimental science that advances from well-thought
out investigations in the laboratory. No serious student should neglect the opportunities
that this course provides to appreciate this fact. Your course includes experiments devised
for you to gain some insight into how laboratory investigations are carried out and how
data are processed and interpreted. To obtain useful results an experiment should be
designed to answer a definite question and the detailed planning should be sufficiently
rigorous to exclude adventitious errors. The course gives you the opportunity to plan some
experiments for yourselves. You should benefit from the practicals in three ways:
(i) You will learn a variety of experimental techniques, all of which are currently used in
biochemical research. The practicals have been designed to complement the lectures
and fit in with their sequence as far as possible. The hands-on experience should link
to the mental framework provided by the lectures, and give you a deeper
understanding and more realistic perspective of the topics discussed.
(ii) You will learn to handle experimental data effectively, and to extract the maximum
information content without falling into the trap of over-interpretation.
(iii) You will be helped when it comes to the data handling questions in the Tripos
examination. A collection of question papers from previous years is available on the
course website. Quizzes based on the previous papers can also be found on Moodle.
The Demonstrators
Demonstrators are there to help you. The senior demonstrator will normally be a member
of the Staff of the Biochemistry Department – often a lecturer in your course. The assistant
demonstrators will be graduate students working for a PhD research degree, or post-
doctoral research workers who have recent and sympathetic memories of the difficulties
felt by studying the subject. Rely on them not only to sort out practical difficulties but also
to help you make sure you understand what the experiments are about and what your
results mean. They may well also be able to help you understand theoretical or lecture
material.
Discussions
As the class size is relatively small, discussion of the practical work will be informal and will
normally take place in the laboratory at the end of the experimental work. Demonstrators
will be on hand at these occasions and you are urged to make the most of these discussions
and take an active part in them.
Safety and care in the laboratory

PLEASE be careful in the laboratory. Attend to your own safety and that of others
around you: this is a statutory obligation – a matter of law.
PLEASE also extend your consideration to the equipment in the laboratory. Most of it is
costly to repair or replace.
Policy, rules, and guidance on safety are given on the pages in this handbook headed 'Safety
in the Biochemistry Part I Teaching Laboratory'. You must read this before coming to the
first practical. In addition, you must also watch the short film about lab safety before
coming to the first practical, which can be found on Moodle.
At the end of the safety pages, there is a declaration form to acknowledge that you have
read the document and will abide by the rules set out. Copies of the form can be
downloaded from Moodle, or will be available at the first practical class. This form must be
signed, and will be collected during the first practical.
Practical sheets
You are given comprehensive notes for each practical. They are colour-coded according to
purpose. The practical notes must be read before you come to the practical. In
addition, there will be a ‘pre-practical’ quiz available on Moodle that can be
completed prior to the practical class, which will help you to complete your
practical most efficiently.
a) Green sheets
The GREEN SHEETS set the context and state the learning objectives for each practical. If
appropriate, they will indicate the lecture handout material that you might want to review
before reading the practical notes.
b) White sheets
The WHITE SHEETS contain the experimental plan and instructions.
These are NOT a recipe to be read for the first time when you are faced with
the experiment.
Make a practice of scanning the white sheets in advance, a day or two before the practical.
Not to take in every detail, which will only make sense when you have the apparatus in
front of you, but to get an overview of the planned experiment. Once in the laboratory, the
senior demonstrator will give a brief introduction and you should then read and follow the
instructions carefully. Take a few minutes to read through the instructions again at the
beginning of the practical – don’t just dive in, no matter how busy everyone else looks.
Always try to think out the principles of what you are doing as you go along, and to
understand what is going on in the procedures you carry out. Ask the demonstrators – or
they may well ask you first!
Probably the most important contribution you can make to the success of your
experiments is in making up reaction mixtures. THINK about what you are adding, so you
add the CORRECT components. MEASURE and DISPENSE the volume of component
reagents ACCURATELY using the semi-automatic GILSON PIPETTES provided. If in doubt
as to how to use a Gilson properly watch the video on the course Moodle site.
c) Blue sheets
The BLUE SHEETS are for processing the data and also contain questions about the
practical to help you understand what you have done, and why. They are a form of self-
assessment – we don’t take them in and give marks, but demonstrators can advise and
comment on your efforts. You will find them helpful when preparing for the end of year
exam, so it is important to get them filled out properly. Going from raw numbers that
come straight off an instrument to a meaningful calculated result is found quite difficult by
most students, and may be tested in the examination. So take advantage of the help
available.
You should complete the Blue Sheets during the practical if that is feasible, or as soon after
as possible after each practical. Bring your calculators to each session. Graph paper is
available if you don’t have your own. Compare your results against the yellow sheets (see
below) or discuss with a demonstrator.
d) Yellow sheets
The YELLOW SHEETS contain specimen results and, where relevant, examples of how to
analyse them. They will generally be handed out either at the end of the practical or at the
appropriate discussion period.
e) Pink sheets
The PINK SHEETS contain important background material related to several practicals and
practical aspects of biochemistry. They are put at the front of the book of practical notes as
appendices to this introduction. They include an introduction to units, to pH and buffers,
and to the principles of spectrophotometry. Try and have a brief look at them before your
first practical. Raise any difficulties with your supervisor or a demonstrator.
Recording your results

You should bring your own notebook or loose-leaf book along to the practical class. In each
experiment plan what observations you will make. Record your results and interpretations
directly into the notebook as you go along rather than on scraps of paper that will surely
get lost. Also record any arguments and conclusions from your data if there is not enough
space for these on the summary sheets.
You should endeavour to complete this writing up of your argument and conclusions during
the experiment itself. There is no need to write out again what is already in your practical
sheets. Concentrate on ensuring that you get down the key arguments and conclusions.
Any writing up after the experiment is not expected to take more than about
an hour for a day's practical, but it is important to do it at once while the
experiment is still fresh in your mind.
The results of an experiment are often best recorded graphically as well as numerically. So
far as possible the graph is best drawn while you are doing the experiment as you go along,
so that in doubtful regions points may be repeated or additional ones obtained. The
conventional way of plotting a graph is to plot the dependent variable (the thing you
measure) on the ordinate (vertical axis) against the independent variable (the thing you fix)
on the abscissa (horizontal axis). Remember to label the axes with the quantity being
plotted and units involved. Remember, too, that the way you plot a graph shows your
interpretation of the experiment. Consult Appendix 1 on “Units” for further guidance.
Your notebooks are not “marked” as part of a “summative assessment” or examination.

Rather they are to help you get to grips with each practical as you do it and write it up so
that you still understand it when it comes to revision later in the year. To help with this, we
ask you to complete the Blue Sheets (see above). If you feel all at sea, or if your Blue Sheets
are full of blank spaces make a point of asking a demonstrator for help.
Moodle and the Practicals

There is a significant amount of information online to support the practical classes, and it is
important that you access this prior to beginning your first practical. The information you
will find is as follows:
• A film that provides essential safety information for the classroom – this must be
watched prior to your first practical class
• Films that provide information about practical lab techniques (E.g. use of Gilson
micropipettes)
• Pre-practical quizzes that are available before the start of each practical class.
• Interactive practicals materials (new for 2018/19) – great for checking you
understand the techniques used in the practical classes
• The various colour coded sheets described above (i.e. green, white, and pink sheets)
• Technique posters that provide a ‘snapshot’ of information that describes commonly
used biochemical techniques. This knowledge is typically assumed in lectures and
practical classes, so use these resources to remind yourself of necessary techniques.
• Tutorial videos to explain biochemical methods (E.g. primer design)
• Materials required for the Journal Clubs (Michaelmas and Lent Terms) and the
Experimental Design Exercise (Lent Term). An additional Experimental Design
exercise is also included on Moodle if you want to give it a go.
Journal Clubs
There are two Journal Clubs, one on a molecular topic and the other more cell biological.
You will be given a published paper, with some guidance notes and questions, to analyse
during the week before the practical session in which you critically evaluate its merits in a
small group directed by one of the Biochemistry staff. Most students find this a challenging
but worthwhile exercise, since it gives exposure to the raw material of the scientific
literature.
Experimental Design Exercise

There will be a teaching session on experimental design in Lent Term. You will be briefed in
advance of the session. You will be allocated to groups and given an outline of a problem to
address. You will be required to design an experimental strategy to investigate the problem
and will report back as a group to a discussion session, led by a member of staff.
Examinations
General advice on examination skills and on the criteria used for marking and classing have
been drawn up by the Faculty of Biology and the Management Committee for the Natural
Sciences Tripos. They can be found by following links from these web sites
http://www.biology.cam.ac.uk/exams/skills
http://www.natsci.tripos.cam.ac.uk/exams
https://www.biology.cam.ac.uk/exams/raven/marking-tripos
Some more specific information relating to the Biochemistry & Molecular Biology
examination is given below. Copies of recent past papers are provided on Moodle. Answers
to the data-handling questions in Paper 3 have been made available to your supervisors; it’s
better for you to try the questions before looking at the answers! Quizzes based on the
data-handling questions in Paper 3 from previous exams can also be found on Moodle.
General University guidance can be found at:

http://www.admin.cam.ac.uk/students/studentregistry/examinations/
Reports from the Senior Examiners are also available on Moodle.

BMB Introduction NST 2018-19
NST Part IB Biochemistry & Molecular Biology

The examination consists of three papers, each of three hours’ duration, and each paper
carries equal marks.
Paper 1 consists of five sections, and students should answer one question from each
section. Sections A and B will be based on lectures in the Michaelmas Term, Sections C and
D on lectures in the Lent Term and Section E on the Easter Term lectures. Each section
will carry equal marks.
Paper 2 consists of two sections. Section A is made up of roughly 14 short-answer factual

questions based on the whole lecture course, and carrying equal marks. Students should
attempt all questions in Section A. Section B consists of four or five essay questions of a
more general and wide-ranging nature than those in Paper 1, from which students need to
answer two. These can be perceived as “open-ended” questions in which the student may
be asked to develop an issue that was not specifically dealt with in the lectures, but is based
on the material taught in the lectures. Sections A and B will carry equal marks.
Paper 3 is concerned with practical techniques covered in the practical classes and journal
clubs, as well as other material in the practical section of the course handbook and
experimental techniques covered in the lecture course. There are three sections. Sections
A and B each contain two to three questions while Section C consists of one longer
subdivided question. Sections A, B, and C will carry equal marks. All questions in Paper 3
should be attempted.
When answering essay questions, take particular care that you have absorbed what the
question is specifically asking for. It is a common fault for candidates to react unthinkingly to
a "trigger word" and simply write all they know in response. Take a little time to reflect,
rather than leaping straight in.
The examiners will have regard to the style and methods of candidates' answers. You
should write legibly and not adopt note form unless specifically requested to do so. Helpful
diagrams are welcome as part of an essay. You are perfectly free to use abbreviations that
are standard scientific vocabulary without definition (E.g. G6P, ATP, DNA, RNA).
18 © Department of Biochemistry, 2018

BMB Lecture Timetable NST 2018-19
BMB Lecture Timetable 2018-19

Course Organiser: Dr Dee Scadden (adjs100@cam.ac.uk)
All lectures start at 10am in the Sanger Building Lecture Theatre, Department of
Biochemistry, Old Addenbrooke’s site. Entrance from Tennis Court Road (see map).
Note that some lectures begin earlier in Term, and end later in Term, than is usual.
This is to allow more time between the end of the course and the examinations.
MICHAELMAS TERM: Genes and proteins; macromolecules in action

First Lecture on Friday, October 5th; Last Lecture on Friday, November 30th
Most relevant
Date No. Title Lecturer
Techniques Posters
Introduction to the course
Oct 5 Dr ADJ Scadden
(in lecture 1)
1, 2, 4, 9, 13, 15, 16, 17,
Oct 5, 8, 10,
5 Gene cloning and manipulation Dr ADJ Scadden 19, 21, 20, 22, 26, 28,
12, 15
29, 30, 31, 33
Oct 17, 19, Post transcriptional control of gene
5 Dr J Mata
22, 24, 26 expression
Nucleic acid structure, protein-
Oct 29, 31
5 nucleic acid interactions and Prof. BF Luisi
Nov 2, 5, 7
transcription
Nov 9, 12, Protein structure, function and
5 Dr M Hyvönen
14, 16, 19 evolution
Nov 21, 23, Enzyme catalysis and protein
5 Prof. F Hollfelder
26, 28, 30 Engineering
LENT TERM: Energy transduction, cell signalling and cell proliferation

First Lecture on Wednesday, January 16th; Last Lecture on Friday, March 15th
Most relevant
Techniques Posters
Jan 16, 18, 21,
6 Control of metabolism Prof. KM Brindle
23, 25, 28
Jan 30, Feb 1, Energy transduction in bacteria,
6 Prof. GC Brown
4, 6, 8, 11 mitochondria and chloroplasts
Feb 13, 15, Transmembrane signalling;
6 Prof. S Lummis
18, 20, 22, 25 molecules and mechanisms
Feb 27, Mar
4 Control of eukaryotic cell growth Prof. DM Carrington
1, 4, 6
March 8, 11, Oncogenes, tumour suppressor Prof. GI Evan &
4
13, 15 genes and cancer Dr TD Littlewood

BMB Lecture Timetable NST 2018-19
EASTER TERM: Biochemistry of Microorganisms

First Lecture on Wednesday, April 24th; Last Lecture on Monday, May 13th, followed by
practical exam discussion.
Most relevant
Techniques Posters
April 24, 26,

3 Bacterial chemotaxis Dr R Monson
29
Bacterial signalling and secretion

May 1, 3 2 Prof. GPC Salmond
systems
May 6, 8, 10,
4 Molecular biology of protozoa Prof. DM Carrington
13

BMB Practical Timetable NST 2018-19
BMB Practical Timetable 2018-19

Practicals are held in the Biochemistry Teaching Laboratory, Hopkins Building, Tennis Court
Road, commencing at 11am, unless stated otherwise. Monday sessions commence at
12 noon. Enter the classroom from the door opposite the Genetics Department (see map
at the start of this handbook).
MICHAELMAS TERM 2018

Senior Most relevant
Dates Practical
Demonstrator techniques posters
Safety
Week 1
Cloning of Oct1 POU domain gene into E. 2, 15, 26
October 4 – 10 Dr D Scadden
coli plasmid. Restriction mapping of plasmid.
Week 2 Expression of POU domain in E. coli. Melting

Prof. B Luisi
October 11 – 17 properties of DNA.
Week 3
Purification of POU domain Prof. N Gay 13, 1, 3, 15
October 18 – 24
Week 4 5, 7, 13, 15, 16, 18, 20,
Electrophoresis mobility shift assay Dr N Standart
October 25 – 31 22, 28
Week 5 Using Online Databases as Tools

Dr D Scadden 13, 26
November 1 – 7 Venue: Craik Marshall Building 1.30 pm
Discussion in lab of weeks 1-5: 11.00 am Dr J Rees
(Monday 12 noon)
Week 6
November 8 –14 Journal Club (2 pm, except Monday 3 Staff
22, 34
pm)
Venue: see Moodle
Week 7 Reactivity and enzyme catalysis
Prof. F Hollfelder
November 15 – 21 Venue: see Moodle.
Week 8 Protein structure by molecular graphics

Dr RW Broadhurst
November 22 – 28 Venue: Craik Marshall Building, 1.30 pm

BMB Practical Timetable NST 2018-19
LENT TERM 2019

Dates Practical
Week 1
Subcellular fractionation Dr M de la Roche 15
January 17 – 23
Week 2
Kinetic analysis of catalysis by chymotrypsin Dr S Weyand
January 24 – 30
Experimental design: 11-1 pm (Monday Staff

3pm start) Prof. M Carrington
Week 3 Venue: see Moodle
January 31 – February
6 Metabolic Control in silico: 2.00-4.30pm
(Monday 12 noon start) Prof. J Griffin
Venue: Craik Marshall Building
Week 4
Mitochondrial oxidative phosphorylation Prof G Brown
February 7 – 13
Week 5 Cell signalling (1): Tyrosine phosphorylation Prof S Lummis/ Dr

15, 33
February 14 – 20 in platelets A Git
Week 6 Cell signalling (1): Tyrosine phosphorylation Prof S Lummis/Dr A

15, 33
February 21 – 27 continued and discussion Git
Week 7 Cell signalling (2): Thromboxane production 10, 37, 38, 1, 19, 35,
Dr M Hyvönen
February 28 – March 6 by platelets 28, 33
Cell signalling (2): Thromboxane production
by platelets: data analysis & discussion Dr M Hyvönen
Week 8 (11am; Monday 12 noon). 10, 37, 38, 1, 19, 35,
March 7 – 13 28, 33
Journal Club (2pm, except Monday 3pm) Staff
Venue: see Moodle
EASTER TERM 2019

Dates Practical
Week 1
Microbial Biochemistry Prof. G Salmond
April 25 – May 1
Week 2
Microbial Biochemistry Prof. G Salmond
May 2 – May 8
Revision Session – see lecture timetable

BMB Reading List NST 2018-19
Reading List
For NST Part IB Biochemistry & Molecular Biology, 2018-2019
Most of these books should be available in your College library, but to give as many
students as possible an opportunity to use them on a regular basis the Department of
Biochemistry also keeps copies in the Part I Section of the Colman Library (in the Hopkins
Biochemistry building, facing Tennis Court Road on the Downing Site). Selected books may
be borrowed overnight from this collection. You are also welcome to make use of the main
collection of books and journals, but borrowing these is not permitted. Part II and Part III
Biochemistry students have priority for seating in the library. The Assistant Librarian is
available to help locating books. Part IB BMB students who wish to use the library outside
the hours of 8.30 am – 5 pm, Monday-Thursday, or 8:30 am - 4 pm, Friday, should ask the
Hopkins building receptionist to programme their University proximity card for access.
There are a number of very good biochemistry text books available – some of which are
quite general and cover similar topics, while others are more specialized. It’s probably a
good idea to primarily use the ‘General Biochemistry Texts’, then use the others to dip in
and out of as required. The use of relevant textbooks is essential for underpinning the
material given to you in lectures – do make the most of the resources available to you.
Recommended books
If you wish to buy, we suggest that you browse first in libraries, book shops or third-years’
book collections.
a. General Biochemistry Texts

There are good companion web sites that give further information about these books, and
contain useful onward links.
• Biochemistry, Berg, J. M., Tymoczko, J. L. and Stryer, L. (Freeman, 8th edition, 2015).
For access to Student Resources see:
www.macmillanlearning.com/Catalog/product/biochemistry-eighthedition-
berg/studentresources - tab
• Molecular Biology of the Gene, Watson, J. D. et al. (Pearson Education, 7th edition).
• Lehninger Principles of Biochemistry, Nelson, D. L. & Cox, M. M. (Freeman, 6th

edition, 2017).
Particularly useful for bioenergetics and metabolism. Good for signalling. For access to Student
Resources see:
www.macmillanlearning.com/Catalog/product/lehningerprinciplesofbiochemistry-
sixthedition-nelson/studentresources - tab
• Molecular Biology of the Cell, Alberts, B. et al. (Garland Science, 6th edition 2014).
The largest and most comprehensive of the cell and molecular biology textbooks, now with a
CD-ROM as well as a revised Problems Book. Will be useful for Part II courses as well as IB.

www.garlandscience.com/product/isbn/9780815345244
• Molecular Biology of the Cell – The Problems Book. Wilson, J. & Hunt, T.
(Garland Science, 6th edition 2014).
b. Books on specific topics
Protein structure, function and evolution

• Protein Structure and Function, Petsko, G. & Dagmar, R., 2008
Enzyme catalysis and protein engineering:

• Introduction to Protein Structure, Branden, C. & Tooze, J. 2nd edition, 1999.
• Biochemistry, Abeles, R.H., Frey, P.A. & Jencks, W.P., 1992.
• Proteins, Creighton, T.E. 2nd edition, 1995.
• From Enzyme Models to Model Enzymes, Kirby, A.J. & Hollfelder, F., 2009.
• Structure and Mechanism in Protein Science – A guide to Enzyme Catalysis and Protein
Folding, Fersht, A., 1999.
• Enzymatic Reaction Mechanisms, Frey, P.A. and Hegemann, A.D., 2007.
Energy transduction
• Bioenergetics, Nicholls D.G. & Ferguson S.J. 4th edition, 2013.
• Molecular Mechanisms of Photosynthesis, Blankenship R.E. 2nd edition, 2014.
Oncogenes, tumour suppressor genes and cancer:

• The Biology of Cancer, Weinberg, R. Garland Science, 2nd edition, 2014.
The most comprehensive cancer biology textbook complete with a CD-ROM. Will be useful for
Part II courses as well as IB.

Other sources of information
a. Journals – well worth consulting

Research-oriented summaries (2-4 pages) of important topics in biochemistry and
molecular biology are contained in the monthly journal Trends in Biochemical Sciences (usually
known as TIBS). A similar journal is Bioessays. The Scientific American usually contains at least
one biochemical article per issue and is well worth a glance. Copies of these journals are
found near the Part I reading area.
b. Web Sites
Ever more useful source of information. We shall just give here two of our favourite
general addresses, which have many forward links.
• Online Mendelian Inheritance in Man
(www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM)
• Swiss Institute of Bioinformatics (www.expasy.ch/)
c. Links to literature sources on Moodle

The Reading list and links to Textbooks and Journals can be found on Moodle in the book
entitled “Course Information”.
• Textbooks via pubmed

• Links to Electronic Journals via the University
• Trends in Biochemical Sciences (TiBS)
• Current Opinions in Chemical Biology
• Current Opinions in Structural Biology

Plagiarism NST 2018-19
Plagiarism
As of October 1st 2018 the university has adopted the definition below:
“Plagiarism is defined as submitting as one’s own work, irrespective of intent to deceive, that which
derives in part or in its entirety from the work of others without due acknowledgement; or, in the
case of self-plagiarism, unless explicitly permitted by regulation, submitting one’s own work that has
already been submitted for assessment to satisfy the requirements of any other academic
qualification, or submitted for publication without due acknowledgement. It is both poor scholarship
and a breach of academic integrity.”
(http://www.admin.cam.ac.uk/univ/plagiarism/students/statement.html)
Although there is no assessed course work for Biochemistry and Molecular Biology it is an
essential part of your scientific training that, in your supervision work and any other
writings, you ensure you are following best practice regarding avoiding plagiarism.
General university guidelines are available at:

http://www.admin.cam.ac.uk/univ/plagiarism/students/statement.html
Faculty guidelines are available at:

http://www.biology.cam.ac.uk/exams/plagiarism and are reproduced below.
Note that the use of essays purchased from any source or copied from other students is
unacceptable regardless of whether the source is acknowledged.
It is an important aspect of academic integrity to cite all sources on which you base your
work (even if it is not copied directly from them), be they published in hard copy or web-
based. Your supervisor may ask you to do this for written work produced during the year,
and this can be useful for your subsequent revision. However, full citation is not expected in
written unseen examinations such as those taken at the end of the year for BMB.
If you still have questions after reading the faculty guidelines below, you should talk to your
Supervisors and /or Director of Studies and/or the Course Organiser.
The following guidance has been issued by the Faculty Board of Biology:
In general, plagiarism can be defined as:
“The unacknowledged use of the work of others as if this were your own original work”
In the context of an examination, this amounts to:

Passing off the work of others as your own to gain unfair advantage.
Such use of unfair means will not be tolerated by the University; if detected, the penalty
may be severe and may lead to disciplinary proceedings being taken against you.

1. The scope of plagiarism

Plagiarism is defined as submitting as one's own work, irrespective of intent to deceive, that
which derives in part or in its entirety from the work of others without due
acknowledgement. It is both poor scholarship and a breach of academic integrity.
Examples of plagiarism include copying (using another person’s language and/or ideas as if
they are a candidate’s own), by:
• quoting verbatim another person’s work without due acknowledgement of the
source;
• paraphrasing another person’s work by changing some of the words, or the order
of the words, without due acknowledgement of the source;
• using ideas taken from someone else without reference to the originator;
• cutting and pasting from the Internet to make a pastiche of online sources;
• submitting someone else’s work as part of a candidate’s own without identifying
clearly who did the work. For example, buying or commissioning work via
professional agencies such as ‘essay banks’ or ‘paper mills’, or not attributing
research contributed by others to a joint project.
Plagiarism might also arise from colluding with another person, including another
candidate, other than as permitted for joint project work (i.e. where collaboration is
concealed or has been forbidden). A candidate should include a general acknowledgement
where he or she has received substantial help, for example with the language and style of a
piece of written work.
Plagiarism can occur in respect to all types of sources and media:

• text, illustrations, musical quotations, mathematical derivations, computer code, etc.
• material downloaded from websites or drawn from manuscripts or other media
• published and unpublished material, including lecture handouts and other students’
work
Acceptable means of acknowledging the work of others (by referencing, in footnotes, or

otherwise) vary according to the subject matter and mode of assessment. Faculties or
Departments should issue written guidance on the relevant scholarly conventions for
submitted work, and also make it clear to candidates what level of acknowledgement might
be expected in written examinations. Candidates are required to familiarize themselves with
this guidance, to follow it in all work submitted for assessment, and may be required to sign
a declaration to that effect. If a candidate has any outstanding queries, clarification should be
sought from her or his Director of Studies, Course Director or Supervisor as appropriate.
Failure to conform to the expected standards of scholarship (e.g. by not referencing
sources) in examinations may affect the mark given to the candidate's work. In addition,
suspected cases of the use of unfair means (of which plagiarism is one form) will be
investigated and may be brought to one of the University's Courts. The Courts have wide
powers to discipline those found guilty of using unfair means in an examination, including
depriving such persons of membership of the University, and deprivation of a degree.

2. How to avoid plagiarism

The stylistic conventions for different subjects vary and you should consult your Course
Organiser or project supervisor about the conventions pertaining in your particular subject
area. Most courses will issue written guidance on the relevant scholarly conventions and
you are expected to have read and to follow this advice. However, the main points that
apply to submitted work (e.g. dissertations, project reports) are:
• when presenting the views and work of others, include in the text an indication of the
source of the material, e.g. 'as Sharpe (1993) has shown,' and give the full details of the
work quoted in your bibliography
• if you quote text verbatim, place the sentence in inverted commas and give the
appropriate reference, e.g. 'The elk is of necessity less graceful than the gazelle'
(Thompson, 1942, p 46) and give the full details in your bibliography as above
• if you wish to set out the work of another at length so that you can produce a counter-
argument, set the quoted text apart from your own text (e.g. by indenting a paragraph)
and identify it by using inverted commas and adding a reference as above. NB long
quotations may infringe copyright, which exists for the life of the author plus 70 years
• if you are copying text, keep a note of the author and the reference as you go along,
with the copied text, so that you will not mistakenly think the material to be your own
work when you come back to it in a few weeks' time
• if you reproduce an illustration or include someone else's data in a graph include the
reference to the original work in the legend, e.g. (figure redrawn from Webb, 1976) or
(triangles = data from Webb, 1976)
• if you wish to collaborate with another person on your project, you should check
with the Course Organiser to see whether this might be allowed and then seek their
permission
• if you have been authorised to work together with another candidate or other
researchers, you must acknowledge their contribution fully in your introductory
section. If there is likely to be any doubt as to who contributed which parts of the
work, you should make this clear in the text wherever necessary, e.g. 'I am grateful to
A. Smith for analysing the sodium content of these samples'
• be especially careful if cutting and pasting work from electronic media; do not fail to
attribute the work to its source. If authorship of the electronic source is not given, ask
yourself whether it is worth copying
Please note that during written answers for unseen examination papers, you will not be penalised
for failures to reference information in this manner.
3. The Golden Rule:

The examiners must be in no doubt as to which parts of your work are your
own original work and which are the rightful property of someone else.
For the University-wide statement on plagiarism, and further information on the topic, please see:
http://www.admin.cam.ac.uk/univ/plagiarism/

Synopsis of the BMB lecture course NST 2018-19
Synopsis of the BMB lecture course

Note: This information is provided at the beginning of the year for your guidance and that of your
supervisors. It is not intended to be a comprehensive list of contents. Lecturers will all issue their
own handouts, and may vary the topics and the order in which they are presented.
Michaelmas Term: Genes and proteins – macromolecules in action

In Michaelmas, the course examines the molecular biology of DNA and protein structure.
How is DNA packaged in cells? How does chromatin structure affect gene expression?
How is genetic engineering actually carried out? How are transcription and translation
regulated? What are the principles of protein design and how can we exploit them through
protein engineering?
§ Dr D Scadden: Gene cloning and Manipulation

These lectures introduce the techniques of gene cloning and manipulation that underpin
much of the work described in the rest of the course. Building on material covered in the
Part IA Biology of Cells lectures, we look at the use of various techniques to ask specific
experimental questions. Examples of topics covered include:
• The polymerase chain reaction and its various applications
• Vectors and hosts that are used in conventional gene cloning
• Investigating how clones may be used experimentally. E.g. for preparing recombinant
fusion proteins, making RNA for in vitro studies, etc.
• Methods for reducing gene expression (e.g. RNAi, CRISPR/Cas9), and for creating
transgenic mice.
§ Dr J Mata: Post-Transcriptional Control of Gene Expression

The production of functional proteins involves multiple processes in addition to
transcription. Although these steps are usually referred to as post-transcriptional, many of
them occur concurrently with transcription. These lectures will introduce the processes
required for the formation of a mature RNA in eukaryotic cells (capping, splicing and 3’ end
processing), translation (in both prokaryotes and eukaryotes) and RNA decay. The basic
machinery that carries out these processes, as well as the mechanisms by which this
machinery is modulated in a gene-specific manner, will be addressed.
§ Prof. B Luisi: Nucleic acid structure, Protein-Nucleic acid Interactions and Transcription
These lectures cover the first step in gene expression – transcription of RNA using genomic
DNA as template. How do RNA polymerases recognise the correct locations at which to
initiate transcription, and how can this be regulated? Six main topics will be covered:
• DNA and RNA structure
• Prokaryotic transcription mechanisms
• Prokaryotic transcriptional regulation
• Packaging of eukaryotic DNA into chromatin
• Eukaryotic transcription – core promoter and general transcription factors (GTFs)
• Eukaryotic transcription – activating transcription factors and enhancers

The overarching theme of DNA-protein interactions – both sequence-specific and non-

specific – runs through all of these topics. At appropriate points, relevant experimental
approaches and techniques will be highlighted.
§ Dr M Hyvönen: Protein Structure, Function and Evolution

Proteins play most of the effector roles in living organisms. They maintain the structures of
cells, of the extracellular matrix and tissues; they catalyze most reactions in cells and
generate mechanical force in the muscles; they are involved in information transfer through
recognition of other molecules and can act as ligands, as receptors, as messengers, and as
transcription factors; they act as receptors, gates and channels in membranes. The aim of
these lectures is to understand the unique principles of protein structure from primary
structure to formation of large oligomeric complexes and molecular machines and to
introduce the methods that are used to study protein structures from optical
spectroscopies through X-ray crystallography and NMR to cryo electron microscopy. We
will also discuss how proteins have evolved and how analysis of protein structure can help
us to understand the evolutionary relationships between different proteins and their
function.
§ Prof. F Hollfelder: Enzyme Catalysis and Protein Engineering

This lecture series focuses on how the peptide and protein structures discussed in the
preceding module can assume functions – and on experiments that delineate the
mechanisms involved. First we develop ideas about enzyme catalysis, mechanism and
kinetics. We look in detail at the cooperative (allosteric) molecular basis of metabolic
regulation. Other protein structures that are discussed include immunoglobulins and their
binding to specific antigens, and the principles of protein folding and stability. Finally we look
at the ‘holy grail’ of protein engineering and mechanistic enzymology – how to create novel,
functional proteins, by rational design, semi-rational approaches, and by directed evolution.
Lent Term: Energy transduction, cell signalling and cell proliferation

The course now builds on the molecular foundations laid in the Michaelmas Term to
develop an integrated view of cellular processes. How do cells make a continuous supply
of energy available for transcription, translation, ion pumping, biosynthesis and a host of
other processes? How is metabolism regulated according to the varying needs of the cell?
What are the mechanisms by which hormones regulate intracellular processes? How is
normal eukaryotic cell growth controlled, and what goes wrong when such control is
pathologically disturbed in cancer?
§ Prof. K Brindle: Control of Metabolism

The aims of these lectures are:
• To examine the different ways in which enzyme activity may be controlled.
• To consider the benefits these different modes of control offer for the regulation of
flux in metabolic pathways.
This discussion takes place in a wider context, as these various modes of control are
employed throughout biological systems. Textbook descriptions of control in the metabolic
pathways tend to assume that the enzymes involved are ‘soluble’ and homogeneously
distributed in the cell cytoplasm. We will see how this is not the case: rather, a high degree
of spatial organisation is critical to the control of these pathways.

Various experimental approaches are described for studying how metabolism is controlled,
with particular emphasis on methods that may be used to study intact systems. These
include:
• Metabolic control analysis, which allows for quantitative determination of the
importance of any enzyme for flux control in vivo.
• Two key non-invasive spectroscopic techniques – fluorescence and NMR – that permit
the study of metabolic events in intact cells and tissues.
§ Prof. G Brown: Energy Transduction in Bacteria, Mitochondria and Chloroplasts

Bioenergetics is the study of how energy is acquired and used in living systems. Recent
discoveries of key structures and mechanisms have greatly enhanced our understanding of
this process. This knowledge is being applied to medicine, nanotechnology, and the energy
industries, informing our attempts to develop renewable biological energy sources. The six
lectures explore how bacteria, plants and animals use light, electrons, protons and ATP to
transduce energy from the sub-molecular to the cellular level. The lectures use an
evolutionary emphasis to make it easier to understand the diversity of bioenergetics
systems in nature.
§ Prof. S Lummis: Transmembrane Signalling: Molecules and Mechanisms

Cells are continuously bombarded by many different types of signal; the ability of these cells
to respond appropriately to such signals is critical for cell survival, adaptation, and
specification of function, whether they are individual amoebae or components of a large,
complex organism such as a human. This lecture course explores how cells monitor the
presence of specific extra-cellular signalling molecules and how these signals then instigate
and drive complex and interwoven intracellular responses. The course will focus on:
• The diversity of signals carrying information to cells; these range from single photons
and small molecules to complex proteins.
• The relatively few mechanisms, usually involving plasma membrane receptors, by which
the cell perceives the signal.
• The means by which the cell decodes 'the message', a process which may be very rapid,
as in neurotransmission, or much slower, as in the signals that regulate gene expression
and control growth.
The lectures will, for example, examine the roles of the ‘second messengers’ that often
mediate part of cell signalling cascades, and will explore how these cascades allow very low
concentrations of initiating signals to generate large responses in their target cells. Special
attention is paid to G-protein coupled responses, and to the multiple roles played by
protein phosphorylation in relaying intracellular signals.
This lecture course will be complemented by two successive practical classes in which
students gain hands-on experience of the techniques used to probe the roles of proteins in
three different cell signalling pathways.
§ Prof. M Carrington: Control of Eukaryotic Cell Growth

The cell cycle is the term used to describe the succession of events that occur to produce
two cells from one. An understanding of the molecular events involved in progression
through the cell cycle is central to solving the larger problems of how the tightly controlled
expansion of cell populations during the development and growth of any organism occurs

and how the loss of regulation of the cycle results in disease – not just cancer but also the
inappropriate growth of normal cells.
The aims of the lectures are:
• To give an understanding of the experimental approaches that can be taken to
investigate the molecular machinery of a complex biological process.
• To explain how the molecular components that regulate cell cycle progression were
identified and how their function was determined.
• To discuss a model of how the ordering of transitions that ultimately lead to cell
division is regulated.
§ Prof. G Evan & Dr T Littlewood: Oncogenes, Tumour Suppressor Genes and Cancer
The next four lectures build on the story of the cell cycle in eggs and yeasts by describing
how normal mammalian cell proliferation is controlled. The focus is on the mechanisms of
normal signalling pathways – growth factors and mitogens, their receptors and the
mitogenic signals they generate inside the cell, and the pathways that then transduce such
mitogenic signals to the various intracellular effectors that precipitate cell growth and
replication. The principal effector responses to mitogenic signalling are transcriptional
activation of proliferation-associated and cell survival genes and repression of growth
suppressing genes, activation of RNA and protein synthesis, and an abrupt shift of
metabolism to biosynthesis and aerobic glycolysis.
These lectures address the question of what happens in diseases, such as cancer, where
control of cell growth, proliferation, survival and migration is lost through activating
mutations in proto-oncogenes, and inactivating mutations in tumour suppressor genes. This
introduction to molecular oncogenesis sets the scene for a more comprehensive analysis of
cancer biology in one of the Part II Biochemistry courses.
Easter Term: Biochemistry of Microorganisms

This final group of lectures considers bacteria and protozoa as model systems and the
course now brings together the themes explored in the first two terms to examine key
questions relating to prokaryotic and protist biochemistry such as motility, chemotaxis and
the importance of protein targeting and other systems in virulence and pathogenicity.
§ Dr R Monson: Bacterial Chemotaxis and Signal Transduction

The field of bacterial chemotaxis and motility encompasses perhaps the best-understood
prokaryotic signalling pathway. We start by using video footage of motile E. coli cells to
define the basic swimming behaviour of bacteria in the unstimulated state. We then look at
how this behaviour is altered when the cells are challenged with chemostimuli, and
demonstrate that the observed changes correlate with the sense of flagellar motor rotation.
The altered bias in flagellar motor rotation brought about by exposure to chemostimuli
causes structural changes in the architecture of the flagellar filaments, and we examine how
these subtle molecular alterations can give rise to substantial changes in the behaviour of
the whole cell.
We also look at how the molecular components of the chemotaxis and motility apparatus
of the cell were discovered, and at the techniques that have been used to piece together
the complex signal transduction pathway that is involved in integrating the multiple
chemosensory inputs received by the cell at any given time into a single output. This signal
transduction pathway involves multiple protein components, transient protein-protein

interactions, phospho-transfer events and other chemical modifications, and its workings
are now beginning to be understood at the atomic level.
We look at how the signalling pathway is assembled, how it works, and how its output
influences the rotational bias of the flagellar motor (and therefore, ultimately, the swimming
behaviour of the cell). Finally, we look at what is known about the flagellar motor itself –
the world’s smallest multi-speed motor, incorporating both forward and reverse gears. The
ingenious methods that have been developed to study this remarkable device are discussed,
including some video footage of the motor in action. Moreover, the study of chemotaxis
and motility is not simply an esoteric branch of microbiology. With the recent completion
of many eukaryotic genome sequences (including the human genome), it has become clear
that homologues of the chemotaxis proteins are widespread in “higher” organisms, so these
findings are likely to yield valuable insights into the function of many other organisms.
§ Prof. G Salmond: Bacterial Secretion Systems

Protein secretion mechanisms are of fundamental importance in bacteria. Prokaryotes are
highly tractable model systems for analysis of the basic principles of protein targeting and
the ability to manipulate such targeting can be exploited in some biotechnological
processes. Furthermore, the ability to actively secrete and regulate the production of
structurally diverse proteins involved in bacterial virulence is a key aspect of pathogenesis in
plant and animal diseases. The lectures summarise the main protein secretion systems in
Gram-negative bacteria, with examples taken from pathogens of animals and plants.
Evolutionary connections between secretory machines is highlighted.
The general nature of bacterial cell surfaces is discussed and the exploitation of prokaryotic
surface molecules that are parasitized as “receptors” by bacterial viruses (bacteriophages) is
highlighted.
In the lab classes associated with these lectures, students conduct experiments on protein
targeting using bacterial mutants generated via transposon insertions that can generate
protein fusions. In addition, global gene regulation and intercellular chemical signaling
(quorum sensing) in a bacterium that makes antibiotics are both addressed.
§ Prof. M Carrington: Molecular Biology of Protozoa

Protozoa encompass over 60,000 species of eukaryote including many that are highly
divergent from animals, there is more evolutionary diversity within the protozoa than
between green plants, metazoa and fungi. The best-studied protozoans are parasites that
cause diverse chronic diseases, such long-term infections provide a model for studying the
complex molecular interactions between pathogen and host.
Trypanosome VSGs have divergent primary, but conserved tertiary, structures to function
in antigenic variation and as a protective coat on the external surface of the plasma
membrane.
Protozoa have evolved a number of novel strategies for overcoming host resistance to
infection and, in this context, lectures will address the unusual strategies for regulation of
gene expression, especially of the Variable Surface Glycoproteins (VSGs) in trypanosomes.
Such studies of parasitic protozoa have provided unique insights into our understanding of
basic molecular processes, for example, the structure and biosynthesis of GPI anchors in
the context of cell surface architecture.

Course Management and Student Feedback NST 2018-19
Course Management and Student Feedback

The Biochemistry & Molecular Biology Course is run by a Management Committee,
consisting of the course organiser and lecturers from the course. The Management
Committee decides broadly on the content of the lectures and practicals, and has the
responsibility for organising and delivering these.
Decisions made by the Management Committee are influenced by the Consultative
Committee, which comprises student representatives (chosen by you) and lecturers
from the course. The Consultative Committee meets at the end of each term (dates below)
and looks at the results of student feedback questionnaires as well as hearing your views
about the course.
The course is revised on a yearly basis in the light of comments made by you in student
feedback questionnaires and by your representatives on the Consultative Committee.
Dates for the Consultative Committee Meetings for BMB:

• Friday November 30th, 11.15am
• Friday March 15th, 11.15am
• Monday May 13th, 11.15am
Minutes of the Consultative Committees and Analysis of Student Feedback

Questionnaires
The minutes of the Consultative Committee meetings and the results of the student
feedback questionnaires can be found on Moodle.
Contact Information:
Contact Name Email Role

Chair of the Academic issues
Management Dr Dee Scadden adjs100@cam.ac.uk relating to the
Committee course
Organisation of
meetings of the
Teaching
Wendy Bundy tchasst@bioc.cam.ac.uk Consultative and
Administrator
Management
Committees
Classroom Organisation of the
Dr Siolian Ball slrb2@cam.ac.uk
Manager practical laboratory

Safety Information
Part I Practical Classroom
Safety in the Biochemistry Part I Practical Teaching Laboratory
These pages MUST be read before your first practical.
The ‘Safety Acknowledgement and Compliance Form’ and the ‘Health Record Form’
MUST be read, then completed and signed. Please note that the forms in this
handbook are for INFORMATION ONLY, and forms for completion will be provided
in your first practical class.
Alternatively, copies of these forms can be downloaded from Moodle in the file “Experimental
Practicals – information for before you begin the course”.
1. Departmental policy
1.1 It is the overriding policy of the University and Department of Biochemistry that
experimental work, whether associated with teaching or research, be done
efficiently and safely. All employees, students and research workers in the
Department are required to observe the provisions of the Health and Safety at
Work etc. Act 1974, the Radioactive Substances Act 1993, the Ionising Radiations
Regulations 1999, and the Control of Substances Hazardous to Health (COSHH)
Regulations 1988, as and when appropriate. Copies of the Acts and Regulations are
available in the University Safety Office; copies of the University and Departmental
Safety Regulations are available in the Teaching Laboratories. Copies of the risk
assessments are available at the end of the Safety Instructions. Copies of the
COSHH assessments will be put up on the notice board in the practical laboratory.
1.2 Safety in the Department begins with the individual's personal responsibility.
However, in addition, each member of the academic staff, each research worker,
and each technician in charge of a section or a laboratory has a statutory duty to
take reasonable care for persons under his/her supervision or visitors to his/her
area.
1.3 It is advisable to inform the Classroom Supervisor if you suffer from any medical
problem (e.g. allergies, asthma, serious hearing or sight impairment etc.) that might
be aggravated by an experiment or might affect your ability to carry out an
experiment.
1.4 The staff involved with practical teaching in the Part I Laboratory have ensured, so
far as is reasonably practical, that students will not be exposed to risks to their
health and safety. Students have a statutory obligation to protect themselves and
others from hazards resulting from their acts or omissions in the laboratories.
2. General
2.1 Smoking, eating and drinking are NOT permitted in the laboratory.
2.2 Outdoor clothing, umbrellas and bags must be placed in the under-bench cupboards
provided for the purpose and not allowed to obstruct gangways or bench tops.
2.3 Suitable laboratory coats must be worn (fastened up) in the laboratory and
removed before leaving. Safety spectacles must be worn when carrying out all
procedures, and gloves of the appropriate type must be worn when necessary.
Long hair must be restrained e.g. by means of a cap, ribbon, headband or net.
BMB Safety Information NST 2018-19
2.4 The notes for each practical should be read before coming to the practical and note
taken of any safety matters. Students are NOT permitted to do any experimental
work unless a supervisor (demonstrator or member of staff) is present.
2.5 The use of unfamiliar equipment (particularly centrifuges, vortex mixers, chart
recorders, spectrophotometers, and oxygen electrodes) and the handling of
potentially hazardous materials will be explained to students. If a student, for some
reason, misses the appropriate explanation, then it is the student’s responsibility to
bring the lack of knowledge to the attention of the Classroom Supervisor, so that
appropriate arrangements can be made to remedy the situation. See 3.2 below.
2.6 Glassware and plastic ware that is being used, for example to make up mixtures,
should be labelled with marker pen; this avoids confusion and will help the
laboratory staff.
2.7 Hazard and other labels that have been fixed to solution containers must NOT be
tampered with or removed.
2.8 Hand-washing facilities are available in the laboratory. Hands should be washed
before leaving.
2.9 Lavatory facilities are available just outside the main entrance to the laboratory.
3. Substances and procedures hazardous to health

3.1 Where a potential hazard (e.g. certain chemicals, biological materials and
procedures) exists in a particular practical, this will be discussed in the talk before
the practical and details of safe working methods will be highlighted in the practical
notes.
3.2 Students must NOT use unfamiliar equipment or procedures without them having
been given instruction. All safety instructions given in the preliminary talk and
practical notes must be adhered to.
3.3 A hazard assessment (COSHH assessment) has been prepared for each practical;
these are available for reference on the notice board situated to the right of the
doorway to the NST IB classroom and exit. Extra copies of the COSHH
assessments for all practicals will be made available either prior or during each
practical session.
4. Waste
4.1 Broken glassware must be placed in one of the special bins provided in the
laboratories where the sign 'BROKEN GLASS DISPOSAL POINT' is displayed. It
must NOT be put into any other waste container.
4.2 Ordinary waste (e.g. tissues, sealing film and its backing paper, gloves) should be
placed in one of the waste buckets provided at the ends of the benches. It must
NOT be put into the bins for broken glass (referred to in 4.1 above).
4.3 Used plastic pipette tips must be placed in the appropriate labelled container on the
bench.
4.4 Certain waste materials (e.g. used lancets, syringe needles, particular chemicals) that
need specific disposal methods must be placed in the appropriate containers

provided during that practical. They must NOT be placed in any other waste
container.
4.5 No liquids should be poured down the sinks. Liquids should be disposed of in the
disposal vessels supplied.
4.6 Benches should be left as waste-free and tidy as possible at the end of each practical
– this reduces potential for accidents and spillages and is of considerable help to the
laboratory staff.
5. Accidents
5.1 All accidents and spillages, including any personal injuries and damage caused to
equipment, must be reported as soon as practicable to the Senior Demonstrator
and Laboratory Manager or any of the other technicians. They will organise first aid
if required and attend to the required written report.
5.2 Spillages must be cleared up immediately and the area decontaminated; they must
NOT be left as a hazard to others.
5.3 A first aid box is located in the Classroom Supervisor’s office area; see the
classroom plan in the practical manual. Consult a Demonstrator or member of the
Technical Staff if you need items from the First Aid Box.
6. Evacuation procedures
6.1 Any fire must be reported immediately to a technician or demonstrator.
6.2 A two-tone siren means evacuate the building immediately, by the designated
routes (see plan).
6.3 In the event of fire do not panic, hesitate, or run; follow any oral instructions
immediately and leave in an orderly manner. Do NOT use the lift.
6.4 The designated fire exits at both ends of the laboratory have clear signs and are
shown on the plan of the laboratory. Students must identify those exits on their
first visit to the laboratory.
7. Summary
Safe working is an attitude of mind, and the result of the sensible application of
acquired knowledge. Always plan experimental work with safety in mind. Anticipate
possible hazards and seek to minimise them, but be alert for the unexpected.
Students should learn to carry out their work safely and maintain their working area
in a safe condition, for the benefit of themselves and others working in the area.
If in Doubt – ASK!!

Please note that the ‘Safety Acknowledgement and Compliance

form’ and the ‘Health Record form’ in this handbook are for
INFORMATION ONLY – forms for completion will be provided in
the first practical class.
Safety Acknowledgement and Compliance Form
NAME ________________________ COLLEGE ______________

(please print)
BENCH/GROUP No ________________
(This is the number on your side of the card at the centre of the bench).
PRACTICAL DAY ____________________
I have read the document entitled Safety in the Biochemistry Part I Practical Teaching
Laboratory. I agree to abide by the rules contained therein.
Signed ____________________________ Date ________________
This form to be handed to Laboratory Manager or her deputy
Form received by Date

Health Record Form – Record of Hazardous Substance Usage
The COSHH Regulations require all individuals working with substances that can cause certain identifiable diseases or adverse health effects to be kept under health surveillance. For most workers this
is confined to maintaining a record of a person’s involvement in such work. Individuals who work with respiratory sensitizers, mercury, latex, arsenic and skin sensitizers will have additional health
surveillance arranged by the Occupational Health Service. As a precautionary measure the University also requires health surveillance for all individuals working with Nanoparticles. Therefore, all persons
working with nanoparticles should use this health record form and register with the University’s Occupational Health Service. For further information on the criteria for health surveillance see the
University’s Hazardous Substances Policy: Policy and Guidance.
Personal Details for Workers in the Department of Biochemistry
Surname: Forenames: Male/Female/Prefer Date of birth:
not to say: (circle)
N.I. Number: Date commenced present job: Permanent address:
Status: Undergraduate student (NST 1B BMB)
Supervisor’s name: Siolian LR Ball Supervisor’s phone no: 333344
Your signature: Date: Postcode: Dept. Tel No:
SUBSTANCE DETAILS
Name of substance Nature of hazard1 Physical state2 Quantity, amount3 Frequency of use4 Control measures5
Ampicillin (sodium Salt) Respiratory sensitiser Solid 100 µg/ml in agar plates Rarely PPE – gloves, lab coat, safety specs
Substance toxic to
Bis/Acrylamide reproduction
Liquid (37:5:1) 10 ml Rarely PPE – gloves, lab coat, safety specs
Impair fertility and toxic
Boric Acid Liquid 8.9 mM in 400 ml Rarely PPE – gloves, lab coat, safety specs
to unborn child
Carbonyle cyanide 4-
(trifluoromethoxy)phenylhydrazone Skin sensitiser Liquid 20 µM in 200 µl Rarely PPE – gloves, lab coat, safety specs
(FCCP)
a-Chymotrypsin Respiratory sensitiser Liquid 4 mg/ml (0.5 ml) Rarely PPE – gloves, lab coat, safety specs
Solid In Polymerised Gel
Gel Red Unknown Up to 45 ml Rarely PPE – gloves, lab coat, safety specs
(<0.01%v/v)
Nitrilotriacetic Acid Disodium Salt Cat 2 Carcinogen Liquid 5 mM (30 ml) Rarely PPE – gloves, lab coat, safety specs
2-Mercaptoethanol Skin Sensitiser Liquid (5% v/v) 10 µl per sample Rarely PPE – gloves, lab coat, safety specs
1:10,000 dilution from
SYBR GoldTM Unknown 20 ml Rarely PPE – gloves, lab coat, safety specs
stock
Respiratory sensitiser.
Virkon Irritating to skin. Can cause Liquid (1-10%) 200 ml Rarely PPE – gloves, lab coat, safety specs
irreparable damage to eyes.
(1) Carcinogen, mutagen, substance toxic to reproduction, respiratory sensitiser (i.e. asthmagen), skin sensitiser, (Relevant Risk Phrases/Hazard Statements: R42/H334, R43/H317, R45/H350, R46/H340,
R49/H350i, R60/H360f, R61/H360d, R64/H362 where listed) (2) Liquid, solid, dust, vapour, gas or nanoparticle (particles of approximately 100 nm or less in at least one dimension) (3) Include amount
and units (4) Daily, weekly, monthly, rarely (5) Fume cupboard, laminar flow bench, local exhaust ventilation (LEV), glove box or other form of containment, personal protective equipment (PPE; specify).
The completed form should be given to the Safety Secretary (Biochemistry), who will contact the Occupational Health Service. This record must be kept for 40 years.

Risk Assessments
Level of risk = [severity of the event] x [likelihood of the event happening]
Severity Likelihood 1 2 3 4 5
descriptors è very low low medium high very high
ê <2% likely 2-5% likely 5-10% likely 10-20% likely >20%
1 (Insignificant)
1 2 3 4 5
None
2 (Minor)
2 4 6 8 10
Minor reversible injury
3 (Moderate)
3 6 9 12 15
Major reversible injury
4 (Severe)
4 8 12 16 20
Irreversible injury or death
5 (Very Severe)
Irreversible injury or death to 5 10 15 20 25
multiple people
Overall Risk Levels Rating Monitoring and Reviewing Guidelines

1-6 Low Should be reviewed annually
8-12 Medium Should be monitored and reviewed 6 monthly
14-20 High Should be constantly monitored and reviewed quarterly
Above 20 Very high Should be constantly monitored and reviewed monthly
This is a guide to how risk is assessed. The control measures we employ (which includes how we behave around dangerous activities and
chemicals) can modify these risks. As you read the risk assessments you will notice that the safety instructions given to you in your protocols
and your training (control measures) can reduce the risk by affecting the likelihood that something bad will happen, rather than the severity
of the result. You must read and ensure you understand the risk assessments and the COSHH (Chemical Hazard) forms for each experiment.
The COSHH forms will be supplied during each experimental session.

CLASSROOM RISK ASSESSMENT FOR STUDENTS Page 43 of 282
Aseptic Technique: Culturing

Assessors: Dr Siolian Ball Date: 22/07/18
DEPARTMENT OF BIOCHEMISTRY Microorganisms
Staff Affected: Staff, Students and academic visitors
Risk level Revised Risk

Activity / Hazard Severity x Likelihood Control Measures Implemented
= (Total risk)
Level
Equipment supplied is of correct type and in good working condition
4x5 Safe operating procedures are described in practical protocol 4x1
1 Use of Bunsen burner/Burns (20) User to use PPE as described in practical protocol (4)
User will be adequately trained and supervised
Non-Pathogenic strains used (Escherichia coli K12 derivatives, Serratia sp ATCC39006,
Handling microbes/Pathogenic 4x5 Chromobacterium violaceum) 4x1
2
Microbial contamination (20) Safe operating procedures are described in practical protocol (4)
User to use PPE as described in practical protocol
Handling microbes/Puncture Equipment supplied is of correct type and in good working condition
injuries from use of cocktail sticks 4x5 Safe operating procedures are described in practical protocol 4x1
3
causing pathogenic microbial (20) User to use PPE as described in practical protocol (4)
infection User will be adequately trained and supervised
Further Information: Review Date: July 2019


Assessors: Dr Siolian Ball Date: 22/07/18 Liquid Handling and mixing

DEPARTMENT OF BIOCHEMISTRY
Risk level
Severity x Revised Risk
Activity / Hazard Control Measures Implemented
Likelihood Level
= (Total risk)
Vortex mixer use/Possible 4x2 4x1
1 Safe operating procedures are described in practical protocol
electrocution (8) (4)
2 Vortex mixer use/Vibration injury (6) User will be adequately trained and supervised (3)
Concentrations and volumes reduced to minimal values
Non-Pathogenic strains used (Escherichia coli K12 derivatives, Serratia sp ATCC39006,
Chromobacterium violaceum)
Exposure to substances hazardous 4x5 3x1
3 Safe handling procedures described in practical protocol
to health (20) (3)
User to use PPE as described in practical protocol and by demonstrators
Refer to relevant COSHH forms
2x2 2x1
4 Liquid Handling/Possible RSI (4)
Safe operating procedures are described in practical protocol
(2)
User will be trained and adequately supervised


Assessors: Dr Siolian Ball Date: 22/07/18 Electrophoresis


= (Total risk)
Level
Gel electrophoresis/Possible 4x2 Safe operating procedures are described in practical protocol 4x1
1
electrocution from gel rig (8) User to use PPE as described in practical protocol (4)
Safe handling procedures described in practical protocol
Gel electrophoresis/Exposure to 4x5 4x1
2 User to use PPE as described in practical protocol
substances hazardous to health (20) (4)
Refer to relevant practical COSHH forms
Gel electrophoresis/Risk of injury 3x3 3x1
from breaking glass plates (9) (3)
Liquid Handling/Possible Safe operating procedures are described in practical protocol
4x5 4x1
4 contamination with substances User to use PPE as described in practical protocol
(20) (4)
hazardous to health User will be adequately trained and supervised


Assessors: Dr Siolian Ball Date: 22/07/18 Homogenisers – glass


= (Total risk)
Level
Use of Glass Safe handling procedures described in practical protocol
4x5 4x1
1 Homogenisers/Exposure to (20)
User to use PPE as described in practical protocol
(4)
substances hazardous to health User will be adequately trained and supervised
Use of Glass Homogenisers/Risk 3x3 3x1
of injury from breaking glass (9) (3)


Assessor: Dr Siolian Ball Date: 22/07/18 Centrifuges


= (Total risk)
Level
1 Use of centrifuge/Electrocution (8) User to use PPE as described in practical protocol (4)
Use of centrifuge/Exposure to 4x5 4x1
substances hazardous to health (20)
(4)
3 Use of centrifuge/Physical injury
(6) User to use PPE as described in practical protocol (4)


Assessor: Dr Siolian Ball Date: 22/07/18 Oxygen Electrodes


= (Total risk)
Level
Use of oxygen electrode 4x2 Safe operating procedures are described in practical protocol 4x1
1
stirrer/Electrocution (8) User to use PPE as described in practical protocol (4)
Use of oxygen electrode/Exposure 4x5 4x1
to substances hazardous to health (20) (4)
Use of chart 4x2 Safe operating procedures are described in practical protocol 4x1
3
recorders/Electrocution (8) User to use PPE as described in practical protocol (4)
2x2 2x1
4 Liquid Handling/Possible RSI Safe operating procedures are described in practical protocol
(4) (2)


Assessor: Dr Siolian Ball Date: 22/07/18 Heat Blocks


= (Total risk)
Level
1 Use of heat blocks/Burns
(8) User to use PPE as described in practical protocol (4)
2 Use of heat blocks/Electrocution (8) User to use PPE as described in practical protocol (4)


Appendix 1: Units NST 2018-19
Appendix 1: Units – prefixes, amount and concentration
Units
Quantitative results MUST be given in the appropriate units. Biochemical calculations often
involve going from raw data to units that enable you to compare sets of measurements.
When doing calculations, being crystal clear about the units at each stage will help you to
keep track of what you are doing and where you are going. If you make a mistake in a
calculation - perhaps dividing by a volume instead of multiplying – the units can alert you to
the error.
Some simple but important points are noted below.
Prefixes
You will be familiar with milligrams (mg; milli = 10-3) and kilograms (kg; kilo = 103) and
cubic decimetres (dm3; deci = 10-1). The full range of prefixes is as follows:
atto (a) = 10-18

femto (f) = 10-15
pico (p) = 10-12
nano (n) = 10-9
micro (µ) = 10-6
milli (m) = 10-3
kilo (k) = 103
mega (M) = 106
giga (G) = 109
tera (T) = 1012
peta (P) = 1015
eta (E) = 1018
The distinction between amount and concentration

Somewhat surprisingly, students often confuse amount with concentration.
Amount
Quantities (amounts) of substances are usually best expressed in moles. A mole is defined
as that amount of a substance that contains as many elementary units (i.e. atoms, molecules
or whatever) as there are carbon atoms in exactly 12 g of the isotope carbon-12. The
symbol for mole or moles is mol, with a prefix if appropriate (e.g. mmol, µmol). Thus, 1 mol
of glucose (C6H12O6) would weigh (6x12) + (12x1) + (6x16) = 180 g.
Moles are used to enable quantities easily to be compared – e.g. to find how many
molecules of a product are formed per second by a molecule of enzyme. Of course, if the
molecular weight is unknown, units of mass (grams etc) must be used. (It would be neither
helpful nor feasible to consider the molecular weight of a sausage or a banana. However,

Appendix 1: Units NST 2018-19
the molecular weight of a defined complex of components such as a ribosome is available

and useful).
Biochemists often express the mass of a molecule in daltons (which is just another name for
unified atomic mass units). So a protein with a molecular mass of 50, 000 atomic mass units
can be described as a 50, 000 dalton protein. Usually, this would be shortened to 50 kDa
(kilodaltons). Another term that can be used is relative molecular mass (Mr), which is also
called the molecular weight. This has no units, because it is relative to 1/12 of a C atom –
check back to ‘A’ level Chemistry. The Mr of our example would be 50, 000.
Concentration
This is amount per unit volume. Biochemists (and chemists of course) often work in molar
concentrations. A solution that contains 1 mol of a substance in a volume of 1 dm3 has a
concentration of 1 molar, symbolised 1 M. Biochemists usually refer to the cubic decimetre
(dm3 ) as a litre (l), and a cubic centimetre as a millilitre (ml, pronounced “mill”).
Thus, you need to become fluent in the types of quantities shown below
1 litre of a 1 M solution contains 1 mol of solute

1 ml of a 1 M solution contains 1 mmol of solute
1 ml of a 1 mM solution contains 1 µmol of solute
1 ml of a 1 µM solution contains 1 nmol of solute
1 µl of a 1 µM solution contains 1 pmol of solute etc.
Never use M to mean number of moles: M means molar, a unit of concentration equal to
moles per litre.
Dilutions
It is frequently necessary to dilute stock solutions when working in a biochemical
laboratory.
Some sample calculations will be made available on the course Moodle site. You will get
practice at this throughout the year and should feel comfortable with this by the time you
sit the examination in June. If you are having problems you will not be alone in this. Please
consult with your supervisor early in the year for help.

Appendix 2: pH and buffers NST 2018-19
Appendix 2: Acids, Bases, pH and Buffers

Why are acid-base concepts important in Biochemistry?
Very simply, cells are largely composed of molecules with functional groups that can alter
their electric charge by loss of protons (acidic groups) or gain of protons (basic groups).
Such molecules include proteins, nucleic acids and phospholipids (components of cell
membranes) as well as metabolites such as the intermediates of glycolysis and the citric acid
cycle. The properties of molecules with ionisable groups therefore can alter with the acidity
of the medium they are exposed to, whether within living cells or in solutions prepared in
the laboratory.
Important points that affect ionisation and hence molecular properties are:
1. The strength of acids and bases – how easily are protons lost or gained?
2. How the electric charge of ionisable groups is affected by the proton concentration
of the ambient solution.
3. How the proton concentration of the ambient solution can be regulated by buffers.
What are protons, acids and bases?

Strictly speaking, a proton is the nucleus of a hydrogen atom that has lost its single electron
– an H+ ion. However in aqueous solution protons are not naked (or “dry”) but become
hydrated by reacting with H2O to form H3O+ ions called hydroxonium ions. For
convenience, biochemists call these hydrated ions simply “protons” (or sometimes
“hydrons”) unless the context makes it important to be more precise.
Acids are donors of protons, and bases are acceptors. For example, in proteins -COOH
groups are acids, and -NH2 groups are bases. A -COOH or other acidic group is said to
dissociate when it donates its proton to water to form the anion, -COO-, which is the
conjugate base of the -COOH acidic group.
R·COOH + H2O = R·COO- + H3O+
This is shown simply as

R·COOH = R·COO- + H+
Similarly, for R·NH2

R·NH2 + H+ = R·NH3+
Here, R·NH3+ is the conjugate acid of the R·NH2 basic group.
Any acid has a dissociation constant, K, which is the equilibrium constant for the dissociation.
Sometimes, a subscript “a” is added, to show that the constant refers to an acid, Ka: For our
carboxyl group example,
K=
and for the amino group

K=
Water itself dissociates, and has a dissociation constant called Kw.
H2O = H+ + OH-
Because the concentration of H2O is effectively constant in ordinary aqueous solution, it is

generally given an arbitrary value of 1 and omitted from equilibrium constants. Hence
Kw = [H+][OH-]
The value of Kw is close to 10-14 M2. (Note that the unit is M2 because of the convention
adopted for water concentration).
Logarithms: definition of pH
Very wide ranges of proton concentration need to be considered in a laboratory context: 1
M HCl contains 1 M H+ and 1 M NaOH contains 10-14 M H+ (see below). This is one
reason why a more convenient and compressed scale of acidity than simple molar
concentration of H+ is used. Of course, this is the pH scale and pH is defined as the
negative logarithm to base 10 of the H+ ion concentration1.
pH = -log10[H+]
This can also be written as the logarithm of the reciprocal of H+ concentration. (Note that
1/[H+] = [H+]-1).
pH = log10
So, a solution at pH 5 would have [H+] = 10-5 M. The higher the pH, the lower the value of
[H+]. A solution of 1 M HCl would have 1 M H+ concentration and pH = 0 (because log101
= 0). In the case of 1M NaOH, the concentration of OH- is 1 M. As explained above, Kw is
10-14 M2 so that [H+] is 10-14 M, corresponding to a pH of 14.
The Henderson-Hasselbalch equation and its uses

The expression for the dissociation constant of acids can be manipulated into a more
convenient form, replacing [H+] by pH, and K by pK. For a general acid, HA
K= Eqn (1)
Rearrange
1Logarithms: if y = 10x, then log y = x. For example, if y = 10-5 then log y = -5, and -log y = 5.
10 10 10

Now take the logarithm of each side. [Note that log (A·B) = log A + log B]
pH = log + log
We give the symbol pK to log(1/K ), to get the Henderson-Hasselbalch equation2.
pH = pK + log Eqn (2)

The Henderson-Hasselbalch equation
For carboxyl groups, = and for amino groups the ratio is .
In general, the less-protonated form (base: proton acceptor) is the numerator of the ratio,
and the more protonated form (acid: proton donor) is the denominator.
The characteristics of the pK

Notice from the definition of K, in equation (1) that the stronger the acid the lower is its pK
value, and vice versa the weaker the acid the higher the pK. For example, acetic acid
(ethanoic acid) with pK = 4.7 (so K = 10-4.7) is a much stronger acid than phenol (pK =
10.0). For bases, the same applies: the higher the pK the weaker is the conjugate acid form,
and the stronger is the proton-accepting conjugate base. For example, the side chain of the
amino acid arginine has a pK = 12.5 and is a hundred-times stronger base than the side chain
of lysine (pK = 10.5).
Notice from equation (2), the Henderson-Hasselbalch equation, that when the pH is equal
to the pK, the concentrations of A- and HA are equal because3 (pH - pK) = log = 0.
So the pK is the pH at which there are equal concentrations of the acid and its conjugate
base, in other words the acid is 50% dissociated.
Curves of pH-dependence
You can see from the Henderson-Hasselbalch equation how the extent of dissociation of an
acid will vary with pH above and below the pK. We can calculate the ratio of at any
pH we like, expressed as a difference from the pK.
At pH = pK - 1, log = -1 so that = 1/10
2This equation dates from 1908/1916 and was originally given for the carbon dioxide/carbonic acid system that
is so important in the acid-base balance of blood. See Physiology.
3If you don’t see that: the ratio of [A-]/[HA] = 1, and the log of 1 = 0 because 1 = 100

At pH = pK , log = 0 so that = 1/1
At pH = pK + 1, log = 1, so that = 10/1
In order to calculate the fraction (or percentage) of the total acid species that is dissociated,
it is necessary to appreciate the difference between the RATIO of [A-] to [HA] and the
FRACTION that A- forms of the total species present (A- and HA).
This simple point is a common source of confusion for students. The total number of
"parts" into which the “whole” is divided is numerator + denominator. So if the ratio is
1/10, there are 1+10 = 11 parts in all, and the fraction that A- contributes to the total is
1/11. Look at the table below to see how it is done. We can build up a curve showing the
pH dependence of the extent of dissociation of an ionising group. Why not try this for
yourself?
pH - pK RATIO FRACTION DISSOCIATED % DISSOCIATED
-1 1/10 1/(10 + 1) = 1/11 = 0.091 9.1%

-0.5 0.32/1 0.32/(0.32 + 1) = 0.32/1.32 = 0.24 24%
0 1/1 1/(1 + 1) = 1/2 = 0.5 50%
+0.5 3.2/1 3.2/(3.2 + 1) = 3.2/4.2 = 0.76 76%
+1 10/1 10/(10 + 1) = 10/11 = 0.91 91%
The general shape of a pH-dependence curve is shown below.
Notice that if we didn't want to bother with pH and pK, and just plotted the percent of
total species present as HA as a direct function of H+ concentration, we would have the
rectangular hyperbola that describes any simple type of equilibrium where two molecules
combine reversibly – in this case H+ and A- associating to form HA.

Properties of buffers
A buffer resists pH changes: if H+ or OH- are added to a buffered solution, its pH changes
less than would the pH of water alone. Any mixture of an acid and its conjugate base can
act as a buffer - for example a mixture of acetic acid and sodium acetate. This is because if
H+ is added, some ions will be removed by protonating the conjugate base (acetate anions);
similarly, H+ removed by combining with added OH- to form water will be replaced by
dissociation of the acid.
R·COOH = R·COO- + H+
Buffers are only effective near their pK values

When H+ is added to a buffer, some A- will be converted to HA so that the ratio of
will fall. Provided that the ratio is quite close to 1 in the first place, then there will be no
large proportional change in either and the pH will not change much. But if A- (or HA) is
the predominant species, the buffering power will be lower because the proportional
change will be larger. In practice, a good buffer requires reasonably large concentrations of
both buffering species, and so its pH must not be too far from its pK.
The following worked example illustrates this principle in action.
Consider a series of buffer solutions, each of volume 1 litre and containing 1 M total
concentration of A- (salt) and HA (acid), but in different proportions. To each buffer, add
100 ml of 1 M HCl (i.e. 100 mmol). Suppose that the pK of the acid is 5.0. Now apply the
Henderson-Hasselbalch equation to see how the composition of the buffer affects the pH
change after addition of HCl.

BEFORE HCl AFTER 100 mmol HCl Change

[A-] mM [HA] mM old pH [A-] mM [HA] mM new pH in pH
200 800 4.40 100 900 4.06 0.34
500 500 5.00 400 600 4.82 0.18
900 100 5.95 800 200 5.60 0.35
Just pure water 7.00 0.09 M HCl 1.05 5.95
Buffers are effective if they are concentrated

The ratio of [A-]/[HA] will alter less for a given quantity of one species changed into the
other as their concentrations increase. Hence, buffers become more effective at resisting
pH changes the higher their concentrations.
The pH of a buffer is independent of its concentration

Provided that [HA] and [A-] remain large compared with [H+] and [OH-], dilution of the
buffer to lower its concentration will not alter the ratio . Hence, dilution will not
affect the pH of a buffer solution, provided it behaves “ideally”; it will only make it a less
powerful buffer. In practice, concentrated solutions are not “ideal” because solute
molecules no longer behave independently. This means that dissociation constants can
change with the total ionic strength of the solution, so small changes in buffer pH do occur
on dilution.
Types of buffer useful for experimental purposes

What criteria influence the choice of a buffer for experimental purposes?
Obviously the buffering capacity must be sufficient to stabilise the pH within an acceptable
range. The pK of the buffer is central to this, but buffer concentration can also be raised
although this may be undesirable because it will increase total ionic strength and osmolarity.
Ionic strength effects will be diminished if the buffer anion or cation is univalent. Other
practical criteria are that the buffer substance should be easily available in pure form, not be
too expensive, and not interfere with the assay method. Thus, it should be optically
transparent to the wavelength used in a spectrophotometric assay, and not interfere with
other substances in assay mixtures by forming strong complexes or insoluble salts with
metal ions.
Buffers should not interfere with reactions being studied. Thus in enzyme assays, buffers
should not contain substances that are substrates, products, inhibitors or activators of the
enzyme. For example, phosphate buffer (H2PO4-/HPO42-; pK = 7.2) would not be suitable
for assaying glucose 6-phosphatase (phosphate is a product) or glycogen phosphorylase
(phosphate is a substrate). You will meet these enzymes in the lectures on carbohydrate
metabolism. When experiments concern membrane-bound organelles, or intact cells it is
important that the buffer should not cross the membranes to cause osmotic problems or
unwanted changes in the pH of compartments. For example, that would rule out acetate
buffers for many purposes because the CH3COOH species is membrane permeant.

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Another commonly used buffer, which you will encounter in several practicals is Tris (pK 8.2).
The name is an abbreviation of tris(hydroxymethyl)aminomethane.
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Structure of Tris
Some Exercises
Working through these should help you get a feel for buffers. The answers are given below.
Q1. What fraction of lactic acid will be present as the free acid at pH 4.5, given that its pK is
3.9?
Q2 (a). What is the pH of a solution of 0.1 M NaHCO3 and 0.08 M Na2CO3, given that the
pK for dissociation of bicarbonate to carbonate is 10.0?
Q2 (b). What will its pH be when 1 ml of 0.5 M NaOH is added to 10 ml of this solution?
Q3. What concentration of HCl must be added to a solution of 0.6 M sodium acetate to
achieve a pH of 5.0? The pK of acetic acid is 4.7.

Answers to exercises
Answer 1: Applying the Henderson-Hasselbalch equation,
4.5 = 3.9 + log
0.6 = log
4 =
So = = 0.2 (20%).
Answer 2(a): Applying the Henderson-Hasselbalch equation to the dissociation
HCO3- = CO32- + H+
pH = 10 + log = 10 + log = 10 + log 0.8 = 10 - 0.1 = 9.9
Answer 2(b): Proceed by converting concentrations into amounts.
10 ml of the buffer contains 1 mmol of HCO3- and 0.8 mmol of CO32-.

1 ml of 0.5 M NaOH contains 0.5 mmol of NaOH.
The 0.5 mmol of NaOH reacts with 0.5 mmol of HCO3- and converts it to 0.5 mmol of
CO32- according to the equation
HCO3- + OH- = CO32- + H2O
The result of this reaction is that the amount of CO32- increases from 0.8 to 1.3 mmol,
and the amount of HCO3- decreases from 1 to 0.5 mmol. Since these are both obviously
dissolved in the same volume (11 ml), their concentrations will have the same ratio as their
amounts, .
Calculate the new pH from the Henderson-Hasselbalch equation
pH = 10 + log = 10 + log 2.6 = 10 + 0.4 = 10.4
Answer 3: The pH wanted is 0.3 above the pK. From the Henderson-Hasselbalch equation,
log is 0.3. Hence, the ratio is 2/1. Therefore to achieve this

ratio by adding HCl to the original 0.6 M sodium acetate (assumed to be fully dissociated),
1/3 of the initial concentration must be converted to acetic acid. This is done by 0.2 M HCl.

Appendix 3: Spectrophotometry NST 2018-19
Appendix 3: Principles of Spectrophotometry

Absorption of light
Water is colourless; it does not absorb visible light, or light in the ultraviolet region of the
spectrum. But many substances dissolve to give coloured solutions (e.g. haemoglobin,
cytochrome c), because their molecules do absorb visible light. Others, like nucleic acids,
proteins, and NAD(H) absorb in the ultraviolet. The higher the concentration of solute, the
more light is absorbed. Spectrophotometry is a simple technique based on this
quantitative relationship that allows concentrations of dissolved substances to be
determined by measuring light absorption of solutions.
For example, reduced cytochrome c is yellow and has absorption peaks at around 550 nm
and 420 nm. This is due to the haem prosthetic group, which is a chromophore (literally,
colour carrier). NADH has peaks in the ultraviolet at 260 and 340 nm, while NAD absorbs
at 260 nm only. Thus the activity of NAD-linked dehydrogenases can be measured by
changes in light absorption at 340 nm.
Quantitative Applications: The Beer-Lambert Law

Imagine a solution of NADH in a spectrophotometer cell (width 1 cm) and that
monochromatic ultraviolet light at 340 nm is shone through it.
Let the intensity of the incident light = Io and of the transmitted light = I
Incident light
of 340 nm
I0
Transmitted light
I
width of cell (l) = 1 cm
The most intuitive measure of how much light has been absorbed is the transmission, I/Io.
This can be expressed as a percentage.
However, it is more useful to use the absorbance, A. This is defined as the logarithm (to
base 10) of Io/I. Note that Io/I is the reciprocal of the transmission.
A = log Io/I

Incident Intensity (Io) Transmitted Intensity Percent transmission Absorbance

Arbitrary units (I) I/Io x 100 log Io/I
100 100 100% 0

100 50 50% 0.3
100 10 10% 1.0
100 1 1% 2.0
Relationship between transmission and absorbance
The reason why absorbance is useful is simply that it is directly proportional to the
concentration of the substance that absorbs the light (NADH in our example). The
absorbance is also directly proportional to the distance travelled by light through the
sample, the path length – in practice the width of the spectrophotometer cell (cuvette)4.
Absorbances much higher than 2 are difficult to measure, since less than 1% of the incident
light is available to be detected by the instrument’s photomultiplier. (Note that some
instruments claim linearity to absorbances of 3 and beyond, but such values should be
treated with caution.)
The constant that relates absorbance to concentration and path length is called the
absorption coefficient, or sometimes the extinction coefficient. When concentrations
are in units of moles per litre (molarity), we have a molar absorption coefficient that is
symbolised eM. (The “e” is the Greek letter epsilon). We can express this relationship as an
equation, where A = absorbance, c = concentration (mol l-1) and l = width of cell (cm). This
expresses the Beer-Lambert law.
A = eM×c×l
The units of eM are M-1×cm-1 (that is, litre×mol-1×cm-1). For NADH, the value of eM is 6220
M-1×cm-1.
The Beer-Lambert law can also be represented as a straight-line graph of A vs c. The slope
is then eM×l. It is most common to use a 1-cm wide cell, and in that case the slope is
numerically eM.
slope = molar absorption coefficient
Concentration (M)
Graphical representation of the Beer-Lambert law
N.B. The absorption coefficient of any substance varies with wavelength, so the Beer-
Lambert law only holds for monochromatic light.
4The reason why absorbance (a logarithm) is linearly proportional to concentration and

width of cell (path length) is that intensity of the transmitted light falls off exponentially as it
passes through the absorbing solution.

Values of eM are known (and can be looked up) for many substances of biological interest,
or they can be determined by making up accurately solutions of the pure substance. It is
then a simple matter to determine the concentration of the substance in a solution from its
absorbance – for example, if a solution of the amino acid tryptophan has an absorbance of
0.167 in a 1 cm cell at 280 nm, we may calculate the concentration from the Beer-Lambert
law, given that eM at 280 nm for tryptophan = 5580 litre×mol-1×cm-1:
A = eM×c×l
Rearranging:
c = A/eM×l
So
c = 0.167/5580 = 3.0 x 10-5 mol l-1
Some practical points about measuring absorbance

1. Spectrophotometer cells can be made of glass or synthetic polymers (such as
polystyrene) and will work down to about 260 nM (the wavelength at which nucleic
acids have peak absorbance). Polystyrene can easily warp though and so it is
conventional to use quartz cuvettes to measure absorbance of wavelengths in the UV
(Ultra Violet) range. The surfaces must be flat and clean, and be separated by an
accurately known path length (often 1 cm). Quartz cells are both fragile and
expensive, so take care if you should need to use them! Only handle cells by their
opaque surfaces.
2. It is usual to set the spectrophotometer to read zero absorbance using a reference
cuvette that contains solvent only. This is because light losses due to the cuvette
material itself and to the solvent need to be allowed for in order to determine the
true absorbance of a solute in the sample solution.
3. For any particular instrument, make sure that you understand the arrangement for
“data output”. A spectrophotometer and linked recorder can usually be set at
different sensitivities (as well as allowing either absorbance or transmission to be
displayed). Typically, you will use a recorder set up so that the full width of the chart
paper corresponds to 1 absorbance unit. The speed at which the recorder moves the
chart paper can also be varied – you might well use a speed of 0.5 or 1 mm per
second. Alternatively, the data may be displayed on a screen on the
spectrophotometer. In this way, you can follow the rate of an enzyme-catalysed
reaction that is consuming or producing NADH by measuring the change in A340 with
time. You will encounter this application in the practical course.
4. It is common to measure absorbance using 96-well format instruments, that may
operate at pre-set wavelengths determined by optical filters in the light path. On more
sophisticated instruments, wavelength can be varied using an inbuilt diffraction grating
monochromator. In the Lent term, you will use ELISA methodology that uses a simple
96-well plate reader and provides absorbance values for each well. Normally, blank
wells or wells containing controls will provide a background that can be subtracted
from other sample readings. The multiwell format has the great advantage that it
allows a range of standards to be measured alongside the test samples.

Michaelmas Weeks 1-4: Basic Techniques in Molecular Biology NST 2018-19
Weeks 1-4 (Michaelmas) – Basic Techniques in Molecular Biology

These first four practicals of the course occupy weeks 1 to 4.
Objectives
We intend to give you some “hands on” experience of some of the simpler procedures that
are central to modern molecular biology and are covered in the lectures by Drs Scadden
and Mata, and Professor Smith this term. You will apply these to the study of the DNA
binding domain (the POU domain, NB rhymes with cow not moo!) from a transcription
factor called Oct-1. You will use PCR to amplify the DNA corresponding to the POU
domain from a longer template, and incorporate a different restriction site at each end to
allow directional cloning. You will then cut the PCR product with suitable restriction
enzymes and ligate it into a vector for blue/white screening. The following week you will
express the POU domain in Escherichia coli. The expression plasmid is called pMAT8 and
contains the DNA encoding the POU domain as a fusion protein with maltose binding
protein (MBP) to aid solubility and a hexahistidine tag (His)6 for purification. The fusion
protein is under the control of a promoter recognized by the RNA polymerase from
bacteriophage T7, whose expression is in turn switched on in the presence of lactose or its
stable analogue isopropyl β-D-1-thiogalactopyranoside (IPTG). In week 3, you will purify the
protein that you expressed the previous week, using an affinity column and in the final week
you will use electrophoresis mobility shifts assays (EMSA) to characterize DNA binding by
the POU domain.
In a different type of experiment, in week 2, while you are waiting for the protein to
express, you will study the melting of eukaryotic DNA and factors that influence the
stability of the double helix.
At the end of these practicals you should be able to perform the following tasks:
• Perform a PCR (polymerase chain reaction) to amplify a desired section of DNA.
• Digest the PCR product with restriction endonucleases and clone the resulting
fragment into a vector.
• Express a recombinant protein in E. coli and purify it using a Ni2+-affinity column.
• Use your purified protein to perform a gel shift assay to investigate its DNA-binding
properties.
• Use spectrophotometry to investigate the melting of nucleic acid double helices by
changes in light absorption at 260 nm.

Michaelmas Weeks 1-4: Basic Techniques in Molecular Biology NST 2018-19
Summary of Schedule

Michaelmas Week 1: Basic Techniques in Molecular Biology NST 2018-19
Week 1
There are several steps in the practical today:

1. PCR to amplify the POU domain from a template DNA molecule that encodes the
domain.
2. Incubate the PCR product with restriction enzymes to generate sticky ends.
3. Separate and visualise the digested PCR products by agarose gel electrophoresis
4. Ligate the digested PCR product into a plasmid vector for analysis.
Week 1 – Introduction and background

PCR
PCR is based on three simple steps: (1) denaturation of the template DNA into single
strands; (2) annealing of oligonucleotide primers to each original strand for new strand
synthesis; and (3) extension of the new DNA strands from the primers.
PCR achieves the repeated synthesis of new DNA strands, exponentially amplifying a
defined region of the starting material, and allowing the birth of a new technology that has
revolutionised daily life in labs and hospitals. To achieve multiple rounds of synthesis, the
DNA templates must be repeatedly denatured, which requires temperatures well above
those that inactivate most enzymes. A heat-stable DNA polymerase from a thermophilic
bacterium, Thermus aquaticus (Taq), is often used in PCR. The polymerase chain reaction is
discussed in some detail in Dr Scadden's lectures and handout. Please consult that for
further background information.
A reminder about primer design for PCR:

The success of any PCR reaction is dependent upon the specificity of primer hybridization to
the targeted site with respect to non-specific regions of the input DNA. The most important
aspect of primer design is to ensure that the sequence chosen for the primer is unique i.e.
that the primer anneals to a single site in the DNA template. These days, one simply
searches the DNA database for homologous sequences (as you will be doing in the
Bioinformatics computer practical).
To amplify double stranded DNA it is necessary to have one primer for each strand. PCR
primers are DNA (unlike in DNA replication in the cell, where RNA primers are used). The
sense strand of a gene is identical to the mRNA sequence (with T in DNA corresponding to
U in RNA of course) and is by convention written 5’à3’ when describing a gene. The
antisense strand is complementary to the sense strand and often is not written out at all in a
gene sequence. To amplify the sense strand the primer needs to be identical to the 5¢ end
of the sense strand; the antisense strand is used as the template for the DNA polymerase,
such as Taq, included in the reaction mixture: à
5' primer 3' sense

5'
3' 5' antisense

Remember, DNA polymerase works in the 5¢ à 3¢ direction i.e nucleotides are added to
the 3¢ end of the primer. To amplify the antisense strand, the primer is designed in the same
way, but is now identical to the 5¢ end of the antisense strand, with the sense strand
being used as the template for the polymerase:
5' 3' sense

5'
primer
3' 5' antisense
Finally, the primers are designed so that they anneal to the template DNA at similar
temperatures.
Restriction endonucleases
Restriction enzymes were so named because their presence in a number of bacteria
restricts the ability of certain bacteriophages to grow in those bacteria. The enzymes do this
by degrading the viral DNA before any harm can come to the cell.
You will be using two restriction enzymes today –

• BamHI (isolated from Bacillus amyloliquefaciens strain H)
• HindIII (isolated from Haemophilus influenzae strain d)
The sequences they recognise and cut are:
Note that these recognition sites are palindromic, so that the resulting overhanging ends
are identical when read from 5’ to 3’. Also notice that these recognition sites contain 6
specific bases, so they are expected to occur once in every 46 base pairs, or roughly 4 kbp.
Amplification of the POU domain

Today you are going to amplify the cDNA encoding the POU domain of Oct-1. cDNA is an
abbreviation for complementary DNA, this means that the DNA is a double stranded DNA
copy of the mRNA encoding the POU domain. Oct-1 is a ubiquitous transcription factor in
mammalian cells that regulates transcription of some common genes. Oct-1 can also
regulate the transcription of cell-specific genes, e.g. immunoglobulin genes in B cells, by
recruiting co-activators (see Prof. Smith’s and Dr Mata’s lectures). Oct-1 is known to bind
to DNA via a small domain of 162 amino acids called the POU domain.

The POU domain of Oct-1 encompasses amino acid residues 282 – 443. Here is the
cDNA sequence that encodes these amino acids:
5’CCCAGTGACCTTGAGGAGCTTGAGCAGTTTGCCAAGACCTTCAAACAAAGACGAATCAAACTTGGA
TTCACTCAGGGTGATGTTGGGCTCGCTATGGGGAAACTATATGGAAATGACTTCAGCCAAACTACCAT
CTCTCGATTTGAAGCCTTGAACCTCAGCTTTAAGAACATGTGCAAGTTGAAGCCACTTTTAGAGAAGT
GGCTAAATGATGCAGAGAACCTCTCATCTGATTCGTCCCTCTCCAGCCCAAGTGCCCTGAATTCTCCA
GGAATTGAGGGCTTGAGCCGTAGGAGGAAGAAACGCACCAGCATAGAGACCAACATCCGTGTGGCCTT
AGAGAAGAGTTTCTTGGAGAATCAAAAGCCTACCTCGGAAGAGATCACTATGATTGCTGATCAGCTCA
ATATGGAAAAAGAGGTGATTCGTGTTTGGTTCTGTAACCGCCGCCAGAAAGAAAAAAGAATCAACCCA
CCAAGCAGTGGATA 3’
Often it is necessary to ensure that the PCR product contains DNA sequences at its ends
that enable it to be cleaved by restriction endonucleases so that the amplified target
sequence can be cloned into an appropriate vector. Any such extra sequences are added to
the 5’ ends of the primers so that they do not interfere with copying the target sequence
(where nucleotides are added to the 3' end of the primer): the rest of the primer is
designed as already described. As the extra sequences are included in the primers they will
quickly be amplified even though they are not in the original target sequence template.
The primers that you will use for your PCR are thus (restriction enzyme recognition sites in
bold):
POU Forward (5’ primer)

5’- CGCGGATCCCCCAGTGACCTTGAGGAGCTTGAGC-3’
POU Reverse (3’ primer)

5’- CGCAAGCTTTATCCACTGCTTGGTGGGTTGATTC-3’
The region of the forward primer in italics is identical to the underlined sequence at the 5’
end of the POU domain DNA sequence. The region of the reverse primer in italics is
complementary to the underlined sequence at the 3’ end of the POU domain sequence.
Measuring sizes of DNA molecules.

The most convenient way to measure the size of a DNA molecule is by gel electrophoresis.
A DNA molecule is uniformly covered with negative charges and will move towards the
positive electrode during electrophoresis. The small molecules thread their way through the
gel matrix more easily than the large ones and so will move faster through the gel.
The gel matrix you will be using is agarose. This is a highly purified form of agar, with its
physical and electrophoretic properties carefully defined. The DNA samples are loaded into
wells at one end of the gel, which is then 'run' in an electric field to separate the fragments.
Traditionally, a dye, ethidium bromide, is included in the gel. This is a flat, polycyclic
molecule capable of fitting between the bases in the DNA (intercalation). Intercalated dye
fluoresces much more strongly under UV light than free dye, so the position of DNA
molecules in your gel can be easily determined. In this practical however, GelRed will be
used to stain the nucleic acids in your gel. GelRed is structurally closely related to ethidium
bromide, and consists of two ethidium subunits that are bridged by a linear spacer. Its
fluorophore, and therefore its optical properties, are essentially identical to those of
ethidium bromide. When exposed to ultraviolet light, it will fluoresce with an orange colour

that strongly intensifies after binding to DNA. The substance is a less toxic and more
sensitive alternative to ethidium bromide.
You will also include a sample of 'standard' sized DNA molecules to use as a size marker. In
this case you will use a 100 bp ladder, which covers a convenient range of molecular
weights.
A graph of mobility against log of fragment size gives a straight line over
a fairly wide range, so you can estimate the size(s) of fragments in your
PCR products by comparison with the standards.
Note that this all relates to linear molecules. Circular molecules

migrate rather anomalously in agarose gels, so that you cannot use
the mobility of an intact plasmid as a quantitative measure of
its size.
Ligation of the PCR product into a plasmid vector

Plasmids are DNA molecules found in a wide range of prokaryotic and some eukaryotic
cells. They are usually independent of the main chromosome, although some, like the F
factor in E. coli, can become integrated into it. Almost all are circular. Sizes vary widely, from
only 1 kbp up to hundreds of kbp. Most confer some useful phenotype, such as antibiotic
production and/or resistance, the ability to kill other bacteria, resistance to heavy metal
poisoning and the ability to break down unusual sugars or other organic compounds.
Plasmids are also widely used in genetic engineering.
The plasmid vector into which you will clone your POU domain fragment is called
pBluescript. Here is a vector map:
The important features are marked on

the map:
MCS: a multiple cloning site (see below)
ampicillin: the gene for ampicillin
resistance
pUC ori: an origin of replication that
allows the plasmid to be maintained
independently in bacteria
P lac: the promoter for the lac operon
lacZ’: one of the lac operon genes that
encodes an enzyme called b-
galactosidase.

The MCS is a cluster of restriction enzyme recognition sites, which in this plasmid includes
sites for BamHI and HindIII. If the PCR product and the vector are both cut with these two
enzymes, they will have complementary sticky ends, which should anneal to each other.
When this happens there will be a gap left in the DNA backbone at the joining site and you
will use another enzyme called DNA ligase in the reaction to seal that gap – hence the
whole operation is called a ligation. When you have successfully ligated the POU domain
fragment into the vector, the resultant plasmid will have the lacZ’ gene interrupted by the
extra piece of DNA. The success of your ligation can therefore be monitored by an assay
for lacZ’. For this you will use a modified version of the sugar galactose called 5-bromo-4-
chloro-3-indolyl beta-D-galactopyranoside (abbreviated to X-gal), which, when cleaved by b-
galactosidase gives rise to a blue product.
Purifying DNA after PCR and restriction enzyme digests

After the PCR you will purify the amplified DNA fragment to remove enzymes, primers up
to 40 nucleotides long and dNTPs. For this you will use a QIAquick spin column®. This is a
silica membrane assembly that efficiently binds single or double stranded DNA of more than
100 bp in a high salt buffer that contains a high concentration of chaotropic salts.
Chaotropic agents disrupt the hydrogen-bonding network of water, destabilizing the DNA-
water hydrogen bonds. The DNA can then establish binding interactions with the silica
surface. Washing with a high percentage of ethanol removes unbound impurities, while
enhancing the DNA binding. The DNA is then eluted with a low-salt buffer.
The same procedure is followed after the restriction enzyme digest, in this case to remove
the small fragments that the restriction enzymes have removed from the PCR products as
well as the enzymes themselves.
The QIAquick Spin Purification Procedure

(PCR reaction or restriction digest)
Buffer PB
Buffer PE
Buffer EB
Pure DNA fragment

Transformation of ligated DNA

After the ligation, it is necessary to amplify the successful plasmid products. The most
efficient way to do this is to put the plasmid into bacteria by a process called transformation,
and let them replicate the plasmid as they grow. E. coli do not readily take up plasmids
unless they are made competent to do so. A common way of making E. coli competent is to
treat them with CaCl2, which works by an unknown mechanism to permit them to take up
DNA when they are subjected to a heat shock. The bacteria can then be plated onto a
medium containing the antibiotic present in the plasmid, to select only those that have taken
up the plasmid.

Week 1: PCR and Cloning – Experimental
This part of the practical will run over two weeks: this week you will generate a
fragment using PCR, which you will then ligate into the plasmid vector pBluescript. You
will then plate out the ligation products on an agar plate that will be incubated at 37 °C
overnight and then stored at 4 °C until next week, when you will be able to estimate the
efficiency of the transformation (number of colonies) and the ligation (number of blue
colonies).
It should be obvious from this that it is critical that you label your plates
clearly to show the day that you do the practical and your bench number.
PCR
The stock reagents for the PCR reaction are as follows:
10x PCR buffer: 100 mM Tris-HCl, pH 9.0, 15 mM MgCl2 and 500 mM KCl.
Primer 1 (POU Forward) – 10 µM
Primer 2 (POU Reverse) – 10 µM
DNA template – 0.2 mg/ml
dNTPs (mix of dATP, dGTP, dCTP, dTTP) – 2.5 mM
Taq DNA polymerase
Key to buffer and reagent solutions:

• DNA – DNA template for PCR
• CT – 10x PCR buffer, 2.5 mM dNTPs, ddH2O
• P – POU F, POU R
(Remember: the PCR primers have restriction sites on the ends – so when cut will give
sticky ended products for directional cloning)
Set up your PCR reaction in a 0.2 ml tube (labelled with your bench number), which
contains the following:
1. 37 µl of CT, which is comprised of:
5 µl 10x PCR buffer

4 µl dNTPs (200 µM final concentration)
28 µl distilled, deionised (dd) H2O
Total 37 µl
2. 10 µl of the primer mix (P), which is comprised of:
5 µl POU F (1 µM final)
5 µl POU R (1 µM final)
3. 2.5 µl plasmid DNA that contains the POU cDNA (“DNA”)
The final volume should be 49.5 µl.

A demonstrator will then add 0.5 µl of Taq DNA polymerase. Mix gently and briefly spin in
the benchtop microcentrifuge (1-2 sec), using an empty 1.5 ml tube with the lid removed as
a carrier for the PCR tube and a second empty tube as a balance.
When your PCR reaction is set up, put your tube in the thermocycler and run the PCR
using the following program, which will already be set up for you:
95 °C 1 min, followed by:

95 °C 20 sec (to denature the DNA)
58 °C 20 sec (to anneal the primers to the template)
72 °C 20 sec (to allow the polymerase to copy the DNA)
for 22 cycles then
72 °C 3 min
Purify the PCR product using a QIAquick spin column

Key to buffer and reagent solutions:
• PB – contains guanidine hydrochloride (a chaotropic agent) and propan-2-ol
• PE – wash buffer containing a high percentage of ethanol
• EB – 10 mM Tris-HCl, pH 8.5
Safety note: wear gloves and take care when handling buffer PB as guanidine
hydrochloride is a severe irritant.
1. Add 250 µl Buffer PB to the PCR reaction and mix. Check that the colour of the
mixture is yellow. PB contains a pH indicator, which will change colour to orange or
violet if the pH is above 7.5. If this happens, consult a demonstrator, as DNA
will not stick to the column at high pH. A small amount (1-2 µl) of 3 M
Sodium acetate, pH 5.2, will be added to reduce the pH to <7.5.
2. Place the QIAquick spin column in a 2 ml collection tube
3. Apply your sample carefully to the QIAquick column. Put the column and the collection
tube into the benchtop microcentrifuge, using a microfuge tube containing a similar
volume of water as a balance, and centrifuge for 60 sec. The DNA should bind to the
column.
4. Discard the flow-through (i.e. the contents of the 2 ml collection tube) into the waste
pot provided. Place the QIAquick column back into the same tube.
5. Carefully add 0.75 ml Buffer PE to the QIAquick column. This will wash off any excess
unbound material. Centrifuge for 60 seconds as in step 3.
Discard the flow-through and place the QIAquick column back in the same tube. Centrifuge
the column for 60 seconds as in step 3. NB: residual ethanol from Buffer PE will not be
completely removed unless the flow-through is discarded before this centrifugation.
6. Place the QIAquick column into a new, clean 1.5 ml tube. Make sure that you cut the lid
off before spinning in the microcentrifuge.

7. To elute the DNA, carefully add 30 µl Buffer EB to the centre of the QIAquick
membrane, let the column stand for 1 minute (use a timer), and then centrifuge for 1
min as in step 3.
Digest the PCR product using restriction enzymes
Key to buffers:
• CutSmart™ (supplied as 10x). 1x Buffer: 50 mM Potassium acetate, 20 mM Tris-

acetate, 10 mM Magnesium acetate, 100 μg/ml BSA
• LB – 6x Loading Buffer: 60 mM Lithium acetate, 60 mM Boric acid (final
concentration 10 mM Lithium acetate, 10 mM Boric acid, pH ~6.7), 60% (v/v)
Glycerol, 0.03% (w/v) bromophenol blue
Add the following to a 1.5 ml microfuge tube:
15 µl purified PCR product (from above)

5 µl of 10x restriction enzyme buffer (CutSmart™)
28 µl H2O
Total 48 µl
A demonstrator will add 1 µl BamHI and 1 µl HindIII to the tube. Mix gently and briefly spin
in the benchtop microcentrifuge using an empty tube as a balance. Incubate the tube in the
37 °C heat block for 30 – 45 min.
Meanwhile, check the size of the PCR amplified DNA on a 0.8% agarose gel
• Add 5 µl of the purified PCR product to a new microfuge tube.
• Add 4 µl 6x Loading Buffer. This contains glycerol to make the sample dense and
stop it diffusing out of the well into the buffer, and a blue dye (bromophenol blue) to
help you judge how far the electrophoresis is going.
• Add 11 µl of H2O to give a final volume of 20 µl.
You should also have a tube containing 20 µl of the 100 bp DNA ladder (labelled M1),
which contains multiple DNA fragments of known size.
Loading the gel is the most difficult part of the whole practical – if in doubt about the
loading, ask a demonstrator for help. It is best to use 2 adjacent tracks in the centre of
the gel, rather than at the side. Draw up the contents of one tube into a Gilson tip.
Without pressing down on the plunger, poke the tip underwater into a well in the gel (over
one of the red lines). Do not jab the tip into the gel itself, keep it in the well. Then
gently depress the plunger to deliver the sample slowly into the well. Don't worry if it won't
all go in.
Connect up the gel apparatus, with the negative terminal (black) at the end where you
loaded the gel. The gel contains the same electrophoresis buffer that it is submerged in (10
mM Lithium acetate, 10 mM Boric acid, pH ~6.7). Switch on and run at ~150 V on constant
voltage until the bromophenol blue dye has moved to the second set of red lines. This
should take approximately 30 – 45 minutes.

Purify the restriction digested DNA using Qiaquick spin column (repeat of the
procedure you performed on previous page)
1. Add 250 µl Buffer PB to the restriction reaction and mix. Check that the colour of the
mixture is yellow.
2. Place a QIAquick spin column in a 2 ml collection tube.
3. Apply the sample to the QIAquick column and centrifuge for 60 sec (balance!).
4. Discard flow-through. Place the QIAquick column back into the same tube.
5. To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 60 sec.
6. Discard flow-through and place the QIAquick column back into the same tube.
Centrifuge the column for an additional 1 min.
7. Place the QIAquick column in a clean 1.5 ml microfuge tube.
8. To elute DNA, add 50 µl Buffer EB1 to the centre of the QIAquick membrane, let the
column stand for 1 min, and then centrifuge for 1 min.
Set up ligations using digested DNA
Key to buffers:
• L – 10x Ligation buffer. (1x buffer composition: 50 mM Tris-HCl, pH 7.5, 10
mM MgCl2, 1 mM ATP, 10 mM DTT).
• TG1 – TG1 E. coli competent cells
• BS – pBluescript plasmid
• UC – Uncut plasmid 20 ng/µl – control
The pBluescript plasmid has already been cut for you and the ends have been treated with
alkaline phosphatase. This removes the 5’ phosphate groups and reduces the chances that
the plasmid will ligate to itself (intramolecular ligation). Even though this has been done, it is
standard procedure to perform a control ligation with no insert DNA. Set up three 1.5 ml
tubes (called Negative Control, Insert and Positive Control) and add the following to them:
PCR Product Ligation

Negative Control Positive Control
(Insert)
2 µl 10x ligation buffer (L) 2 µl 10x ligation buffer (L) 1 µl uncut plasmid (UC)
2 µl pBluescript plasmid 2 µl pBluescript plasmid 9.5 µl ddH2O
5.5 µl H2O 2 µl digested DNA
3.5 µl H2O
The demonstrator will add 0.5 µl T4 DNA ligase to each of your tubes (except the positive
control). Mix gently and spin down as before and leave at room temperature for at least 30
minutes. While the ligations are taking place, put the agar plates in the 37 °C oven to
ensure that they are dry (hence spreadable).

Meanwhile, switch off the agarose gel when it has finished running. Remove gel (on plastic
tray) from the gel tank, and place it in a plastic tray provided to carry it to the UV-light box.
Your gel will then be placed on the UV-light box to visualize the DNA. DNA is readily
visualized as the gel contains GelRed.
Note: GelRed is more sensitive and less toxic than Ethidium Bromide.
Nevertheless, do wear gloves when handling the gel, and avoid getting GelRed
on your hands (or anywhere else). Note that any spills or drips from either your
gel or running buffer should be cleaned up immediately with tissue.
A demonstrator will take a photograph of the gel. How big do you expect the PCR product
to be? Is it the correct size? Use the semi-log paper provided to determine the size
of your PCR product.
Transformation of E. coli TG1 cells

In experiments with bacteria you must avoid contamination from bacteria in the air. Aseptic
techniques for the following procedures will be demonstrated to you. In addition, ensure
that you have your lab coat buttoned and that you wash your hands with soap before
leaving the laboratory.
The TG1 cells to be used for transformation will be brought to the lab during the
afternoon, and are kept on dry ice. Thaw in your ice bucket just before they are needed.
Take each of your samples (ligations and control) and put them into your ice bucket. Taking
care to keep everything on ice, carefully add 100 µl of TG1 competent cells to each tube
and mix gently by shaking the tube from side to side. DO NOT VORTEX.
Incubate the tubes on ice for at least 15 minutes.
Heat shock the cells by incubating them in the 42 °C heat block for EXACTLY 1 minute.
Using a Gilson pipette, transfer the transformed bacteria onto the surface of three agar
plates (labelled + or – Control and Insert). Then, using a new sterile plastic spreader for
each plate, gently distribute the cells across the whole of the plate. Be sure to keep the
spreader flat, so that you do not damage the agar surface and try to keep the plate as sterile
as possible. Ask the demonstrators to show you how to do this. The plate contains
ampicillin, 0.1 mM IPTG to induce the lac promoter and 40 µg/ml X-gal for blue/white
selection).
Once you have put the lids back on the plates, turn them over so that the agar is on top.
This prevents condensation from the lid dripping onto the plate surface. Your plates will be
incubated overnight at 37 °C and then stored at 4 °C for you to look at next week. Make
sure you have labelled them with your bench number, your name and the day
that you do your practical as well as whether it is the positive control, the control with
no insert or the real ligation.



Notes

Week 2
There are three parts to the practical today:
1. Expression of the POU domain in E. coli.
2. Assessment of the efficiency of the ligation that you performed last week.
3. Investigation of the melting of nucleic acids at high temperature.
Protein Expression
The Oct-1 POU domain will be expressed in an E. coli strain called BL21(DE3). BL21 is often
used for protein expression because it is deficient in two proteases (Lon and OmpT) that
might otherwise degrade the recombinant protein, in this case to POU domain. The plasmid
vector contains the POU domain coding sequence under the control of a promoter that is
recognised by the RNA polymerase from the bacteriophage T7. This T7 promoter is used
as it is small, the T7 RNA polymerase binds to and initiates within a 17 bp sequence, and has
a high rate of initiation and processivity so it makes a lot of mRNA. T7 RNA polymerase is
not normally present in E. coli and the (DE3) part of the name means that there is a DNA
insert in the bacterial chromosome, which encodes the T7 polymerase under the control of
a highly efficient promoter derived from the lac promoter so we can switch on its
expression with IPTG, the lactose analogue. See Dr Scadden’s lecture notes for a
description of the other features of bacterial strains commonly used in molecular biology.
Expressing a foreign protein in large quantities is quite a strain for a bacterial cell and it is
common for cell division to slow down once protein expression has been induced. For this
reason, induction by IPTG is carefully timed, so that the bacterial cultures are in late log
phase, that is, they are growing quickly but that they have not yet run out of nutrients in the
medium. At this stage, there will be plenty of cells in the culture to produce protein. The
density of bacteria in a culture can be estimated by measuring the apparent absorbance
(technically turbidity due to the light scattering) at 600 nm in a spectrophotometer. Once
they have been induced, the bacteria will be left to grow for three hours to express the
protein. After this time, most of the nutrients in the medium will have been used up and
there will be little extra protein produced.
Nucleic acid structures

All nucleic acids are composed of chains of nucleotides linked by phosphodiester bonds.
The sugar-phosphate 'backbone' of the nucleic acid, from which the bases project, may be
coiled in various configurations to give secondary structure to the molecules.

5' 5'
deoxyribose 1'- adenine ribose 1'- adenine
3' 3'
P P
5' 5'
deoxyribose 1'- guanine ribose 1'- guanine
3' 3'
P P
5' 5'
deoxyribose 1'- thymine ribose 1'- uracil
3' 3'
P P
5' 5'
deoxyribose 1'- cytidine ribose 1'- cytidine
3' 3'
P P
single stranded DNA single stranded RNA
In double stranded DNA, the form found in all prokaryotes and eukaryotes, two antiparallel
strands are wound together to give a B-form helix, with the negatively charged sugar-
phosphate backbone on the outside and the complementary base pairs (A–T, G–C) on the
inside.
The base pairs are planar and roughly perpendicular to the axis of the helix. This double
helical structure is stabilized by hydrogen-bonding between complementary bases and by
hydrophobic interactions between adjacent base pairs (base stacking). In the absence of
counterions, repulsion between negatively charged phosphate groups keeps the molecule in
the extended form, so that DNA in solution behaves as an inflexible rod, hence the high
viscosity of DNA solutions.
5' 3'
deoxyribose 1'- adenine thymine - 1' deoxyribose
3' 5'
P P
5' 3'
deoxyribose 1'- guanine cytidine - 1'deoxyribose
3' 5'
P P
5' 3'
deoxyribose 1'- thymine adenine - 1'deoxyribose
3' 5'
P P
5' 3'
deoxyribose 1'- cytidine guanine - 1'deoxyribose
3' 5'
P P
double stranded DNA
Most RNA molecules are single stranded. This does not mean that they lack secondary
structure, but that secondary structure is a result of interactions within one molecule (i.e.
intramolecular). The single stranded molecules fold up on themselves with limited base-

pairing (A–U, G–C) occurring within a single molecule. The primary sequence of bases will
determine the position and size of the stems and loops, with base-pairing occurring in the
stem and non-base-paired regions at the ends as loops. In non-base-paired regions base
stacking may occur which will stabilise the structure.
U G
C A
U U
A - U
U - A
G - C
G - C
C - G
U - A
U - A
A - U
......AGGCUGCA C - GGCGGAAAA......
All bases found in nucleic acids have a characteristic absorption of light in the ultraviolet
region, and nucleic acids have a maximum absorbance at around 260 nm. Both hydrogen
bonding and base-stacking interactions decrease this absorption by the bases. So the
absorbance of intact, native, nucleic acid molecules is less than that of the nucleotides of
which they are composed.
On disrupting the secondary structure of DNA either by heating or by exposing it to

strongly alkaline pH, the absorption of light at 260 nm (A260) rises to approximately 1.4
times its original value. This is known as the hyperchromic effect. The extra hydroxyl group
on the 2’ position in the backbone of RNA makes it sensitive to alkali hydrolysis, so changes
in RNA structure are induced by heating.
The change in the structure of the DNA that produces this effect is a separation of the two
strands (dsDNA to ssDNA). No hydrolysis of the backbone need take place in order to
produce this increase in absorbance. The DNA is said to have melted; the transition
occurs over a fairly narrow range of temperature due to the cooperative nature of the
unwinding process (i. e. once a small region of dsDNA has separated to ssDNA the energy
required for the next region to separate is reduced). The process is fully reversible under
controlled conditions and the 're-annealed' DNA regains all physical and biological
properties. The change in absorption at 260 nm of nucleic acids can be used to follow
changes in the conformation of nucleic acids, for instance as a result of increasing
temperature. Under standard conditions the temperature at which half the DNA is melted
is known as the melting temperature (Tm). You might consider why the Tm can be used as
an estimate of the percentage GC in any given DNA sample (this can vary between 30% and
70% depending on the organism of origin).
In the experiment today the effects of NaCl, MgCl2 and urea upon the melting of dsDNA
will be investigated. Also the melting of DNA and RNA will be compared. The structure of
urea is shown below:
O
H C H
N N
H H


Week 2 – Basic Techniques in Biochemistry – Experimental

Check the bacterial cultures for the expression of the POU domain first and then
investigate the DNA melting while you are waiting for the expression. You can count the
colonies on your plates whenever you have a spare 10 minutes.
1. Expression of the Oct-1 POU domain in E. coli.
There is a 250 ml conical flask for each group in the orbital incubator. It contains 50 ml of
LB (a rich medium for bacteria) and 100 µg/ml ampicillin. This flask was inoculated with 5 ml
of an overnight culture of E. coli containing the plasmid that includes the DNA for the Oct-1
POU domain under the control of a T7 promoter.
1. When you are ready to start, check that the wavelength of the spectrophotometer is set
to 600 nm. A demonstrator will show you how to use the spectrophotometer. Zero the
spectrophotometer with a plastic cuvette containing 1 ml of LB (this is your blank).
2. Take the conical flask out of the incubator, remove 1 ml of culture and place it in a
cuvette. Measure the absorbance of the culture at 600 nm. If the absorbance is between
0.6 and 0.8 go to step 3. If your absorbance has not yet reached the lower limit, put the
flask back in the 37 °C incubator for a short time (consult a demonstrator to see how
long) and then measure again. Discard the 1 ml of culture into Virkon disinfectant.
3. Once the absorbance of the cultures is 0.6-0.8, induce expression of the Oct-1 POU
domain with 50 µl of 1M IPTG, under sterile conditions, and return to the 37 °C shaker
for ~3 hours.
4. After ~3 hours, transfer ~35 ml of the culture into a centrifuge tube and pellet the cells
in the benchtop centrifuge for 10 minutes at 4,000 rpm. Make sure you label the tube
with your name, practical day and bench number. You will need another tube to balance
the centrifuge, either by spinning at the same time as another group or by using a tube
containing the same volume of water.
5. Carefully pour off the supernatant into a beaker containing Virkon. Make sure you do not
disturb the pellet.
6. Transfer the remaining culture into the same tube and spin again for 10 minutes at 4,000
rpm, again ensuring that you balance the tube in the rotor appropriately. Pour off the
supernatant into a beaker containing Virkon.
7. Place your tube on ice, making sure that it is clearly labelled with your name, practical
day, and bench number. The pellet will be stored at -20 °C and returned to you next
week.
In experiments with bacteria, ensure that you have your lab coat
buttoned and that you wash your hands with soap before leaving the
laboratory.

2. Assessing the efficiency of the ligation that you performed last

week
You should have three plates (the positive and negative control plates and the ligation plate,
which contains the products of the ligation containing insert) that you spread last week:
they have been stored for you at 4 °C. Count the number of blue and white colonies on
each of the plates (if you have too many to count, estimate the total by dividing the plate
into 4 equal sections with a marker pen, then count the colonies in one quadrant and
multiply by 4). Now calculate the percentage of blue and white colonies on each plate. How
can white colonies arise on the control plate? How can blue colonies arise on the ligation
plate?
3. Investigation of the unfolding of DNA and RNA at high

temperature
Note the temperature of the heat block. Always use a thermometer; do not rely on the
thermostat setting. If it is above 25 °C, turn off the heat block for at least 15 minutes,
before turning it on again. The on/off button is at the rear of the machine.
You are provided with three of the following five buffers for the melting experiment:
1) TE
2) TE plus 4 mM sodium chloride.
3) TE plus 2 mM magnesium chloride.
4) TE plus 100 mM sodium chloride.
5) TE plus 2 M urea.
TE is 10 mM Tris-HCl, 1 mM EDTA, pH 7.5
Preparing the nucleic acid solutions

Dilute the stock DNA before making any measurement. You are given a stock of
herring testis DNA solution of approximately 2 mg/ml in TE. Take 40 ml of each buffer and
separately add 0.35 ml (350 µl) of stock DNA to each, mixing well. Take great care to mix
well as the DNA solution is often viscous and will not mix if you only give it a gentle
swirl – put the top of the tube on and invert a few times
You are also provided with a solution of yeast RNA at approximately 10 mg/ml in TE. Place
40 ml of TE buffer in a 50 ml tube, add your entire RNA stock solution (50–100 µl) to it and
mix well.
Set up the spectrophotometer

Ask a demonstrator to show you how to use your spectrophotometer.
Using the plastic Pasteur pipette or 1 ml Gilson provided, transfer approximately 1 ml of TE
buffer into a quartz cuvette to serve as a blank. Place the cuvette in the spectrophotometer.
Ensure that the wavelength is set at 260 nm. This blank will usually be the same for all three

buffers, but you should check that your other buffers have no absorbance at 260 nm – if
either does, correct your later measurements accordingly.
Initial absorbance readings and denaturation of double-stranded

DNA in alkali
Take a second quartz cuvette and add about 1 ml of DNA solution in TE. Place this cuvette
into the spectrophotometer, and read the absorbance at A260. It is also instructive to
measure the absorbance of the DNA at 280 nm. Do not forget to re-zero on the TE
blank when you change the wavelength. The ratio of A260/A280 should be about 2. If
the ratio is much lower than this, it indicates that the DNA is contaminated with protein.
Nucleic acids have an absorption maximum at 260 nm, whereas proteins show an
absorption peak at 280 nm, which is mainly due to the aromatic amino acids. Re-set the
wavelength to 260 nm and re-zero with the TE blank.
Double-stranded DNA can be melted not only by heating, but also by alkali (at low
temperature). To verify this, add 0.03 ml (30 µl) of 3 M NaOH, mix well, and repeat the
measurement.
Remove the cuvette(s) from the spectrophotometer, and pour out the sample. Take care
to remove all the solution including any large adhering droplets. Change solutions on the
bench not in the spectrophotometer. This avoids the DNA solutions being splashed over
the inside of the machine. Repeat the A260 measurement for the other nucleic acid
solutions. The solutions should be in the range 0.3 to 0.6. If not, adjust the DNA
concentrations accordingly. The A260 for RNA should be about 0.2.
Nucleic acid melting experiment

You are now ready to start the melting experiment. During the experiment regularly
check that the cuvette is clean: the urea solution is concentrated and given the
opportunity will crystallise out on the cuvette, so interfering with the readings. The
procedure involves warming the solutions in a water bath, and taking A260 readings at noted
temperatures. Each reading takes 1 ml that you will throw away after the
measurement, so plan how many readings to take – you don’t want to run out of
solution at 75 °C.
Keep your cuvettes on top of the heat block so that they do not cool down and change the
temperature of the solution. If you wash your cuvettes with H2O, you will cool them down
so remember to reheat them by placing on top of the heat block, when they’re dry, for at
least 2 minutes.
Place each of your solutions into 2 ml microfuge tubes in the heatblock at the first
temperature point (room temperature) and put a screw cap on to reduce evaporation.
Adjust the heat block to the temperature you want by using the up/down keys at the front
of the block. Start the block by pressing ‘Start’. Measure the temperature by following
temperature changes recorded by the thermometer placed in the heat block.

Record the temperature and immediately remove a sample and measure the new A260.
Discard this sample, and repeat for each solution. Try to record the A260 at 10 °C intervals
for each DNA sample and 5 °C intervals for RNA over the lower temperature range. Note
that you cannot go down in temperature, so you must do the low temperature points first.
When 60 °C has been reached continue to try to obtain readings at 5 °C intervals.
Always take care, and try not to touch the heat blocks directly. Be very
careful at higher temperatures as the gloves may melt onto your skin.
Plot your results as the experiment proceeds and estimate Tm from your graph for each of
the solvents.
The most useful way is to use the starting A260 as A0 and the A260 at
temperature T as AT then plot AT/A0 against temperature.

Linear graph paper

Linear graph paper

Notes

Notes

Purification of His-tagged Proteins

You have only one task in today’s practical, to take the bacterial cell pellet from last week
and extract your protein of interest in a relatively pure form.
The first task is to disrupt the bacterial cell walls to get at the protein inside. Many research
labs use sonication (application of ultrasound) for this, but it is not suitable for a practical
class. You will use a commercial solution called BugBuster® that is a mixture of detergents
that perforate the cell wall without denaturing the proteins (which most detergents do).
You will help the process along by adding the enzyme lysozyme, which as you know from IA
Biology of Cells, breaks the peptidoglycan layer in bacterial cell walls. The final addition to
the lysis buffer is the nuclease, DNase I. When the cell walls are disrupted and the contents
of the cell are released the solution becomes very viscous due to the large amount of DNA
present. DNase I cleaves the DNA into smaller pieces and prevents the high viscosity.
The protein that you expressed last week is a fusion protein that includes:
(His)6 – maltose binding protein (MBP) – Oct1 POU domain
Both the His and MBP tags are present to aid protein purification but today you are going to
utilise the His tag. At high pH, histidine sidechains chelate to metal ions, such as Ni2+ (why?).
Ni2+ ions are immobilized on Sepharose, which is an inert, cross-linked polysaccharide
polymer in the form of a small column. When the bacterial cell lysate is passed over the
column, the His-tagged protein sticks to the Ni2+ and most other proteins, nucleic acids etc.
will pass through. After washing to remove excess unbound material the His-tagged protein
can be eluted from the Ni2+ by adding a high concentration of imidazole, which also binds
tightly to Ni2+. You are going to use a His SpinTrap® column today, which is suitable for
small-scale protein production. It comprises 50 µl of Ni2+-Sepharose in a small column that
fits into a 1.5 ml microfuge tube and can be centrifuged.
Wash buffer 1
Wash buffer 1 & 2
Elution buffer

After the affinity purification, you will perform a Bradford assay to estimate the
concentration of protein that you have produced. For this you will use the dye Coomassie
Brilliant Blue G250, which is green-brown when free in acid solution, and becomes blue
upon binding to protein. You will use the spectrophotometer to measure the intensity of
the blue colour developed by a series of protein solutions of known concentration. This
allows a standard curve to be constructed against which your samples can be compared and
quantified.
You will monitor the success of your protein purification using gel electrophoresis, where
molecules are separated according to their size by applying a voltage across a fixed
conducting medium. For large molecules like proteins (whose Mr values can lie between 1
kDa and 1 MDa), a porous medium of aqueous polyacrylamide is used.
Polyacrylamide gels are prepared by dissolving acrylamide monomer, [CH2=CH-CO-NH2],
and a cross linking agent called N,N’-methylenebis-(acrylamide), [(CH2=CH-CO- NH-)2
CH2]. In the presence of a suitable catalyst, gels are formed, consisting of a three
dimensional network of long chains of [-CH2- CH2 (-CO- NH2)]n- cross-linked at intervals
by methylene groups.
The pore size of the gel can be modified by varying the acrylamide monomer concentration.
For example, gels containing 5% acrylamide will separate polypeptides of 100-400 kDa Mr
while gels containing 15% acrylamide separate polypeptides of 10-100 kDa Mr.
In the presence of the strong ionic detergent sodium dodecyl sulphate (SDS) CH3-(CH2)11-
O-SO3- Na+ proteins are completely unfolded. Most proteins bind equal amounts of anionic
dodecyl sulphate per gram (1.4 g per g of protein) and so behave as polyanions with a
constant ratio of negative charge to mass. Any inherent differences in charge due to the
amino acid side chains are thus obscured and the proteins behave like rigid rods in solution.
Provided that disulphide bonds have been reduced (by, for example, b-mercaptoethanol),
multimeric proteins are also completely dissociated into their subunits by SDS.
During electrophoresis in a polyacrylamide gel that contains SDS, the proteins move at
speeds determined only by the size of their SDS–protein complexes (i.e. length of
polypeptide chain and therefore molecular mass). For a wide range of protein sizes, the
speed of migration falls linearly as the logarithm of the Mr of the protein rises, because
longer chains are more retarded by the gel medium.
You will run molecular weight markers at the same time as your unknown samples. Plotting
the migration distance vs the log of Mr should yield a straight line that can be used to
estimate the size of the proteins in the samples. Today, you will use the markers
qualitatively, to help you figure out which band in the gel sample corresponds to your
protein of interest.
Molecular weight markers used for the

SDS-PAGE in today’s practical.

Week 3 – Protein Purification – Experimental
Key to buffer and reagent solutions
BLD: BugBuster, containing 0.2 mg/ml lysozyme, 20 µg/ml DNase I, 20 mM imidazole

WB1: Wash Buffer 1: 20 mM sodium phosphate, pH 7.4, 500 mM NaCl
WB2: Wash Buffer 2: 20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 20 mM imidazole
EB3: Elution Buffer: 20 mM sodium phosphate, pH 7.4, 500 mM NaCl, 500 mM imidazole
SB: 2x Sample Buffer: 0.26 M Tris-HCl, pH 6.8, 10% (w/v) SDS, 40% (v/v) glycerol, 5% (v/v)
b-mercaptoethanol, 0.05% bromophenol blue
PDB – Protein Dilution Buffer: 100 mM Tris-HCl, pH 7.5, 1.6 mM EDTA, 50 mM NaCl,
10% glycerol (N.B. MAY NOT BE NECESSARY).
Cell Lysis
Last week you made a pellet of cells that has been stored at -20 °C. These have been placed
on ice on your bench.
1. Make sure that your pellet is thoroughly defrosted.
2. Add 1 ml of BLD to the centrifuge tube. Thoroughly resuspend the pellet by pipetting up
and down using a Gilson pipette.
3. Using a Gilson pipette, divide the suspension equally into two microfuge tubes. Label
them L1 and L2, there should be approximately 0.7 ml in each. Leave at room
temperature for 30 minutes.
Preparation of His SpinTrap Column

Meanwhile, prepare the column for the purification of your protein:
1. The His SpinTrap column is in your microfuge rack.
2. Invert and shake the column to resuspend the beads in the storage buffer.
3. Loosen the cap 1/4 of a turn and break off the bottom closure. Cut off the lid from a 1.5
ml microfuge tube and place the column into the tube.
4. Place the column and microfuge tube into the microcentrifuge, using an empty tube as a
balance tube. Press the pulse button on the microcentrifuge for 10-20 seconds to
remove the storage buffer. Discard the flow-through in the microfuge tube into the
beaker provided and not the sink! And replace the column.
5. Apply 600 µl of WB1 to wash the column.
6. Pulse for 30 seconds in the microcentrifuge and discard the flow-through in the tube.
Your column is now ready for use.

Purification of the His-tagged protein

At various stages during the protocol you will take samples for analysis by SDS-PAGE. Label
1.5 ml microfuge tubes for the gel samples as follows: S1, S2, W1, W2, W3, W4, W5, W6,
E1, E2.
1. Once the 30 minute lysis is complete, centrifuge your two lysate samples, L1 and L2, in a
microcentrifuge for 10 minutes at high speed. Make sure the His SpinTrap column
is NOT in the microcentrifuge during this spin! A pellet will form at the bottom of
the L1 and L2 tubes.
2. Carefully remove the tubes from the microcentrifuge. Take 10 µl of the supernatant
from L1 and add to the first gel sample tube, S1 and 10 µl from L2 and add to sample
tube S2. Add 90 µl of 2x gel sample buffer (SB) to each of S1 and S2.
3. Carefully remove 600 µl of supernatant from L1 without disturbing the pellet and add it
to the top of the His SpinTrap column. Put the column into the microcentrifuge, ensuring
that it has an empty tube as a balance and is sitting in an empty tube to collect the flow-
through. Pulse for 10-20 seconds as before. Discard the flow-through into a beaker
containing a weak solution of Virkon. Remove 600 µl of supernatant from L2 without
disturbing the pellet and add it to the His SpinTrap column. Pulse for 10-20 seconds and
discard the flow-through into Virkon. If there is a small amount of supernatant left in L1
and L2, carefully remove it with a 200 µl Gilson pipette (yellow tip) taking care not to
disturb the pellet, add it to the His SpinTrap column and pulse for 10-20 seconds,
discarding the flow-through.
4. Wash the His SpinTrap column by applying 600 µl of WB1 to the top of the column and
pulsing for 10-20 seconds in the microfuge. Take a 10 µl sample of the flow-through and
add it to the gel sample tube ‘W1’. Discard the remainder of the flow-through. Add 10 µl
of SB to sample tube W1 and put the tube aside for analysis later. Repeat twice more,
using sample tubes W2 and W3.
5. Wash the His SpinTrap column once more by applying 600 µl of WB2 to the top of the
column and pulse for 10-20 seconds in the microfuge. Take a 10 µl sample of the flow-
through and add it to the gel sample tube W4. Add 10 µl of SB to sample tube W4 and
put the tube aside for analysis later. Discard the remainder of the flow-through. Repeat
twice more, using tubes W5 and W6 for gel samples.
6. Place the His SpinTrap column into a clean 2 ml microfuge tube, discarding the previous
flow-through and the tube into Virkon. Apply 200 µl of EB3 to the top of the column to
elute the protein. Pulse in the microcentrifuge for 10-20 seconds. Do not discard the
flow-through (eluant). Take the eluant and put it into a clean 1.5 ml tube (labelled
Elution). Take a 10 µl sample of the eluant and add it to sample tube E1, adding 10 µl of
SB. Place the tube containing the eluant on ice.
7. Place the HisSpinTrap column back into the tube. Apply another 200 µl of EB3 to the
column and pulse in a microcentrifuge for 10-20 seconds. Take a 10 µl sample of the
eluant and add it to sample tube E2 along with 10 µl of SB. Remove the remainder of the
eluant and add it to the eluant from step 6.
8. Place the pooled eluants on ice for the remainder of the practical.

Analysis of the protein by SDS-Polyacrylamide Gel Electrophoresis

1. You should have 10 samples collected during the purification and you will have a
microfuge tube marked M provided on your bench that contains protein molecular
weight markers. Place all 11 samples into the 95 °C hot-block for 2 minutes making
sure the safety caps are in place. Please leave the caps in the tray next to the heat block
when you’re done with your incubations.
2. Place the samples in the microcentrifuge and spin at high speed for 5 minutes.
3. Load 15 µl of each sample onto the gel into individual adjacent wells using a 20 µl
Gilson pipette (yellow tip). Get a demonstrator to show you if you are unsure of how
to do this. Load the samples in the order in which you collected them i.e. S1, S2, W1,
W2, W3, W4, W5, W6, E1, E2 with M at end.
4. After making sure that the electrophoresis power supply is switched off, place the lid of
the tank onto the base (taking care not to spill the buffer). Connect the power supply
and the gel tank with the power leads. Switch on the power supply and adjust the
voltage to 200 V and ensure that the switch is on constant voltage. Run the gel until the
dye front just reaches the bottom of the gel (about 30 min).
Estimating the concentration of the protein produced

While your gel is running, you will measure the concentration of your protein using a
Bradford assay. The change in colour is measured as Coomassie dye binds to protein and
turns blue. A standard curve is constructed with known amounts of protein and the
amounts of protein in the samples can then be estimated.
1. A stock solution of 20 mg/ml (=20 µg/µl) BSA (Bovine Serum Albumin) has been
provided for you. First make two serial dilutions from this stock. Place 5 µl of 20 mg/ml
BSA into a new 1.5 ml tube and add 95 µl of distilled water to make a 1 mg/ml solution.
Now take 10 µl of the 1 mg/ml dilution into a new 1.5 ml tube and add 90 µl of distilled
water to make a 0.1 mg/ml solution. Label the new tubes with their concentration.
2. Set up a series of 8 cuvettes, 6 of which will contain a different amount of BSA and the
7th and 8th to contain the sample you wish to test (this is the protein solution that you
have stored on ice). Using the table below for quantities, add the water and the
Bradford reagent to each cuvette first and finally the protein (BSA or test sample). Mix
by covering the top of the cuvette with parafilm and inverting gently.
SAFETY NOTE: WEAR GLOVES WHILE HANDLING THE BRADFORD
REAGENT AND MIXING THE TUBES
Final amount of Volume of BSA Volume of Volume of

BSA per tube (µg) needed distilled water Bradford reagent
0 0 µl 20 µl 980 µl
1 10 µl of 0.1 µg/µl 10 µl 980 µl
2 20 µl of 0.1 µg/µl 0 µl 980 µl
5 5 µl of 1 µg/µl 15 µl 980 µl
10 10 µl of 1 µg/µl 10 µl 980 µl
20 20 µl of 1 µg/µl 0 µl 980 µl
- 5 µl Sample 15 µl 980 µl
- 10 µl Sample 10 µl 980 µl

3. Leave the cuvettes to stand for 10 minutes.

4. Check that the spectrophotometer is set to 595 nm. Place 1 ml (1000 µl) sample of
distilled water into a cuvette and zero the spectrophotometer. Now read the
absorbance of your 7 samples, making a note of the readings in each case.
5. Plot a standard curve on the graph paper provided. Plot µg BSA on the x-axis and A595
on the y-axis. The plot should be linear within the range of BSA used. From the graph,
read off the amount of protein present in the 5 µl sample in µg. Calculate the
concentration of protein that you have purified in µg/ml.
6. You will need 100 µl of an ~100 µg/ml protein sample for the band shift
assay.
If your protein concentration is close to, or less than, 100 µg/ml (ask a demonstrator),
you can use your solution directly in the assay. If your protein is too concentrated, you
will need to make a dilution. For example, if your protein concentration is 500 µg/ml,
you will need to add [(100/500) x 100] µl of protein solution (i.e. 20 µl) and then add
PDB to make the volume up to 100 µl.
You must dilute your protein now. If it is frozen in too high a

concentration it will precipitate out of solution once thawed, and
be ruined.
Staining and photographing the SDS-Polyacrylamide Gel

Once the blue dye has reached near the bottom of the gel turn off the power supply and
then carefully remove the gel tank cover.
SAFETY NOTE: WEAR GLOVES WHEN HANDLING THE GELS.

ACRYLAMIDE IS ABSORBED THROUGH THE SKIN AND IS NEUROTOXIC.
The gels are quite fragile so some care is required. Carefully remove the gel, sandwiched
between glass plates, from the apparatus after removing the two clips which clamp it in
place. Lay the gel sandwich on the bench (notched glass plate upwards) and gently prise the
notched plate off by inserting a spatula between the glass plates, midway between the top
and bottom of the plate and levering it up. The gel should stick to the lower plate with
spacers.
Hold the glass plate with the gel upside down over the plastic tray and gradually peel off the
gel with the spatula letting the gel fall into the plastic tray. Pour 10 ml of Instant Blue Stain
into the plastic dish, then place the dish into the shaking waterbath. You should see the
bands developing, stained a bright blue colour against a clear background.
Once the bands are sufficiently bright, discard the staining solution into the ‘Waste Stain’
pot. Do not put it down the sink.
A demonstrator will help you to photograph your gel.

Linear graph paper

Linear graph paper

Notes

Notes


Electrophoresis mobility shift assay (EMSA)
This week you will take the protein that you purified last week and test its ability to bind to
DNA. The Oct-1 POU domain binds to a specific 8 bp segment of DNA, ATGCAAAT,
called the octamer, in certain promoters (see Dr Mata’s and Prof. Smith’s lectures). The
POU domain itself is composed of two subdomains: the homeodomain (PouH) and the POU
specific domain (PouS). Homeodomains use a helix-turn-helix motif to bind DNA and are
found in several other proteins. The POU specific domain, as its name suggests, is only
found in POU domain proteins. The structure of the complex of the Oct-1 POU domain
with DNA has been solved by X-ray crystallography and it shows that both the subdomains
bind to DNA. Today you will investigate the relative importance of the two binding sites for
the affinity of the interaction.
Oct-1 POU domain binding to DNA.

PouS is on the right and PouH is on the
left. These subdomains bind on
opposite faces of the DNA double
helix and are connected by a flexible
linker.
You will use the electrophoresis mobility shift assay (EMSA), also known as a gel shift or gel
retardation assay, to investigate the binding of the POU domain to DNA. As you know
already from the previous weeks, electrophoresis separates molecules on the basis of their
size and charge. In the EMSA, you will look at the change in size when a small piece of DNA
binds to the POU domain. The POU domain-DNA complex is larger than the DNA on its
own and so the bound DNA is retarded in the gel. When a gel is run with free DNA and
the complex DNA in separate lanes, it is possible to see the difference in the mobility of the
bands. The migration of the DNA in the gel is detected by staining with Sybr Gold, a
proprietary, asymmetric cyanine dye that exhibits >1000-fold fluorescence enhancement
upon binding to nucleic acid – which is considerably (>x100) more sensitive than ethidium
bromide (week 1).
It is important that the gel system used for EMSA is non-denaturing, otherwise the protein-
DNA complex will not stay together while the gel is run. Thus SDS will not be included in
the polyacrylamide gel or in the buffers this time.
The dissociation constant (Kd) is a useful measure to describe the strength of binding (or the
affinity) between the protein (P) and DNA (D). The interaction between P and D can be
described by the equilibrium:
P + D PD

The balance between the concentration of the free protein ([P]) and the concentration of
the bound complex ([PD]) depends on the strength of the interaction. At equilibrium, the
dissociation constant is defined as:
Kd=[P][D]/[PD]
In practice, reactions are performed with an excess of protein, so the change in free protein
concentration is negligible and the input protein concentration can be used as [P]. Then the
Kd equals the protein concentration when 50% of the DNA is bound.
You will perform gel shift assays today with the following oligonucleotide sequences:
5’CCAGGGTATGCAAATTATTAAGGGC
3’GGTCCCATACGTTTAATAATTCCCG
The above is the so-called wild type (wt) sequence, with POU-binding nucleotides in bold.
You will compare POU binding to the wt sequence with one of three mutated dsDNA,
altered as below:
Mutation 1 (PouH mutation)

5’CCAGGGTATGCAAGCTATTAAGGGC
3’GGTCCCATACGTTCGATAATTCCCG
Mutation 2 (PouS mutation)

5’CCAGGGTCGGCAAATTATTAAGGGC
3’GGTCCCAGCCGTTTAATAATTCCCG
Mutation 3 (1+2 combined)

5’CCAGGGTCGGCAAGCTATTAAGGGC
3’GGTCCCAGCCGTTCGATAATTCCCG
Or you will compare the wt with a random dsDNA of the same length.
These oligonucleotides were synthesized as single stranded DNA (just like the primers you
used for PCR) and were annealed by heating to 85 °C and then slowly cooling.

Week 4 – Electrophoresis Mobility Shift Assay – Experimental
Key to buffer and reagent solutions

AB – 5x band shift assay buffer: 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT,
250 mM NaCl, 50 mM Tris-HCl, pH 7.5.
DNA – annealed oligonucleotides at a concentration of 0.58 µM.
LB – 10x gel loading buffer: 250 mM Tris-HCl, pH 7.5, 40% glycerol, 0.1% bromophenol
blue.
Heparin – 1 mg/ml
You purified your POU protein last week and should have diluted it to a concentration of
100 µg/ml. This has been stored at -80 °C and is an ice bucket on your bench.
1. Label 6 microfuge tubes 1-6 and place on ice. Then, using the wt DNA, add the following
from the table, adding the protein last:
1 2 3 4 5 6
Tube
7 8 9 10 11 12
DNA (µl) 2 2 2 2 2 2
AB Assay Buffer (µl) 3 3 3 3 3 3
Water (µl) 10 9 8 6 2 0
Protein (µl) 0 1 2 4 8 10
Do the same for the mutant/random DNA, this time with tubes labelled 7-12. We will
allocate mutant or random DNA to each group. Some groups will add, to all of their 12
tubes, 1 µl of heparin, a highly-sulphated glycosaminoglycan.
2. Flick the tubes gently to mix, then briefly (1-2 sec) spin them in the microcentrifuge to
make sure all reagents are at the bottom of the tube. Leave the tubes at room
temperature for 30 minutes to allow the protein and DNA to bind.
3. Whilst your protein is binding, pre-run the gel. Mark the wells of the gel onto the glass
plate, and remove the comb from the gel. Place the lid of the gel tank onto the base and
connect the power supply to the gel tank. Switch on the power supply and adjust the
voltage to 90 V, ensuring that the power supply is set to constant voltage. Run the gel
for 10 minutes. This step removes all traces of ammonium persulphate (used to
polymerize polyacrylamide gels) and ensures a constant gel temperature.
4. Once the 30 min protein-DNA incubation is complete, add 1 µl of LB to each tube, flick
gently to mix then spin briefly in the microcentrifuge.
5. Make sure that the electrophoresis power supply is off, then remove the lid of the tank
from the base, taking care not to spill the buffer. Load 10 µl of each sample onto the gel
into individual adjacent wells using a clear tip. Get a demonstrator to show you if you
are unsure of how to do this. Load the gel samples in order 1 to 12.

6. Place the lid of the tank onto the base (taking care not to spill the buffer). Connect the
power supply and the gel tank with the power leads. Switch on the power supply, adjust
the voltage to 90 V and ensure that the switch is on constant voltage. Run the gel until
the dye front is about 3/4 of the way down the gel (about 45 min).
Safety note: wear gloves when handling the gels. Acrylamide is

absorbed through the skin and is neurotoxic.
7. The gels are quite fragile so some care is required. Carefully remove the gel,
sandwiched between glass plates, from the apparatus after removing the two clips
which clamp it in place. Lay the gel sandwich on the bench (notched glass plate
upwards) and gently prise the notched plate off by inserting a spatula between the glass
plates midway between the top and bottom of the plate and levering it up. The gel
should stick to the lower plate with spacers.
8. Pour 20 ml of SYBR Gold Stain into the plastic tray. Carefully hold the glass plate with
the gel upside down over the plastic tray and gradually peel off the gel with the spatula
letting the gel fall into the plastic tray. Place the dish in a shaking incubator. Make sure
you cover your tray with foil as the stain is light sensitive. The bromophenol blue will
lose its colour as the stain develops. Allow the gel to stain for 30 minutes.
9. After 30 minutes, discard the staining solution into the ‘Waste Stain’ pot. Do not put
it down the sink.
10. A demonstrator will help you to photograph your gel.
Safety Note:
As SYBR Gold also binds to double stranded DNA we are instructed to treat it
in the same manner as Ethidium Bromide.
Ethidium Bromide is a mutagen. Even though SYBR Gold is not yet proven to
be one, wear two sets of gloves when handling the gel and avoid getting SYBR
Gold on your hand, clothing, or anywhere else. Carry your gels on the plastic
trays provided. There are very clear university guidelines on how to handle
SYBR gold/Ethidium Bromide and these are posted on the classroom notice
boards.

Notes

Notes

Basic Techniques in Molecular Biology – Question Sheets NST 2018-19
Question Sheets – Practicals 1 to 4
Week 1 – Questions
(Give one-line answers to the following questions)
Why do Bacillus amyloliquefaciens and Haemophilus influenzae not find their own DNA
digested by the enzymes they contain?
How do you know that the Oct-1 POU domain was inserted in the correct orientation?
How might you confirm this?
What potential problems might you encounter using Taq polymerase for PCR? How are
these problems overcome? Is it likely to be a problem in the PCR in this practical?
Why is it important to treat the vector preparation with alkaline phosphatase prior to
ligation? Where does alkaline phosphatase come from?
How are E. coli cells made ‘competent’ so they will take up DNA? What other methods
might be used to transform E. coli with DNA?

You have just finished an arduous experiment using a new set of primers designed to amplify
the b-globin gene but you have ended up with no PCR product. Suggest five reasons why
your experiment might have failed (other than forgetting to add any of the components).
(i)
(ii)
(iii)
(iv)
(v)
You have performed another arduous experiment and now you obtain a myriad of PCR
products where you expected only one band. Suggest a reason why this might have
happened and how you would go about testing your hypothesis.
What is the theory that allows one to predict the annealing temperature of a primer? (Look
it up).

The following problem, which was set in the 2002 NST IB Practical examination,
illustrates several important aspects of PCR.
A number of PCR reactions were carried out attempting to optimise the amplification of a
particular segment of chromosomal DNA. The reaction conditions were as follows:
Reaction Chromosomal Primers Annealing Mg2+
number DNA added added temperature concentration
(i) Yes short pair 55 °C 1.5 mM
(ii) Yes long pair 55 °C 1.5 mM
(iii) Yes short pair 65 °C 1.5 mM
(iv) Yes long pair 65 °C 1.5 mM
(v) Yes short pair 45 °C 1.5 mM
(vi) Yes one long 55 °C 1.5 mM
primer only
(vii) Yes none 55 °C 1.5 mM
(viii) No short pair 55 °C 1.5 mM
(ix) Yes short pair 55 °C 3.5 mM
(x) Yes short pair 55 °C 7.5 mM
Taq DNA polymerase was added to all reactions, and amplification was for 25 cycles using
the following program: 94 °C for 1 minute (the denaturation or melting step); the stated
annealing temperature for 50 seconds; 75 °C for 30 seconds (the polymerisation step).
The short primers were each 15 residues long, and the long primers 30 residues. The
short and long forward primers were co-terminal at their 3’-ends, and the short and long
reverse primers were likewise co-terminal at their 3’-ends. Primers were added in large
excess.
At the end of the reaction the products were separated by agarose gel electrophoresis in
a gel containing ethidium bromide, and the products visualised by the fluorescence of the
bound ethidium under UV illumination. The result is depicted below. However, the
reaction tubes had been carelessly muddled up before loading the samples on the gel.
Match the lanes (1) – (10) with the reactions (i) – (x) described above, explaining your
reasoning. Note that for some lanes it may not be possible to unambiguously assign that
lane to a single unique set of reaction conditions.

Notes

What percentage of the colonies on your negative control plate were white?
What percentage of the colonies on your ligation plate were white?
How can blue and white colonies arise on the control plate?
How could blue colonies arise on the ligation plate?
Nucleic Acid Melting

From your graph of AT/A0 against temperature determine the TM of dsDNA in TE buffer
alone and in the presence of 0.1 M NaCl and 2 M urea.
Rationalise the effects of NaCl and MgCl2 upon the stability of the dsDNA.
Explain the effects of urea upon the stability of dsDNA.
What is different about the melting profile of tRNA compared with dsDNA? Explain the
differences that you observe.

Estimate the molecular weight of the His6-MBP-POU domain fusion protein that you
have produced. How does this compare with the expected size (N.B. His6-MBP contains
398 amino acids; an average amino acid has a molecular weight of ~110 Da)?
What is the concentration of your purified protein sample?

In mg/ml:
In mmol/litre:
Estimate the degree of purity of your protein (in %). What assumptions are you making?
How much pure protein could you make from 1 litre of E. coli culture?
What could be the nature of the other proteins that co-elute with the POU domain?
What is the difference between the two wash buffers and what is the difference between
the contaminants that these buffers are removing?
What steps would you suggest to purify the POU domain further?
How does the separation achieved by gel electrophoresis differ from that of gel filtration?

What is the approximate Kd of the purified POU domain for the octamer motif?
What is the approximate Kd of the purified POU domain for the mutant octamer
motifs/random DNA (obtain results of the other 2 mutants from neighbouring groups)?
Mutant 1
Mutant 2
Mutant 3
Random DNA
What can you conclude about the relative importance of PouH and PouS subdomain binding
to the octamer motif?
What other techniques can be used to measure and detect specific interactions between
DNA and DNA-binding proteins?
Which of these techniques are applicable to RNA-binding proteins?

Michaelmas Week 5: Using Online Databases as Tools NST 2018-19
Week 5: BMB Experimental Exercise – Using Online Databases as

Tools
This exercise will guide you through using some online tools that would be used in the lab
to answer specific experimental questions or to analyse data. There are numerous
approaches that could be used to do the task set out here – the methods outlined are just
examples of the way you may choose to approach the question.
Your lab is interested in a specific protein (Adenosine Deaminase that Acts on RNA –
ADAR1) that plays a novel role in stress granules in mammalian cells. Stress granules are
cytoplasmic foci that form during stress conditions, and which enable to cell to survive
stresses. Although it is known that stress granules contain an assortment of proteins and
RNA, the function of ADAR1 in stress granules is not clear. To gain some insight into the
role of ADAR1 in stress granules, various experiments have been performed, including an
immunoprecipitation assay that has been used to identify any human proteins that interact
with recombinant ADAR1. The results of this experiment are shown here:
This experiment showed that a protein of unknown identity (~100 kDa) interacted
specifically with ADAR1 under stress conditions – the corresponding protein band was
excised from the polyacrylamide gel and peptide sequences were determined using mass
spectrometry. This gave rise to the following peptides:
1) VHFTAERSSYYK
2) TIHLSSIRPPRLEGENTQDK
3) DTPDEPWAFPAR
4) AGNFIGWLHIDGANLSVLLVEHALSK
5) ETCLITFLLAGIECPR
6) ALLLPDYYLVTVMLSGIK
7) PLYDIPYMFEAR
8) SSYYKSLLSAEEAAK

Summary of what you will achieve in this exercise – you will aim to:
• Determine the origin of the peptides – what is your protein of interest?
• Find the nucleotide sequence encoding your protein
• Identify the cDNA encoding your protein and the ORF
• Design primers for cloning the ORF into an expression plasmid
• Find siRNAs to knockdown your protein of interest
1. Investigating the origin of the peptides

The first thing you want to do is to determine the identity of the protein(s) that the
peptides come from. There are various websites you can use to do this. One such site is
NCBI (National Center for Biotechnology Information – http://www.ncbi.nlm.nih.gov/ ).
First choose one of the peptides listed above (start by looking at

the first peptide: VHFTAERSSYYK), and then use the ‘BLAST’
(Basic Local Alignment Search Tool) function to look for proteins
that contain that peptide sequence. BLAST can be found within the
list at the top right hand side of the page above.
BLAST finds regions of similarity between biological sequences. The
program compares nucleotide or protein sequences to sequence
databases and calculates the statistical significance.

Click on “BLAST”, and you will go to the following window, where you will have the choice
of doing either a Nucleotide BLAST or a Protein BLAST.
Click on “Protein Blast”, and you will be taken to this page:
Enter your query sequence into the box (‘Enter Query Sequence’) – this can be entered as
the accession number(s) (e.g. Genbank Accession number), gi(s), or FASTA sequence(s) –
see glossary at end). Or in our case, we will just enter one of the peptide sequences (‘bare
sequence’).
Once you have done that, you should look at the various search parameters. First, decide
which database you want to search from the top drop down menu. We will use the ‘Non-
redundant protein sequences (nr)’ for this exercise – this database contains non-redundant
sequences (i.e. with duplicated sequences removed) from both non-curated (E.g.
GenBank/GenPept, trEMBL) and curated databases (E.g. RefSeq, SwissProt, PIR, PDB)).

Once you have done that, you can choose whether to search the entire data base, or
whether you want to limit your search to a particular organism (‘Choose Search Set’).
If you leave the box blank, the search will be unrestricted. As we are looking for a human
sequence, we could restrict the search to only look at human proteins – so type ‘Homo
sapiens’) in the box, as follows:
You can also alter the algorithm for the search if necessary – click on the ‘?’ symbol to find
out more about these functions. We are going to use ‘blastp (protein-protein BLAST)’ to
search the database to find proteins that contain the peptide sequence.
When searching with such a short sequence, you will see the message that ‘Your search
parameters were adjusted to search for a short input sequence’. It will take a few moments
for the results of your search to be generated.

When the search is complete, you will see a graphical summary of the proteins matching the
peptide sequence (with colour-coded alignment scores), and below that you will see the list
of proteins that match your query sequence.
Q1: What is the top hit?
Q2: What is the identity score (you will find this information in the 5th column on the right
hand side)?
Click on the top hit to see the alignment between your query sequence and the protein
identified. It is important to look at this to see the matches between amino acids –
sometimes the identity may be less than 100%, but this is the result of conservative amino
acid changes (E.g. Glu (E) for Asp (D)).
Then click on the ‘sequence id’ (above the alignment) to find out more about the protein.
Make a note of the protein name and the GenBank Accession number (‘Accession’), which
you will find toward the top of the page (combination of 3 letters and 5 numbers – see
glossary). You will need this information later.
Q3: How many amino acids are shown for the protein of interest?
Q4: How long would the mRNA be encoding that number of amino acids?
You next want to check whether the other peptide

sequences obtained also match this sequence – that
is important in order to determine whether the
protein identified is the protein of interest. Go to the
box on the right hand side of the page labelled
‘Analyze this sequence’, then click on ‘Run BLAST’.
This will take you back to the search screen seen previously. This time however, the
GenBank Accession number for the protein sequence identified will already be in the search
box.
At the bottom of that box, click on ‘align two or more sequences’.

This will give you a second sequence box for the ‘Subject Sequence’.
You can copy one of the other peptide sequences into the ‘Enter Subject Sequence’ box –
then carry out a BLAST search to check whether the sequence is contained within the
protein sequence identified (look at the alignment). Do this for each peptide sequence in
turn until you are sure that most or all of the peptides are derived from this protein.
Note: if you were using this search function for sequences that have NCBI identifiers (E.g.
accession or gi numbers) or are using FASTA-formatted sequences, multiple query sequences are
allowed – but it is important to include the list of identifiers (accession or gi numbers) as one per
line, or the group of FASTA sequences with each beginning line and starting with the “>” sign.
Q5: If you had started with a different peptide for your initial BLAST search, would you
have got the same initial result? If not, why?
Having checked whether the peptides are all derived from the same protein, this will enable
you to decide whether the protein identified via the BLAST searches corresponds to the
protein band that interacted with your protein of interest (ADAR1) in the
immunoprecipitation assay. If you are confident that this is the case, your next steps will aim
to look more closely at the interaction between the proteins. To do this, it will next be
necessary to clone the DNA encoding the protein identified.

2. Finding the nucleotide sequence encoding your protein

To find the nucleotide sequence encoding the protein you have identified, you need to go
back to the page that you looked at earlier that gave information about the protein. One
way of doing this is to enter the GenBank Accession number noted above into the search
box at the top of the front page of NCBI (http://www.ncbi.nlm.nih.gov/ ).
I.e.
In the dropdown menu at the left of the search box, scroll down and select ‘Protein’, and
then ‘Search’ – this will take you back to the page showing the details of the protein you
have identified.
Towards the top of this page, there is information about the name of the protein, the
number of amino acids determined, the organism, references etc. Further down there is
information about any domains that have been identified within the protein (via homology),
and then the actual amino acid sequence is given. Copy the protein sequence you have
identified for reference later on (E.g. copy and paste into a word document).
If you click on the accession number (2 letters, 6
numbers) toward the top of the page next to the
text ‘DBSOURCE’, this will take you to the page
showing the mRNA (cDNA) that encodes the
protein shown. Make a note of the GenBank
accession number for the mRNA, as you will need this later.
Q6: What is the difference between the mRNA sequence and the genomic sequence?
Q7: Why is it important to think about that when you are trying to obtain the sequence
encoding the protein of interest?
If you look towards the top of this page you will see the length of the mRNA encoding your
protein of interest (in bases).
Q8: How does this number compare with the predicted length of the mRNA encoding your
protein of interest, as calculated above? If it is not the same, how can you explain the
discrepancy?

Note: If you look further down the page, it gives some references for the sequence, which tells us
that the cDNA sequence shown has been identified as a part of a large sequencing project at the
RIKEN Genomic Sciences Centre (http://protein.gsc.riken.go.jp/ ), where cDNAs have been cloned
into a pBluescript II cloning vector (pBluescript II SK plus) and subsequently sequenced. This means
that the sequences given may not be annotated correctly – and if you look at the information given,
it tells us that there is ‘non-experimental evidence, no additional details recorded’ and that the ‘start
codon is not identified’. You need to bear this information in mind when you start analyzing the
cDNA sequence for cloning and expression.
3. Identifying the Open Reading Frame (ORF)

At the bottom of the page you will see the nucleotide sequence encoding your protein of
interest. Copy this sequence, as you will need it later. As noted above, this sequence is
longer than expected to encode the protein you have found. It is therefore important to
identify the Open Reading Frame (ORF) within the sequence that encodes the protein of
interest.
To do this, we can use the ExPASy Bioinformatics Resource Portal to translate the
sequence (http://web.expasy.org/translate/ ).
Copy the complete cDNA sequence from the NCBI page, and paste it into the box on the
ExPASy site, as shown above. You can alter the output format from ‘Verbose’ (‘Met’, ‘Stop’,
spaces between residues) to ‘Compact’ (‘M’, ‘–’, no spaces), or to include the nucleotide
sequence. There is also the option to change the genetic code used for translation, but for
this exercise we will just use the standard code. Once you have chosen the options you
prefer (choose either ‘Verbose’ or ‘Compact’), click on the ‘Translate Sequence’ button at
the bottom of the page.
You will now see a page where the nucleotide sequence has been translated in all 6 possible
reading frames.

Q9: Why is it important to do translate the cDNA in 6 frames?
Q10: How will you know which one is correct?
Have a look at the results, and see which reading frame is most likely to be correct – use
the protein sequence noted above to help. You already know the approximate size of the
protein you are looking for – you know this from the protein sequence obtained in silico,
but also from the initial gel showing the results of the immunoprecipitation assay (above).
Q11: Which reading frame is correct?
If you compare the translated sequence to the amino acid sequence obtained earlier using
NCBI, you will see that the translated sequence appears to be truncated at the N-terminus
by 54 amino acids. Make a note of the first and last few amino acids of the translated
protein (determined using ExPASy).
Q12: Why does the translated sequence not include the first 54 amino acids?
Q13: How could you verify that the translated sequence is correct?
Assuming that the translated sequence you have obtained is correct, you go on with your
analysis.
4. Identifying the cDNA sequence corresponding to the ORF
As the ORF is encoded by only part of the cDNA sequence you obtained above, you need
to identify the part of the cDNA sequence that corresponds to the ORF.
You can do this by inspection – go back and translate the cDNA again in ExPASy but this
time choose the ‘output format’ to ‘Include the nucleotide sequence’.

Once you have the translated sequence, scroll down until you find the correct reading
frame, as determined above, then find:
• The start codon (ATG; use the first few amino acids noted above to help you).
• Next find the stop codon at the end of the ORF (TAA) (use the information you
have regarding the length of the sequence to help you with this – and check that the
amino acid residues preceding the TAA are the same as those noted above).
Use this information to trim the excess 5’ and 3’ nucleotides off the cDNA sequence
obtained from NCBI (that you made a copy of earlier) – you should then have a sequence
that only encodes the ORF for your protein of interest. If you want to be sure you’ve done
this correctly, use the translate tool from ExPASy to translate your final cDNA sequence –
you should get a translated sequence that only contains the amino acids corresponding to
the ORF.
5. Cloning the cDNA encoding the ORF into a suitable expression vector
The next step of your experiment is to clone the cDNA encoding the ORF of your newly
identified protein into an expression vector suitable for expression in mammalian cells (E.g.
HeLa cells – see glossary). The vector you choose is p3xFLAG-CMVTM-7.1(Sigma-Aldrich;
‘p3xFlag’), which contains a sequence encoding a triple Flag tag (epitope tag) – this will
ultimately be fused to your protein of interest (see diagram below).

Q14: Looking at the overview of the plasmid, above, will this generate a fusion protein
(between the 3xFlag tag and your protein of interest) with an N- or C-terminal 3xFlag tag?
Q15: Is the relative location of the 3xFlag tag important when designing your cloning
strategy? Why?
You initially design a strategy for cloning the cDNA into p3xFLAG. This will be carried out
as follows:
• PCR primers will be designed for amplification of the cDNA encoding the protein of
interest. Restriction enzyme recognition sites will be incorporated into the primers
to enable directional cloning into p3xFLAG.
• The template for PCR will be generated by Reverse Transcription (RT; using an
oligo(dT) primer or random hexamer primers), where RNA harvested from HeLa
cells is used as a template for the RT reaction.
Alternatively, it may be possible to order a clone from the I.M.A.G.E. consortium
(see glossary) that contains your sequence of interest, which can then be used as a
PCR template. Information regarding the availability of I.M.A.G.E. clones can be
found via NCBI or equivalent databases.
6. Designing Primers
a) Firstly, using the cDNA sequence determined above, design a pair of primers (Forward
and Reverse) that will be used to amplify the entire ORF (starting with the ATG start
codon, and ending with the TAA stop codon). The primers should be written 5’-3’.
Forward primer sequence:
Reverse primer sequence:
Using the map of the Multiple Cloning Site (MCS; below) and the cDNA sequence
determined above encoding your novel protein, you choose restriction sites that are
suitable for cloning your cDNA into the expression plasmid. In this case, EcoRI and BamHI
restriction sites are chosen for cloning.
Q16: What do you need to think about when choosing restriction sites for cloning?

b) You then incorporate the restriction enzyme recognition sites into the primer
sequences you designed above. i.e.
When you make a fusion protein, you must ensure that the reading frame is
maintained between the tag and the protein of interest.
Q17: Do you need to add any additional nucleotides to your primer sequences in order to
maintain the reading frame?
Q18: Why is this necessary?

c) Write down the primer sequences (5’ to 3’), including any modifications you need to
make to maintain the reading frame:
Q19: If the tag was C-terminal rather than N-terminal, what additional changes to the
primers would be made (in addition to modifications related to frame)?
Q20: When adding restriction enzymes onto primers, additional residues are often added 5’
of the restriction enzyme recognition sequences. Why is this necessary?
New England Biolabs Inc. are a common source molecular biology reagents used in the
lab, and they also have numerous online ‘tools and resources’ that are helpful when
designing cloning strategies (https://www.neb.com/tools-and-resources).

Shown below is part of a table taken from the New England Biolabs online resources, which
has information regarding the efficiency of cleavage for different restriction enzymes when
cutting close to the end of DNA fragments (https://www.neb.com/tools-and-
resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments ).
Q21: Using this table, how many nucleotides should be added to each of your primer
sequences?

d) Using this information, write down the modified primer sequences (5’ to 3’):
7. RT-PCR
Using your new primers, you carry out the RT-PCR reaction in order to amplify the insert
DNA for cloning into p3xFlag. You subsequently digest the amplified DNA with EcoRI and
BamHI, and then clone it into the p3xFlag vector that has similarly been digested with EcoRI
and BamHI.
Q22: How could you check that your final clone is correct?
You thereby obtain your expression vector that will be used to express your 3xFlag-tagged
protein of interest in mammalian cells, which will be used to study the interaction between
ADAR1 and its newly identified interacting protein partner. These analyses will in turn
provide insights into the role of ADAR1 and the novel protein in stress granules.
8. RNAi to knockdown your protein of interest

In addition to overexpressing your protein of interest, it is often informative to investigate a
biological process in the absence of your protein. ‘Knockdown’ of your protein in
mammalian cells (E.g. HeLa cells) is often achieved using RNA interference (RNAi; see Dr
Scadden’s lectures). You therefore need to design or acquire some siRNAs (small
interfering RNAs – see Dr Scadden’s lectures) that are optimised to target the mRNA
encoding your protein. There are numerous suppliers of siRNAs (E.g. Dharmacon, Sigma,
Qiagen), and there are now a large number of ‘pre-tested’ siRNAs available to target many
human mRNAs. If an siRNA is not available, a custom siRNA can be designed using various
websites that use suitable algorithms for siRNA design.
For this exercise, you will use Dharmacon

(http://dharmacon.gelifesciences.com/ ), which
specialises in products related to gene
expression, to obtain siRNAs to knockdown the
protein interacting with ADAR1.

To look for suitable siRNAs, click on the image entitled

‘RNA Interference’, which will take you to the page
showing various products used for RNAi. Of these, you
should click on the ‘siRNA’ option
(http://dharmacon.gelifesciences.com/rnai-and-custom-rna-
synthesis/sirna/ ).
There are various options for siRNAs depending on what cell type you use, what sort of
sequence you are trying to target (E.g. mRNA or non-coding RNA), whether you want the
siRNAs to be modified (which reduces off-target effects), etc. Sometimes the choice you
make (E.g. ONTARGETplus versus siGENOME siRNAs) may simply come down to cost.
For this example, you can select either ON-

TARGETplus siRNA (modified siRNAs to reduce
off-target effects) or siGENOME siRNA.
This will then take you to a page with various

options for siRNAs – to find whether there are
siRNAs already available to target the mRNA
encoding your protein, click on ‘Product
Information and Gene Search’.
You can then ‘Start your gene search’ using the human sequences available – this will take
you to a page that asks for a ‘common gene identifier’ – here you need to enter the
GenBank accession number for the mRNA that you made a note of earlier (Section 2), then
press ‘Search’. Alternatively, you could just type in the gene name, but this may give more
complex results due to common abbreviations for gene names or due to other genes
sharing some parts of the gene name.

Once the search is complete, you will get a list of gene(s) that match the accession number
(or gene name) – check the gene you are expecting is shown – but note that many genes or
proteins are described by multiple different names. If it is correct, click on the symbol at the
left-hand side of the box, and this will take you to a page that shows you more information
about the mRNA, and what siRNAs are available.
For example, if you searched for eIF4E protein, you would see the following information:
To see the siRNAs available, click on the symbol, which will show you various options
for purchasing either a ‘SMARTpool’ of siRNAs (which are a mixture of 4 siRNAs – the use
of 4 different sequences simultaneously should increase the chance of getting good
knockdown) or individual siRNAs, at various concentrations. The use of individual siRNAs
may be desirable to reduce costs, or to target a specific bit of the mRNA sequence (E.g.
ORF or 5’/3’ untranslated region (UTR)).
You are thus able to order siRNAs that will be used to knockdown your protein of interest,
and thereby gain further insights into its function in mammalian cells, and its potential role in
stress granules.
Q23: How would you determine the efficiency of protein knockdown?

Q24: What could you do if the knockdown is inefficient?
Other databases
Other online databases can be used to look at other features of the novel ADAR1-
interacting protein you have identified:
These are just a couple of examples, but there are many other resources that may be used.
(i) http://www.uniprot.org/ – use the GenBank accession number above to find your
protein of interest, and then scroll through the page to find out more about the
protein.
Example:
• Function – Gene Ontology (GO) analyses
• Domains and Repeats
• 3D structure
• etc.
Finding out as much as you can about your new protein will contribute to determining its
cellular function.
(ii) http://www.ensembl.org/index.html – use the name of your new protein to search

within ensembl. Alternatively, ensembl could similarly be used to perform BLAST
searches etc. Ensembl contains a wealth of information, including:
• Analysis of gene expression – e.g. tissue specificity

• Splice variants
• Comparative Genomics etc.
• Gene Ontologies
• etc.

Glossary
A. GenBank
GenBank ® is the NIH genetic sequence database, an annotated collection of all publicly
available DNA sequences (Nucleic Acids Research, 2013 Jan;41(D1):D36-42). GenBank is part
of the International Nucleotide Sequence Database Collaboration , which comprises the
DNA DataBank of Japan (DDBJ), the European Molecular Biology Laboratory (EMBL), and
GenBank at NCBI. These three organizations exchange data on a daily basis.
The GenBank database is designed to provide and encourage access within the scientific
community to the most up to date and comprehensive DNA sequence information.
Therefore, NCBI places no restrictions on the use or distribution of the GenBank data.
However, some submitters may claim patent, copyright, or other intellectual property rights
in all or a portion of the data they have submitted.
B. Accession Numbers
An accession number in bioinformatics is a unique identifier given to a DNA or protein
sequence record to allow for tracking of different versions of that sequence record and the
associated sequence over time in a single data repository.
The International Nucleotide Sequence Database Collaboration DDBJ/EMBL/GenBank all
receive sequence submissions, assign accessions, and exchange data so that all three groups
represent the total collection. The accession assignment process is managed by prior
agreement within the collaboration on which group will 'own' which accession prefix.
C. GenBank Accession Numbers

The format for GenBank Accession numbers are:
Protein: 3 letters + 5 numerals
(Example: AAF78782 corresponds to eIF4E from Pisaster ochraceus)
Nucleotide: 1 letter + 5 numerals OR 2 letters + 6 numerals
(Example: AF272369 corresponds to eIF4E from Pisaster ochraceus)
D. GI (gi) numbers
GI number (sometimes written in lower case, "gi") is simply a series of digits that are
assigned consecutively to each sequence record processed by NCBI. The gi number bears
no resemblance to the Accession number of the sequence record. The gi number provides
a unique sequence identifier which is independent of the database source (E.g. nucleotide
sequences from DDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and many
others.)
Historical Note: GI stands for "GenInfo Identifier." GenInfo was an early system used to access
GenBank and related databases.
E. FASTA sequence format

In bioinformatics, FASTA format is a text-based format for representing either nucleotide
sequences or peptide sequences, in which nucleotides or amino acids are represented using

single-letter codes. The format also allows for sequence names and comments to precede
the sequences.
Example:
>MCHU - Calmodulin - Human, rabbit, bovine, rat, and chicken
ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTID
FPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREA
DIDGDGQVNYEEFVQMMTAK*
F. HeLa cells
A HeLa cell is a cell type in an immortal cell line used in scientific research. It is the oldest
and most commonly used human cell line. The line was derived from cervical cancer cells
taken on February 8, 1951 from Henrietta Lacks, a patient who died of her cancer on
October 4, 1951. The cell line was found to be remarkably durable and prolific which
warrants its extensive use in scientific research.
G. I.M.A.G.E Clones
The I.M.A.G.E. Consortium was initiated in 1993 by four academic groups on a collaborative
basis after informal discussions led to a common vision of how to achieve an important goal
in the study of the human genome: the Integrated Molecular Analysis of Genomes and
their Expression Consortium's primary goal is to create arrayed cDNA libraries and
associated bioinformatics tools, and make them publicly available to the research
community.
Utilizing high speed robotics, the I.M.A.G.E. consortium have arrayed over nine million
individual cDNA clones into 384-well microtiter plates, and have created sufficient replicas
to distribute copies both to sequencing centers and to a network of five distributors located
worldwide. Investigators who identify a clone of interest then contact any one of the
distributors to obtain these clones – free of restriction – for further study.
(also see Dr Scadden’s lectures for information about I.M.A.G.E. clones)

Notes

Notes

Michaelmas Week 6: Discussion of Practicals from Weeks 1-5 NST 2018-19
Week 6: Discussion of Practicals from Weeks 1–5
Two activities will take place in Week 6, Michaelmas Term:

(A) A discussion of Practicals from Weeks 1–5 (11am (except Monday –
12pm))
Dr Jo Rees (jsr31@cam.ac.uk)
The discussion will involve an overview of all the practical knowledge gained in weeks 1-5,
with an opportunity to apply this to a theoretical protein.
More information about the discussion session will be provided in week 4 of the
practical.
(B) Journal Club

Journal clubs will be at 2pm every day except Monday (3pm).
Please see Moodle for more information regarding groups and venues.

Michaelmas Week 7 NST 2018-19
Week 7 Practical
Details about the practical to be held in Week 7 of Michaelmas

Term will be provided prior to these sessions.

Michaelmas Week 8 – Protein Structure by Molecular Graphics NST 2018-19
Week 8 – Analysis of Protein Structure by Molecular Graphics
Objectives
The aim of this practical is to give you an overview of the current understanding of protein
structure, using molecular graphics software as a tool to aid the learning process.
Visualising and manipulating molecules with a sophisticated program, the ICM–Browser, will
help you learn how to analyse protein architecture in a hierarchical way.
When this practical has been completed you should be able to:
• Recognise common elements of secondary structure, super-secondary structural
motifs, protein folds and tertiary structures and relate them to possible protein
functions.
• Use the ICM–Browser to view and manipulate protein structures.
Introduction
The complex nature of protein structures makes them extremely difficult to represent in
two dimensions. Models must be built in three dimensions for a full appreciation of their
structures and hence their functions. This can be done in two ways: by building physical
models or by generating an interactive computerised graphical representation. Physical
models have some benefits over computer models in that they are truly three dimensional,
but computer models are far easier to analyse, distribute, manipulate and represent.
A protein model is stored on a computer as a file containing a set of Cartesian co-
ordinates, in which each atom of the protein is defined by x, y and z co-ordinates in three
dimensional space. The bonds between the atoms are generally not stored in this file, but
rather calculated by the computer algorithms used to display the structure. The precise
way in which a structure is displayed using computer graphics can be varied so as to
facilitate the observation of particular features of the protein being investigated.
The aim of this practical is for you to learn how to view protein structures and recognise
features which often occur in them, rather than merely perceiving a collection of rather
uninteresting and apparently random lines!
In the practical you will use an interactive molecular visualisation program, the ICM–
Browser, to view protein structures presented in a series of electronic documents.
Molecular graphics software of this sort is very easy to operate and is widely used in
research. It enables you to generate a range of interactive pictures of molecules
that highlight different aspects.
The practical is designed to guide you through a hierarchical approach to protein
structure. It is divided into the following sections:
1) Analysing the covalent connectivity of the polypeptide chain
2) Analysing the secondary structure of proteins
3) Analysing protein super-secondary structures
4) Analysing functional motifs
5) Analysing the tertiary structure of proteins
6) Analysing the structural basis of protein function

As you progress through the sections, the protein structures you analyse will become
increasingly complicated and may only be comprehensible if you have fully appreciated the
previous sections.
The whole practical will be made available for internet access on the BMB Moodle site
after the week’s practical classes are finished. This should make it possible for you to go
back and work through sections of the practical in more detail at a later date using
computer facilities in your college. Details of how to install your own copy of the ICM–
Browser are provided on the CamTools site.
References
Books
• In Focus: Protein Structure (1993) Darby, N.J. & Creighton, T.E., IRL Press.
• Protein Structure (1998) Branden, C. & Tooze, J., 2nd edition, Garland.
• Proteins – Structures and Molecular Properties (1993) Creighton, T.E., 2nd edition,
Freeman.
• Introduction to Protein Architecture (2001) Lesk, A.M., Oxford.
• Protein Structure and Function (2009) Petsko, G.A. & Ringe, D., Oxford.
• How Proteins Work (2011) Williamson, M., Garland
Websites
• More details about the ICM–Browser
http://www.molsoft.com/icm_browser.html
• PyMOL – a different state-of-the-art open source molecular visualization system: a free

standalone program (available for PC, Mac or Linux) that can produce publication quality
ray-traced graphics
http://pymol.sourceforge.net
• Search for structures deposited in the Protein Data Bank

http://www.pdb.org
http://www.ebi.ac.uk/pdbe
• Hierarchic databases of protein structures

http://scop.mrc-lmb.cam.ac.uk/scop
http://www.cathdb.info

Section 1: Analysis of the covalent connectivity of the polypeptide chain

On the desktop of your workstation, open the folder with the tile
“MolGraph_Practical”, and double click on the file “Section_1.icb”. This will launch the
ICM–Browser, which will display two panels, one containing text, and the other showing a
graphical model of a short polypeptide chain.
Section 1
1(a) Manipulating an object in the graphical window
First, on the uppermost bar of icons, toggle off the “FOG” icon, which is switched on by
default. You can improve the quality of the graphics on screen by selecting the “A” icon (to
switch on antialiasing, thereby minimising lines with jagged edges), and also the
neighbouring circular icon, to toggle on “high quality graphics”. If the browser appears to
run slowly at any stage, you can toggle off these antialiasing and high quality options.
If necessary, click on the link labelled “click here” to emphasize the polypeptide backbone
by marking it with thick bond vectors; side-chain bonds between heavy atoms will be
represented by thin lines, and bonds to hydrogen atoms by even thinner lines. You can
click on the same link at any stage to reset the view displayed in the graphics panel.
Next, learn how to use the mouse to manipulate the image:

(i) Rotation around the x and y axes.
With the cursor inside the graphics panel, click and hold the left mouse button. Move
the mouse up-and-down to rotate about the horizontal x axis and left-to-right to rotate
about the vertical y axis.
(ii) Translation along the x and y axes.
The margin at the bottom of the graphics panel is dedicated to translation in the xy plane.
Move the cursor down until it changes shape (into a four-way arrow), click and hold the
left mouse button, and then practice dragging the object around.

(iii) Zooming along the z axis.

The left hand margin of the graphics panel is dedicated to moving the object closer or
further away along the z axis. Move the cursor left until it changes shape (into a magnifying
glass), click and hold the left mouse button, and then zoom in and out.
(iv) Rotation about the z axis.
The top margin of the graphics panel is dedicated to rotation around the z axis. Move
the cursor up the screen until it changes shape (into two semi-circular arrows), click and
hold the left mouse button, and then rotate the object clockwise and anticlockwise.
(v) Clipping planes.
For large molecules, it is often convenient to view part of an object in a "slab", hiding any
atoms that lie in front and behind a pair of "clipping planes". These can be manipulated by
clicking and dragging the cursor in the right-hand margin of the graphics panel. Play with the
three settings in the upper, middle and lower segments of the margin to discover how they
work.
1(b) Interrogating an atom

Clicking on an atom with the right mouse button will bring up a small
menu. For now, concentrate on the first line, which gives a report on
the atom you just selected. The report will indicate: the name of the
structure file (e.g. “a_5aas.m”); the residue type (e.g. “A”, meaning
alanine); its number in the primary sequence (e.g. 1); and the element
type and name of the atom (e.g. “n”, meaning a backbone amide
nitrogen atom).
1(c) Residue numbering

1.1. Ascertain the amino acid sequence of the penta-peptide and fill this into the
boxes below, using the “three-letter code”.
(Hint: switch on automatic labelling of the residues by clicking on the link for part (c))
1.2. Draw out the covalent structure of the penta-peptide, following on from the
first residue shown below. You may want to refer to the Appendix to look up the
structures of the remaining residues. (Hint: there’s no need to get the stereochemistry right –
for now just focus on the covalent structure of the peptide backbone and the side-chains)

1(d) Hydrogen bonds

1.3. Click on the link for part (d) to display the single hydrogen bond present in the
penta- peptide. Which atoms of which residues are linked by this hydrogen bond?
.................................................................................................................
1(e) Backbone dihedral angles
Click on the link for part (e) to display a set of green

circles which represent measurable backbone and
side- chain dihedral angles. To switch on the values of
these angles, click and hold on the “VAR φ 2” icon
in the second bar at the top of the graphics panel.
This will bring up a square menu; click on the “Label
Style” option to bring up a secondary menu, and
choose the “φ-62” style. This will display the name of
the angle followed by its value.
1.4. The partial double bond character of the peptide bond, which links the
carbonyl carbon of residue (i) with the amide nitrogen of residue (i +1), constrains the CO,
NH and both adjacent Cα atoms to lie in the same plane. Two planar orientations are
possible, in which adjacent Cα carbons are fixed in either the cis or the trans configuration:
Which of these configurations do the residues in the penta-peptide possess? Why do

you think this might be? (Hint: list the four values of ω measured in the graphical panel)
.................................................................................................................
.................................................................................................................
...............................................................................................................

1(f) Different representations

Next, try out various different representations of the penta-peptide (such as wire,
xsticks, ribbon, CPK, etc.) by using the links in section (f).
Wire: this basic representation emphasizes the different types of bonds in molecules; it is
good for small molecules such as drugs, but is probably not the best choice
for looking at large protein assemblies.
Xsticks: this ball-and-stick representation provides a “true” idea of the bonding present,
but provides too much detail to help in understanding overall protein structures;
however, it is useful for highlighting specific features of, for example, enzyme active
sites.
Ribbon: this shows alpha-helices, beta-strands and connecting “loop” regions in their
customary “cartoon” forms, providing a useful way of following the secondary
structure and topology of a protein structure. Unfortunately, in this case there
are not enough amino acid residues present for the graphics software to recognize
a genuine alpha-helix, so it has decided that the whole penta-peptide must be a
worm- like “loop”. This representation will be much more useful in later, more
complex examples.
CPK: this space-filling representation shows the different atoms with spheres that
represent their true volumes, and avoids giving the impression that protein
structures are full of empty space; however, the display is now too crowded to
convey an accurate idea of how the protein works. CPK stands for “Corey-
Pauling-Koltun”, after the authors who first suggested this representation, along
with the standard atomic colour scheme of white for hydrogen atoms, blue for
nitrogen, black for carbon, and red for oxygen, etc.
Surfaces: these representations are solvent-excluded surfaces, formed by rolling a
sphere the same size as a water molecule around the protein to indicate the
volume that solvent molecules cannot penetrate; they are useful for giving a feel of
what protein surfaces and binding sites look like to molecules they interact with.
1(g) The workspace panel

From the “Windows” label on the bar at the top of the ICM–Browser,
select the “Workspace Panel” option. A new panel will appear on the
left, but you may need to resize this, along with the adjacent “Html
Documents” panel, to see all of its contents. The Workspace Panel offers
a view under the bonnet of the program.
The penta-peptide structure can be found under the “objects” heading.
Within this, you will find the amino acid sequence in one letter code:
“AENFQ”.
Use the cursor to select a couple of residues, such as
“AE”. The graphical display will highlight the selected
atoms with green crosses and the connecting bonds will
turn green. Change the colour of the selected atoms and
bonds by choosing a shade from the colour palette on the
second icon bar at the top of the program window.
Initially, only the atoms will appear to change colour. However, deselect
the atoms by clicking anywhere on the white space in the Workspace Panel
and you will see that the bonds have also changed colour. What other
transformations can you make using different icon button commands?

1(h) PDB (Protein Data Base) files

The atomic coordinates of the penta-peptide you have been looking at are stored as a text
file in a format specified by the Protein Data Bank, a computer-based archival database for
macromolecular structures. The database was established in 1971 by Brookhaven National
Laboratory, New York, as a public domain repository for resolved crystallographic
structures. In 1999, the Protein Data Bank moved to the “Research Collaboratory for
Structural Biology” (http://www.rcsb.org/pdb/). The database now contains structures
elucidated by additional experimental methods, such as nuclear magnetic resonance
spectroscopy and electron microscopy.
The format of a typical protein database (PDB) file is shown below. After a long header,
which includes remarks concerning details of authorship, publication and the identity and
preparation of the protein studied, the x, y and z coordinates of the atoms of each residue
are listed. The unit for the coordinates is the Ångstrom (1 Å = 10 –10 m = 0.1 nm).
Section 2: Analysis of protein secondary structure – alpha helices
Click on “File” and then “Quit” to exit the “Section_1.icb” file. Next, go back to the
“MolGraph_Practical” folder and double click on the file named “Section_2.icb”, which
should show an α-helix that has been excised from the protein ROP. As before, the
polypeptide backbone is highlighted using thicker bonds.
2.1. Click on the link for part (b) to display the hydrogen bonds as dotted lines
interspersed with small spheres. Choose a residue near the middle of the α-helix. If a
hydrogen bond starts at the carbonyl oxygen atom of residue (i), what is the sequence
number of the residue that donates the amide hydrogen to the hydrogen bond? How many
residues are there in a single turn of the helix?
................................................................................................................

2.2. Are the hydrogen bonds linear or bent?

(Hint: to measure the O-H-N angle across a hydrogen bond, select
the angle measurement tool from the second icon bar at the top of
the window. Then, click on three atoms, such as the carbonyl C of N10, the amide H of F14,
and the amide N of F14. The value of the bond angle will then be displayed in degrees. To remove
any unwanted measurements, select the nearby icon that displays a red cross).
................................................................................................................
.
The figure above shows the definition of the three backbone torsion angles of residue (i):
j (for the angle made by the three bonds and four atoms C'i–1–Ni –Cαi –C'i), ψ (for
Ni –Cαi –C'i –Ni+1) and ω (for Cαi –C'i –Ni+1–Cαi+1). The side-chain conformation is described
by c1, c2, etc., where c1 is the torsion angle around the bond between Cα and Cβ. A useful
way of analysing the backbone conformation of a polypeptide is to plot j against ψ for each
residue, creating what is known as a Ramachandran plot. Only certain values of j and ψ
are permitted, because many rotations about these bonds lead to unfavourable steric
interactions between atoms of the same and neighbouring residues. A Ramachandran plot
for the α-helix shown in the Section 2 file is displayed below, with the “allowed regions”
shaded grey. Note how the values of j and ψ for the α-helix cluster into a small region of
the plot.

2.3. Click on the link for part (c) to display to display circles that represent the backbone
and side-chain dihedral angles, and then switch on the values using the “VAR j 2” icon, the
“Label Style” option, and the “j-62” style. List the backbone and side-chain dihedral angles
for residue Met-11.
................................................................................................................
................................................................................................................
................................................................................................................
.
2.4. Click on the link for part (d) to display the structure without distracting side-chains.
Of the 30 backbone carbonyl oxygen and amide nitrogen atoms in this helix, only 7 amides
(excluding the N-terminal amine) and 7 carbonyls are NOT involved in hydrogen
bonds. Where in the helix are these?
................................................................................................................
................................................................................................................
2.5. Taking into account the answer to the last question, how would you expect the free
energy of formation of a helical turn between residues 1-5 to compare with that for forming
one between residues 5-9 or 10-14? (Hint: remember that ΔG = ΔH – TΔS.)
................................................................................................................
................................................................................................................
2.6. Why do you think that proline residues are very rarely found in helices? When they
are found in helices, can you account for why they tend to appear at the ends of these
structures?
................................................................................................................
................................................................................................................
2.7. Click on the link for part (e) to display the structure in ribbon format and coloured
from blue at the N-terminus to red at the C-terminus. Is the coiling of the helix left-handed
or right-handed?
(Hint: imagine the helix is a screw and you are screwing it into a piece of wood; given that your
right hand twists more naturally clockwise and your left hand twists more naturally anti-
clockwise, would it be easier to use your left or right hand to turn the screw?)
................................................................................................................
.

2.8. Click on the link for part (f) to display the first few turns of the
helix in xsticks format, oriented so that you are looking along it with
the N-terminal end facing you. You may find it useful to move the
residue labels to the ends of the side-chains by holding the cursor
down on the “RES A5” button in the second icon bar at the top of
the window, and then selecting “Shift to Sidechain Tips”.
The helical wheel diagram below represents this view. Fill in the
nature of each of the amino acid residues indicated (i.e. +ve/–ve, polar,
hydrophobic, etc.; refer to the Appendix if you need to).
N.B.: ignore residue 1, as it is not in an α-helical conformation.
2.9. What do you notice about the distribution of hydrophobic and hydrophilic residues in
the helical wheel representation?
................................................................................................................
.
................................................................................................................
.
2.10. What explanation can you suggest for this distribution (taking into account the fact
that the helix is part of a larger globular protein)?
................................................................................................................
.
................................................................................................................
.
................................................................................................................
.

2.11. An α-helix has a net dipole moment. Why do you think this is, and which way do you
think it is pointing?
................................................................................................................
.
................................................................................................................
.
Section 3: Analysis of protein secondary structure – beta strands
The file, “Section_3.icb” shows two regions of polypeptide chain that are hydrogen bonded
together to form an anti-parallel β-sheet. The distribution of the j and ψ angles is now very
different from that displayed by an α-helix (compare the figure below with that on page
146).
Notice that in an antiparallel β-sheet the hydrogen bonds are almost parallel to each
other, are perpendicular to the direction of the strand, and form large and small hydrogen
bonded 'rings'. These hydrogen bonded 'rings' are related by a two-fold rotational
symmetry axis perpendicular to the plane of the sheet.
(Hint: to spot this pseudo-symmetry, try executing a rotation of 180° around the z axis)
3.1. Click on the link for part (b) to display a pair of β-strands with parallel contacts. To
answer the following question, you may need to toggle back and forth between the links for
part (a) and part (b). Can you spot three differences between the hydrogen bonding
patterns in the parallel β-sheet and the anti-parallel β-sheet? How might these differences
affect the relative stabilities of parallel and anti-parallel β-sheets?
................................................................................................................
................................................................................................................
................................................................................................................
................................................................................................................
Click on the link for part (c) to display the side-chain atoms of the antiparallel β-sheet in
a space-filling representation. Hydrophobic side-chains (i.e. either Val, Ile, Leu, Phe, Trp,

Met, Ala or Cys) are coloured in blue. Notice how the side-chains of adjacent residues
in the sequence of each strand point in approximately opposite directions, so that the
polypeptide backbone appears to be sandwiched between two side-chain layers. You
might want to make this more apparent by selecting “Shift to Sidechain Tips” here.
3.2. Compare the properties of the amino acid side-chains on either side of the β-sheet:
what do you observe? Why might this be?
................................................................................................................
................................................................................................................
................................................................................................................
Section 4: Super-secondary structure – recurrent structural motifs in globular

proteins.
The way in which the β-strands and/or α-helices of a protein are connected together
in space is referred to as the topology of the protein. In general, the arrangement of these
units of secondary structure is such that distinct layers of structure are formed. It is
between these layers of structure that the hydrophobic cores of proteins lie, protected
from water to a large degree by the framework formed by the polypeptide backbone.
Certain topologies that are local with respect to the sequence tend to recur in a large
number of different proteins, even where there is no evidence of an evolutionary
relationship. These are commonly referred to as super-secondary structures. The figure
below shows schematic representations of some of the more common super-secondary
structures.
Topology Diagrams:
i +2
i +1 i
i +3

Section 4: Super-secondary structures

Open the file “Section_4.icb” to view what is perhaps the simplest example of super-
secondary structure: a β-hairpin in which two β-strands are linked via a tight β-turn (see
panel (a) on the previous page).
4.1. Examine the hydrogen bonding in the β-turn. How distant in sequence are the two
groups that form the hydrogen bond at the apex of the turn? How does this periodicity
compare with that of the α-helix you were examining in Section 2 (i.e. in question 2.1)?
................................................................................................................
.
................................................................................................................
.
................................................................................................................
Click on the link for part (b) to display the α-helical equivalent of the β-hairpin (see
panel (a) in the “Topology Diagrams” above). Although there is obviously no main chain
hydrogen-bonding between the two α-helices, they are closely packed in an aligned fashion.
Such arrangements of α-helices are often referred to as coiled-coils and in some proteins the
interacting helices can be very long (e.g. leucine zippers and fibrous proteins such as α-
keratin or myosin).
The link for part (c) reveals an elaboration on a simple β-hairpin called a β-meander (see
panel (c) in the “Topology Diagrams” above). β-meanders consist of an array of β-
strands, each linked by β-turns (or sometimes more extensive loops), in which β-strand
(i) is always hydrogen-bonded to β-strand (i+1). This page shows a three-stranded
meander, but many more strands can occur in a β-meander.
4.2. The β-meander shown here is part of a larger globular protein in which there are two
β-sheets that pack together to form a 'sandwich', the filling being the hydrophobic core. By
examining the structure, decide whether residue 18 is likely to be on the
hydrophilic exterior or the hydrophobic interior.
(Hint: Click on the link for part (d) to view the side-chains coloured according to hydrophobicity,
and then select “Shift to Sidechain Tips” once again)
...............................................................................................................
The link in part (e) will show you a more complex topology that is extremely common in a
number of apparently unrelated proteins, commonly referred to as a 'beta-alpha-beta' unit
(see panel (e) in the “Topology Diagrams” figure above). Here the sequence of the protein
contains first a β-strand, then an α-helix, and then another β-strand which is hydrogen-
bonded to the first such that they form a parallel β-sheet.
4.3. Beta-alpha-beta units have a handedness, just like α-helices and β-sheets, and the
handedness is always the same. By following the path of the polypeptide backbone of the
entire unit, can you assess the handedness of the beta-alpha-beta unit?
(Hint: orient the molecule so that the blue strand at the N-terminus travels across the screen from
left to right, with the α-helix below and the red C-terminal strand behind, i.e. into the screen)
................................................................................................................

The beta-alpha-beta motif is an example of a multi-layer structure: the helix is part of one
layer, while the β-strands participate in a second layer. The connections between the
β- strands and the α-helix cross from one layer to another and are therefore referred to as
cross-over connections. With extremely few exceptions, cross-over connections involving β-
strands are always of the same handedness.
An intriguing topological arrangement of β-strands that recurs in a large number of
proteins is the Greek key motif. The simplest definition of a Greek key is that it consists of
an array of four consecutive β-strands in which β-strand (i) is hydrogen-bonded to β-strand
(i+3), as shown in panel (d) in the “Topology Diagrams” above. Sometimes all four strands
lie in the same layer of structure (making a 4,0 Greek key); sometimes three strands are in
one layer and one is in a different layer (a 3,1 Greek key); alternatively, two of the β-
strands form a β-sheet in one layer, while the other two form another β-sheet in a
separate layer (a 2,2 Greek key). The term Greek key is coined from the fact that two-
dimensional representations of the motif make a pattern similar to one that is often found
on ancient Greek vases. Click on links (f) and (g) to view examples of 3,1 and 2,2 Greek
keys, with the relevant strands coloured in yellow.
4.4. A common variation on the four-stranded Greek key is the five-stranded, or “double”
Greek key (see panel (f) in the “Topology Diagrams” above). The double Greek key motif
can be found in immunoglobulins, electron transport proteins, viral coat proteins, inhibitors
and toxins, as well as enzymes.
To view an example of a double Greek key coloured from blue at the N-terminus to red at
the C-terminus, click on link (h). The five consecutive β-strands in this motif are arranged
in space such that two different pairs of β-strands have strand (i) hydrogen bonded to
strand (i+3), creating two overlapping single Greek key arrangements: one for strands 1 to
4, and another for strands 2 to 5. The β-strands in the double Greek key topology form
two distinct β-sheets (one with three strands and one with two) in two separate layers.
As a result, some of the β-strands in the same β-sheet are connected via β-strands
in the opposite β-sheet by cross-over connections. How would you classify the two
Greek keys on this page using the scheme outlined above – e.g. do strands 1 to 4 form a
4,0 Greek key? What about strands 2 to 5?
...............................................................................................................
Section 5: Functional motifs
Before moving on to discuss the tertiary structures of proteins, it is worth
discussing another group of recurring super-secondary structures that are all functional
motifs. These local topologies are found in a number of proteins with quite unrelated
functions, but which bind the same substrate (be it DNA, Ca2+, NADH, etc.). Here you will
briefly examine three types of functional motif: the zinc finger and helix-turn-helix motifs
from two DNA-binding proteins and the Rossmann fold from a nucleotide-binding protein.
Open the file “Section_5.icb” to view the glucocorticoid receptor bound to DNA (PDB
code: 1GLU). In fact this view displays a homo-dimer of the zinc finger DNA-binding
domain of GR. These particular zinc fingers contain four cysteine residues (shown in xsticks
format) co-ordinating each zinc atom (shown as red spheres). Other varieties of zinc
finger may have two cysteines and two histidines co-ordinating each zinc atom.

5.1. How many zinc atoms are bound per monomer?
................................................................................................................
5.2. Which groove in the DNA (i.e. major or minor) does this protein seem to interact
with?
................................................................................................................
5.3. Do the zinc atoms appear to play a direct role in interacting with the DNA?
................................................................................................................
Click on link (b) to display the 434 Cro repressor bound to DNA (PDB code: 3CRO). This
protein is also a homo-dimer. Helix-turn-helix motifs are shown in green and yellow, one in
each protomer. The green coloured section is termed the “recognition helix” and the
“positioning helix” is shown in yellow.
5.4. Which groove in the DNA does the recognition helix interact with? Do any residues
interact with the other groove?
(Hint: to see additional interactions with the DNA, click on link (c))
................................................................................................................
................................................................................................................
Link (d) displays the enzyme lactate dehydrogenase (PDB code: 9LDT). An arrangement of
β-strands and α-helices known as the Rossmann fold (see panel (g) in the “Topology
Diagrams” above) is picked out in the ribbon representation. This fold is found in a
number of enzymes and is involved in binding nucleotides; in this case it binds to NADH,
highlighted in both xsticks and CPK space-filling spheres). It has been suggested that the
recurrence of this fold may be the result of convergent as well as divergent evolution.
The Rossmann fold is made up of two overlapping beta-alpha-beta units, although most
textbooks refer to the Rossmann fold as a supersecondary structure in its own right.
Section 6: Analysis of the tertiary structure of proteins

Just as with secondary structure and super-secondary structure, the tertiary structures of
proteins can be grouped into a number of distinct classes depending on their overall folds.
Two levels of tertiary structure classification are used, one which reflects the usage and
distribution of secondary structure in the protein, and another more detailed classification
that is topology-based. The former (due to M. Levitt and C. Chothia) groups all globular
proteins into 5 distinct classes:

(i) α, in which the vast majority of the protein is α-helical (e.g. myoglobin)
(ii) β, where the vast majority of the protein is β-sheet (e.g. immunoglobulin domain)
(iii) α+β, in which there are distinct contiguous α-helical and β-sheet regions in the
sequence of the protein (e.g. lysozyme)
(iv) α/β, where the sequence of the polypeptide chain alternates between α-helices and
β-strands (e.g. triose phosphate isomerase)
(v) cross-linked irregular domains, which are small protein regions, often rich in
cysteine residues; binding to metal ions or disulphide bridges are used to cross-link
different parts of the domain together, and as a result these proteins often possess
irregular secondary structure (e.g. ferredoxin).
More detailed topological descriptions of the overall tertiary folds of proteins can be
used to identify different structural families. Understanding these classifications is useful, as
modern databases which hold protein structures can require users to define the types
of fold they are interested in. Several hierarchical classifications of the entire set of known
protein structures are available on the internet (e.g. SCOP, “Structural Classification of
Proteins”, based in Cambridge; or CATH, “Class, Architecture, Topology, Homology”,
based in London).
The following web pages illustrate examples of proteins with particularly well known
tertiary folds: a "four-helix bundle", a "TIM-barrel" and an "immunoglobulin fold". For each
of these proteins, use the protein structural analysis skills you have acquired so far to class
the protein as being either α, α/β, etc., and to identify any super-secondary structures.
Open the file “Section_6.icb” to view cytochrome c', a four-helix-bundle protein

(PDB
code: 2CCY), coloured from blue at the N-terminus to red at the C-
terminus.
6.1. The figure below shows a simplified top view of the protein, such that each circle
represents one of the four helices. Show the connectivity between the helices in the
sequence on the diagram. Use bold lines to represent connections between helices at
the top of the protein and dotted lines to show connections at the bottom. The result will
be a two- dimensional topology diagram of the protein. This procedure is worthwhile
because different four helix bundle assemblies can be connected in different ways.

6.2. Do you recognise the prosthetic group in this protein?

(Hint: right click on the green sphere and on an atom in the prosthetic group to get more
information.)
................................................................................................................
What can you say about the environment that the prosthetic group is in?
(Hint: click on link (b) to view a representation showing atoms in CPK space-filling mode, with
non-polar residues shaded in purple, and polar residues in yellow. This view uses a clipping
plane to slice through the structure. You can move the clipping plane backwards and forwards
using the control in the lower portion of the right-hand margin of the window.)
................................................................................................................
................................................................................................................
Link (c) shows a TIM barrel from the protein triosephosphate isomerase (PDB code:
1TRE). This is one of the most elegant structural motifs (and for enzymes one of
the most common).
6.3. To which structural class does this protein belong (α, α/β, etc.)? What is the repeated
super-secondary structural motif in this protein?
................................................................................................................
6.4. Link (d) reveals the structure of a “constant” domain from the heavy chain of
immunoglobulin G (PDB code: 1IGT). To which structural class does this protein belong
(α, α/β, etc.)? Which super-secondary structural motifs can you find?
................................................................................................................
................................................................................................................
Large proteins are generally found to be made up of a number of smaller globular

units, called domains, that are linked together by lengths of polypeptide chain. A domain is
rather hard to define: they can be independent units that are simply tethered by flexible
polypeptide linkers, or they can interact structurally to varying degrees. A domain typically
includes only residues that are consecutive in the peptide chain, and such domains can
often be separated from each other by proteolytic cleavage. Well-defined domains are
often considered as autonomous folding units, because such individual units will fold
independently of each other. However, other domains are not so structurally independent.
Link (e) illustrates protein quaternary structure, showing the four crosslinked polypeptide
chains of immunoglobulin G (PDB code: 1IGT). Label the different chains on the diagram
on page 18 (blue and magenta correspond to the “light chains”, while the “heavy chains”
are displayed in red and green). Note how each chain is composed of a number of domains
that possess the same immunoglobulin fold you examined in link (d).

6.5. On the diagram below, sketch the inter-subunit cysteine bridges to show how the
different chains are covalently linked.
(Hint: click on link (f) to identify the residues involved in cysteine bridges, and discriminate
between intra-subunit and inter-subunit disulphide bond connections.)
Section 7: Analysis of the structural basis of protein function

In this section you will examine not just the structure of a protein but also its function.
The graphics may at first seem distressingly complicated! However, give your eyes a minute
or two to adjust and remember to use the various means of displaying structures that you
have learned so far to simplify the views where necessary.
Open the file “Section_7.icb” to view the structure of chymotrypsinogen (PDB code:
2CGA), the zymogen or inactive precursor of the digestive enzyme chymotrypsin.
Chymotryspin belongs to a family of serine proteases that includes trypsin and elastase; all
three of these enzymes are found in the mammalian pancreas and have very similar
structures.
7.1. To which structural class does the protein belong (i.e. α, α/β, etc.)? Chymotrypsinogen
possesses two similar domains (residues 1-123 and 124-245), which are related by an
approximate two-fold rotational symmetry. How could this kind of structure evolve?
(Hint: click on link (b) to rotate the structure by 90° around the x axis, and then click link
(a) to move back again. What do you notice?)
................................................................................................................
.
................................................................................................................
.
Link (c) highlights the cysteine residues of chymotrypsinogen, which are all paired to form
cysteine bridges, cross-links between different regions of the protein backbone.

7.2. How many bridges are there, and which residues do they connect? On the diagram
below, show how the cysteine bridges form cross-links between different regions of the
polypeptide backbone. According to the definition of the domain boundaries suggested
in question 7.1, are any bridges formed between the two domains?
(Hint: the domain boundaries are residues 1-123 and 124-245.)
................................................................................................................
................................................................................................................
7.3. Cysteine bridges are common in extracellular proteins, but not in intracellular
proteins. Why do you think this might be?
................................................................................................................
................................................................................................................
Chymotrypsinogen is converted into an enzymatically active form, called π-chymotrypsin,

when the peptide bond between residues 15 and 16 is cleaved by trypsin. Further cleavages
are then made by another π-chymotrypsin molecule, removing residues 14, 15, 147 and
148, to make a stable form of the enzyme, which is called α-chymotrypsin. The locations of
these residues can be observed by following link (d).
7.4. Comment on the location of the cleavage sites. Will any of the three new polypeptide
chains be connected by cysteine bridges?
(Hint: use your answer to question 7.2.)
................................................................................................................
................................................................................................................
Click on link (e). The activation of the zymogen involves localised conformational changes.
Cleavage between residues 15 and 16 creates a positively charged backbone amino-
terminal group on residue Ile-16 (marked with a yellow sphere), which turns inwards and
forms a salt bridge with the side-chain of Asp-194 in the interior of the molecule
(marked with a magenta sphere). This electrostatic interaction triggers other changes,
creating a cavity for binging to the substrate that is not fully formed in the precursor
chymotrypsinogen.

7.5. How far will the amino nitrogen of Ile-16 have to move in order to interact with the
carboxyl side-chain of Asp-194?
(Hint: activate the distance measurement tool from the second icon bar at
the top of the window. Clicking on a pair of atoms will then display the
distance between them in Ångstroms. Estimate the distance by clicking on
the “N” of Ile-16, followed by the “OD” of Asp-194.)
................................................................................................................
Link (f) shows an overlay of cartoons of chymotrypsinogen (in green) and activated α-
chymotrypsin (in red), which can be used to identify where conformational changes have
occurred. The side-chains of Asp-194 and Ile-16 are highlighted in both structures.
7.6. How far does the new N-terminus at Ile-16 move in order to interact with Asp-194?
(Hint: note the distance between the red and green spheres for Ile-16.)
................................................................................................................
7.7. What else changes? (Hint: Look at the side-chain of Asp-194.)
................................................................................................................
.
................................................................................................................
.
Link (g) displays important parts of the active site of chymotrypsin in orange, with a
peptide substrate shown in purple (PDB code: 8GCH).
7.8. Comment on the location of the orange active site residues.
................................................................................................................
.
................................................................................................................
.
Chymotrypsin acts as a hydrolase, cleaving the peptide backbone by hydrolysis and
catalysing the overall reaction:

The serine proteases acquired their name because they contain a serine residue with an
unusually reactive side-chain, which attacks the carboxyl group of the substrate. The
reactivity of this residue (Ser-195 in chymotrypsin) depends on the arrangement of the
serine side-chain with two other polar side-chains, all approximately in a straight line. This
so-called "catalytic triad" has Ser-195 at one end of the line, His-57 in the middle, and Asp-
102 at the far end, all highlighted in orange in link (g). These side-chains form a charge
transfer network in which Asp-102 polarizes the imidazole ring of His-57, which then acts
as a proton shuttle, attracting the hydrogen ion from Ser-195 while it makes a nucleophilic
attack on the substrate, forming a tetrahedral oxyanion intermediate (see the scheme
below).
7.9. Click on link (h) and comment on the possible role of the nearby backbone amide
atoms from residues Gly-193 and Ser-195, which are highlighted using yellow CPK spheres.
..........................................................................................................
The serine proteases have no absolute specificity, but they do have strong preferences for
cleaving the peptide bonds adjacent to certain amino acid residues. All three enzymes,
chymotrypsin, trypsin and elastase, have a "specificity pocket" that accommodates the side-
chain of the residue of the substrate that precedes the bond to be cleaved. The specificity of
each enzyme depends on the size and nature of three crucial residues that line the edges of

the pocket, 189, 216 and 226 (using chymotrypsin residue numbering), which are highlighted
using xsticks and CPK representations in link (i). The structure is of a complex between α-
chymotrypsin and a peptide substrate. The tryptophan side-chain of the substrate binds into
the specificity pocket, which is illustrated here using an “accessibility surface”. The surface is
coloured according to ligand binding properties: white – neutral surface; green –
hydrophobic surface; red – hydrogen bonding acceptor potential; and blue – hydrogen bond
donor potential.
7.10. Account for the sequence cleavage preferences shown in the table below:
Enzyme: Side-chain preference: 189: 216: 226:

Chymotrypsin Large & hydrophobic (Phe, Tyr, Trp) Ser Gly Gly
Trypsin Positively charged (Lys, Arg) Asp Gly Gly
Elastase Small & hydrophobic (Ala) Ser Val Thr
...............................................................................................................
...............................................................................................................
...............................................................................................................
...............................................................................................................
...............................................................................................................
Link (j) shows a complex between bovine α-chymotrypsin (in pink and grey) and a serine
protease inhibitor protein (in purple), the third domain of turkey ovomucoid (PDB code:
1CHO). The complex has an association constant, Ka, of 3.2x1011 M-1. The inhibitor acts
by forming a hydrogen bond to the side-chain of Ser-195, the crucial residue in the active
site of the enzyme. Mutating residue Leu-18 of the inhibitor to methionine lowers the Ka
to 1.8x1011 M-1, while a further substitution of Gly-32 of the inhibitor to aspartic acid
reduces the Ka to 4.0x108 M-1.
7.11. Can you account for these results on the basis of the structure of the complex?
...............................................................................................................
...............................................................................................................
...............................................................................................................
Section 8: Some interesting structures

If you have reached this far, you might like to take a look at a few more protein structures.
Link (a) shows the porcine RNase inhibitor (PDB code: 2BNH), a leucine-rich
repeat protein. This is perhaps one of the most aesthetically pleasing proteins in the
database: being a scientist shouldn't preclude one from finding some protein structures
beautiful!

Link (b) displays the structure of a nucleosome (PDB code: 1AOI), the fundamental unit for
DNA packaging found in chromatin. The DNA double helix is wrapped around a
central protein core that contains eight histone molecules.
Link (c) shows the first of an infamous pair of viral proteins: haemagglutinin from Influenza
A (PDB code: 5HMG), which binds to polysaccharide chains on the surface of target cells
and then mediates fusion of the virus envelope with membranes of the host cell. Link (d)
shows its partner in crime: neuraminidase (PDB code: 2BAT), which clips off the ends of
polysaccharide chains that would otherwise prevent new virus particles from escaping the
infected cell. Together, these molecules control the infectivity of the virus, hence the
designation of dangerous mutant strains with names like H1N1.
To view HIV protease (PDB code: 1HIV) click on link (d). Unlike human enzymes, this
dimeric aspartate protease can cleave polypeptide chains between a phenylalanine and a
tyrosine or proline. Drugs that disable it have been designed by analysing structures like
this one using molecular graphics.
Finally, link (e) displays a theoretical model of a section of a lipid bilayer and some
surrounding water molecules. This should be considered alongside the structure of an
integral membrane protein, such as bacteriorhodopsin (PDB code: 1QHJ) shown in link (f).
This protein is a member of an important family called the seven-transmembrane (7TM)
receptors, which regulate many physiological processes and transduce signals from
hormones, neurotransmitters, chemokines, calcium ions, and even photons. Most of the
medicines sold throughout the world target 7TM receptors either directly or
indirectly. Link (g) shows a hot-off-the-press structure of a G-protein coupled receptor
(the β2- adrenergic receptor) bound to a heterotrimeric G-protein (PDB code 3SN6).
Structures of this sort promise to reveal crucial insights into signal transduction pathways.
What next?
Why not set up the ICM-Browser on a computer back in your college and explore some
protein structures by yourself using the list of useful web sites given on page 2? You can
use the program to make your own high quality images. Scientific publications of structural
studies are increasingly using software of this sort (including an internet plug-in named
ActiveICM, which is based on the ICM-Browser used here) to supplement more traditional
text-based descriptions with interactive displays of notable features and functional
aspects of proteins. For more details, you can read the following open-access articles:
“A new method for publishing three-dimensional content.” (2009) Raush, E., Totrov, M.,
Marsden, B.D. & Abagyan, R. PLOS One, e7394.
“SGC – Structural biology and human health: a new approach to publishing
structural biology results.” (2009) Lee, W.H., Atienza-Herrero, J., Abagyan, R. &Marsden,
B.D. PLOS One, e7675.
A series of interactive articles can be accessed at the website of the Structural
Genomics Consortium: http://www.thesgc.org/iSee/
The ICM-Browser itself possesses several other powerful features that there wasn’t time
to look at in this practical session, including a PDB search engine, a protein sequence
alignment tool, and the ability to superimpose similar protein structures, to display electron
density maps from X-ray crystallography studies, and to display the nuclear Overhauser
effect distance restraints obtained in solution NMR experiments.

Notes

Appendix 4: The 20 common amino acids NST 2018-19
Appendix 4: The 20 common amino acids:
Listed in order of decreasing hydrophobicity (more or less).



Lent Week 1: Subcellular Fractionation NST 2018-19
Week 1 (Lent) – Subcellular Fractionation of Mammalian Tissues

Glutamate Dehydrogenase, Lactate Dehydrogenase and Malate Dehydrogenase
Activity of the Fractions
Objectives
This practical is intended to show you how the level and subcellular distribution of enzymes
between mitochondria and cytoplasm can be investigated, starting with intact organs. You
will learn how to calculate enzyme content of total homogenates from raw experimental
data. A second objective is to investigate the tissue distribution of isoenzymes using non-
denaturing gel electrophoresis.
At the end of this practical you should be able to perform the following tasks:
• Prepare a homogenate of rat organs and fractionate it by differential centrifugation.
• Carry out a spectrophotometric assay of NAD-linked dehydrogenases.
• Determine whether enzyme activity is latent.
• Calculate the total enzyme content of the organ homogenates and the recovery of
enzyme activity.
• Prepare and load non-denaturing polyacrylamide gels and carry out an
electrophoretic separation.
• Stain gels to reveal the location of lactate dehydrogenase isoenzymes.
Background
Cells contain a number of components, including organelles and various membrane systems
as well as the fluid cytoplasm. The purpose of today's practical is to demonstrate that
different enzymes may be localised in various parts of a cell; such differences in enzyme
distribution may explain the different metabolic activities of the subcellular compartments.
Fractionation
After gentle disruption of cells, it is often possible to isolate, in a fairly pure state, some of
the intracellular organelles. The procedure normally adopted for mammalian liver is one
involving homogenisation of the organ in cold isotonic sucrose (0.25 M) followed by
differential centrifugation of the homogenate. Intracellular components sediment at different
rates, mainly due to their differences in size:
Component Approximate diameter (nm)
Whole liver cells 20, 000
Red blood cells 8, 000
Nuclei 6, 000
Mitochondria 750
"Microsomes" (membrane fragments) 10 - 150
Ribosomes 10 - 15
A low-speed centrifugation can be used to remove unbroken cells, red blood corpuscles
and nuclei; a subsequent higher-speed centrifugation will sediment the mitochondria; a very
high-speed spin could sediment the microsomal fraction. The same treatment will give a
similar fractionation of kidney components. When brain is studied there is an extra
component, the "synaptosomes". These are vesicles, artificially created by the

homogenisation, being pinched off nerve endings with some cytoplasm trapped inside.
(Note: for tougher, more muscular, tissues, other procedures may be necessary).
In today's practical we can use the bench centrifuge (which will give a centrifugal force of up
to 3,000 x gravity, although we do not need this top speed) to spin down debris, red cells
and nuclei; the supernatant from this centrifugation can then be spun at 10,000 x gravity in a
larger centrifuge. This second spin will sediment most of the mitochondria as a pellet,
leaving as the supernatant the so-called "post-mitochondrial" material, which contains
components smaller than mitochondria – i.e. mainly the final cytoplasmic contents but
including the microsomes. Having collected these two fractions – the one mainly
mitochondria, the other mainly cytoplasm – they can be examined for three well-
characterised oxidative enzymes, glutamate, lactate and malate dehydrogenases. We will try
to determine the quantitative subcellular distribution, and the recovery.
One important point about subcellular fractionation – the intracellular organelles are very
sensitive to mishandling, their membranes being easily ruptured. It is essential that all
fractionation steps are carried out in an isotonic medium The sucrose-Tris medium
provided, which is 0.25 M sucrose, containing 2 mM EGTA, (a metal-chelating agent) and
0.01 M Tris-chloride (a buffer at pH 7.4) has the same osmotic potential as the contents of
the mitochondria, thus preventing swelling damage. The material must at all times be kept
ice cold (buckets of crushed ice are provided for you to stand your vessels in).
Enzyme Assays
General
The assays for the three enzymes will be carried out using a spectrophotometer to give a
measure of the rate of the enzyme-catalysed reaction. Since the reaction rate may be taken
to be proportional to the amount of enzyme present, the assays will give a quantitative
measure of enzyme in the two subcellular fractions. We will refer to this as enzyme activity.
How is this different from the specific activity of an enzyme?
In the assays described below, a variety of reagents are used. When an enzyme is located
within a vesicle or organelle (e.g. a synaptosome or mitochondrion) it may not be possible
to detect unless steps are taken to break down membranes which form permeability
barriers between the enzyme (inside) and the substrates (added outside). In such instances
the enzyme is said to be "latent". The addition of the non-ionic detergent Triton to give a
final concentration of about 0.05% can disrupt membranes and may thus reveal latent
enzyme activities.
If, however, the membrane surrounding the organelle is permeable to the added substrates,
then an enzyme within an organelle can be assayed fairly quantitatively without the necessity
for membrane disruption. Enzymes that are not found within organelles, or those that are
associated with organelles but with their catalytic surfaces exposed to the exterior, would
not be expected to show this phenomenon of latency.
Dehydrogenase assays
Lactate, malate and glutamate dehydrogenases all catalyse reversible reactions, using the
coenzyme NAD as a hydrogen acceptor.
Lactate dehydrogenase catalyses:

CH3·CHOH·COOH + NAD ⇔ CH3·CO·COOH + NADH + H+
lactate pyruvate

while malate dehydrogenase catalyses:

COOH·CH2·CHOH·COOH + NAD ⇔ COOH·CH2·CO·COOH + NADH + H+
malate oxaloacetate
The glutamate dehydrogenase reaction is:

COOH·CH2·CH2·CHNH2·COOH + NAD + H2O ⇔
glutamate
COOH·CH2·CH2·CO·COOH + NH3 + NADH + H+

2-oxoglutarate
In each of these cases the activity could be measured by following the production of NADH,
which absorbs light in the near ultra-violet and can therefore be quantified
spectrophotometrically. A more suitable assay for the dehydrogenases, however, is
provided by measuring the reactions they catalyse in the reverse direction. This is because
(i) the rates of the enzyme-catalysed reactions are 3-4 times faster in this direction and (ii)
the equilibrium constants of many of the NAD-linked dehydrogenases favour the generation
of NAD and reduced substrate, rather than NADH and oxidised substrate.
The assays you will use today therefore involve adding pyruvate or oxaloacetate or 2-
oxoglutarate and ammonia to a solution containing NADH and a sample of tissue fraction,
and measuring the rate at which the ultraviolet absorption decreases. The
spectrophotometer is set to transmit light of a wavelength of 340 nm, where NADH
solutions, but not NAD solutions, absorb maximally – See Appendix 3 on
“Spectrophotometry”. The concentrations of the reactants used are sufficient to saturate
the enzymes being studied. We know the molar absorption coefficient of NADH (6220 M-
1cm-1), so we can determine absolute enzyme activities in units of katals. (A katal is the
amount of enzyme that converts 1 mole of substrate per second – see “calculations” section
of white sheets.)
Study of isoenzymes by electrophoresis

Some enzymes exist in more than one form in tissues (isoenzymes). Differences between
isoenzymes may be large or small (but they carry out the same catalytic reaction, so they
must have a basic similarity in their active centre arrangements). These differences can
sometimes be exploited to demonstrate the existence and number of isoenzymes present.
Lactate and malate dehydrogenases exist in isoenzymic forms in mammals.
LDH is the product of two major genes, encoding LDH subunit polypeptides designated A
and B. The enzyme is a tetramer, so 5 possible isoenzymes exist (A4, A3B1, A2B2, AB3, and
B4). The gene products (polypeptides A and B) differ in their electrical charge; the
tetramers generated from them also differ in charge and can therefore be separated by
electrophoresis. You can try to show the different LDH isoenzymes in a variety of rat tissue
extracts by subjecting them to polyacrylamide gel electrophoresis (PAGE) and then staining
the gel with a substrate mixture that turns purple in regions where LDH is found. The gels
are run without the detergent SDS, and so the proteins are not unfolded and denatured but
retain their native tertiary and quaternary structures.


Subcellular Fractionation – Experimental

The division of labour for the practical is as follows:
Each bench has two groups:
• GROUP A will carry out the tissue homogenisation and fractionation.
• GROUP B will make the gel and resolve the LDH isozymes.
Both groups will carry out the enzyme assays using the spectrophotometer.
Firstly, organise yourselves for the work:

In the main experiment we are primarily concerned with the intracellular location of the
enzymes, although the approximate total amount of enzyme present can also be
determined.
• GROUP A will be provided with either half a liver, a heart, a pair of kidneys, a brain
or a pair of testes.
• GROUP B will make the gel to resolve the LDH isozymes at the same time as
GROUP A carries out the fractionation.
The timetable for the groups should be:
• Making gels; obtaining and homogenising tissues 0 hour
• Low speed spin of extracts + 0.5 hour
• Loading gels; high speed spin of extracts + 1 hour
• Running gels; assaying enzymes + 2 hours
• Staining gels; calculating results + 3.5 hours
1. Procedure for cell fractionation (Group A)

Before starting operations, pre-cool the homogeniser, the bottle of sucrose-Tris-EGTA, and
two empty 50 ml beakers by standing them in a bucket of crushed ice.
The organs will have come from rats that have previously been used to supply aortae for
medical research into smooth muscle pathologies.
Take the relevant organ, and blot them on filter paper; the liver is supplied halved (for
sharing), and the kidneys should have the transparent capsule layer and white hilum
removed. Trim off any obvious bits of blood vessel, fat or other extraneous material. Place
the organs in a beaker containing just enough sucrose to cover them.
Cut the half-liver (or pair of kidneys or the brain) into small pieces with scissors. From
experience it has been found that the most efficient way of doing this is to wedge the
beaker upright in the ice-bucket, and to chop rapidly at the organ with scissors vertical,
holding each handle with a separate hand (ask for a demonstration if this is unintelligible).
When the organ is well chopped, pour the contents of the beaker into a homogeniser (do
not allow water from the outside of the beaker to get into the homogeniser). Add more
sucrose-Tris-EDTA to the homogeniser until the level is just over halfway up the narrow
portion. Total volume at this stage is about 35 ml.
Immerse the homogeniser in ice. Insert the plunger (make sure it is clean); do not, at first,
attempt to push the plunger all the way to the bottom of the homogeniser, but rather by
short strokes break up the tissue a little at a time until the bottom can be reached easily.

Then give 6 slow complete strokes to complete the homogenisation. Homogenisation is a

very critical operation and should be done with the utmost care. Be especially careful not to
raise the plunger too fast, otherwise a vacuum will be created which will play havoc with the
mitochondria and may even cause the homogeniser to implode. The desirable consistency is
similar to milkshake with no ‘bits’.
Measure the volume of the homogenate using a measuring cylinder and note the value. Set
aside about 1 ml of the homogenate in a snap-top centrifuge tube and put into the ice
bucket (for later analysis – this is Sample 1 – homogenate), and divide the rest into two
polypropylene 50 ml centrifuge tubes. Make sure the level in the two tubes is the same
(carefully balancing by eye), by pouring from one to the other if necessary. Wash out the
measuring cylinder with the STE solution (you will need the cylinder later). Load the
balanced tubes into the bench centrifuge, opposite to one another. Switch on centrifuge (if
it isn’t already ‘on’), bring the speed to 3,000 rpm on the screen, using the speed control
knob. This speed will impose a centrifugal force of 1,500 g. Set the time control knob to 3
minutes, press the start button. The centrifuge will stop automatically and the lid will open.
Remove your centrifuge tubes from the instrument carefully. This low speed of
centrifugation should have centrifuged down debris (consisting of tissue fragments, some
unbroken cells and large cell membrane fragments), red blood cells and some mitochondria
(pinkish, red pink from the bottom of the tube upwards, if you hold the tube up to the
light). The precipitate is not wanted, but the supernatant fluid from your two centrifuge
tubes should be poured carefully (including all liquid plus the lightly packed mitochondria)
into a single clean centrifuge tube. Measure the volume of this 1st supernatant fraction using
a measuring cylinder and note the value. Set aside about 1 ml of the 1st supernatant fraction
in a snap-top centrifuge tube and put into the ice bucket (for later analysis – this is Sample
2 – 1st supernatant fraction). How is Sample 1 different from Sample 2?
Make sure that your centrifuge tube is labelled for identification. Take the tube to the
communal ice bucket provided; it will be taken by a demonstrator for centrifugation in a
refrigerated centrifuge to spin at 10,000 x g for 10 minutes.
----------------------------------------break--------------------------------------------
After the high-speed centrifugation, your tube will be returned to you. Handling it carefully,
decant off the supernatant into a measuring cylinder. Keep the precipitate as well. Measure
the volume of the supernatant in the cylinder. The precipitate in your centrifuge tube should
be stirred up with a little fresh sucrose-Tris-EGTA; scraped from the bottom with a glass
rod and transferred to the (sucrose washed) homogeniser. Resuspend the pellet using a
couple of strokes in the homogeniser, and finally transfer to another measuring cylinder.
Make up this resuspended pellet to exactly the same volume as that of the supernatant.
Record the volume of each fraction (you will need this information for the calculations).
These fractions are: post-mitochondrial supernatant fraction (Sample 3) and
mitochondrial-enriched fraction (Sample 4).
The samples you will now have aliquoted into 1.5 ml tubes are as follows:
Sample 1 – homogenate – this is the dounced tissue prior to centrifugation

Sample 2 – 1st supernatant – the cleared version of the 1st homogenate
Sample 3 – the supernatant fraction after centrifugation of Sample 2.
Sample 4 – the pelleted and resuspended fraction after centrifugation of Sample 2.

Assays for enzymes can be carried out, as detailed below; each group should be able to
investigate all three enzymes (in principle only six assays are required: three for cytoplasm,
and three for mitochondria but you should also try to determine how much of the LDH
enzyme was in the whole homogenate and look into the business of "latency" and the Triton
effect.
Therefore, the measurements for 3 different enzyme activities are:

1) Measurement 1 – Total activity – measure total activity of the homogenate
(Sample 1) by first treating with Triton.
2) Measurement 2 – Sample 2 activity – measure the total activity of the 1st
supernatant fraction by first treating with Triton.
3) Measurement 3 – cytoplasmic activity (Sample 3) – activity of the post-
mitochondrial fraction.
4) Measurement 4 – Latent cytoplasmic activity: (Sample 3) – treat the cytoplasmic
sample with Triton.**
5) Measurement 5 – Mitochondrial activity (Sample 4).
6) Measurement 6 – Latent mitochondrial activity (Sample 4) – mitochondrial
fraction that has been Tritonised.**
** Ask a demonstrator how these measurements can be combined with previous ones.
2. Procedure for Enzyme Assays

a) Lactate dehydrogenase
Because we would like to have an idea about the percent recovery of LDH, MDH and
GDH, try to determine how much LDH is in your set-aside 1 ml sample of "whole
homogenate". First Tritonise it (add 10 – 20 µl of the 3% stock Triton) and stir it up a bit by
mixing but don’t froth it up – that tends to denature enzymes. The activity of LDH will be
high, so you must use a small volume (10 µl) of the homogenate for the assay.
Carry out the following LDH assay measurement for the whole homogenate sample and
repeat for the other samples and other two enzymes:
Kinetic measurements of enzyme activity on the spectrophotometer
Start-off by turning on the Computer and connected spectrophotometer. Log-on to
computer as user. The Password will be BCSS followed by the last four digits on serial
number.
Wait until spectrophotometer has self-calibrated, then open the Resolution software,
machine connects automatically and will display “Remote Terminal” on digital display.
Prepare a blank cuvette lacking NADH (3 ml of water in a cuvette will do). Put the cuvette
in the cuvette holder, ensure orientation is correct.

Prepare for kinetic measurements and run the blank as follows:
APPLICATIONS – KINETICS
ACQUIRE (screenshot) – WAVELENGTH (screenshot) – enter 340 nM, TIMING

(screenshot) – 300 sec
TAKE REFERENCE – this will set the blank Absorbance to 0.

The green START button will now be active.
Add 3 ml of LDH/MDH cocktail to a cuvette.
START – the progress curve should proceed over time at a steady absorbance of about
0.8 Au. Monitor for 30 sec.
Add 10 µl of a sample (50 µl if you are using testes) to the cuvette, mix in by 3-4 strokes of
the stirrer, and follow the progress curve (absorbance change) for about 30 sec – does the
progress curve remain stable?
Now start the reaction by adding and mixing in 0.05 ml (50 µl) of 60 mM pyruvate solution,
and resume measurement of the absorbance – does the progress curve show a steady reduction
in NADH absorbance?

Adjust the amount of sample added in a new run:

• Example of too much enzyme –
1st arrow is addition of the enzyme, 2nd arrow is addition of the pyruvate, 3rd arrow is
the progress curve showing rapid reduction of NADH. Note, post-run the guide
arrows appear allow you to dictate what part of the curve you will be calculating the
rate from.
• Example of too little enzyme –

1st arrow is addition of the enzyme, 2nd arrow is addition of the pyruvate, 3rd arrow is
the progress curve showing slow, unsteady reduction of NADH.

When run is done, (or press STOP), click yes to save results. Adjust guidelines to get the
linear part of the kinetic run.
Obtain a slope of the line as follows: ADD LINEAR SECTION – LINEAR FIT – OK.
EXPORT each sample run as an Excel file to the “student data” folder on the desktop.
Opening the file with Excel will show you all of the data and the slope, which corresponds
to the rate of the progress curve in: change in Absorbance per Second.
Proceed to the next measurement: for every new run – APPLICATIONS – KINETICS –
START.
Repeat the whole experiment with a sample of the other fraction. To determine whether
there is any latency in the mitochondrial and supernatant fractions, also carry out assays in
the presence of Triton. The most convenient way to do this is to add enough Triton, to an
assay that is running but has not gone too far, to give a final concentration of 0.05% Triton
(i.e. add 50 µl of a 3% stock Triton solution, and stir). "Tritonise" the assay in the case of
both fractions.
b) Malate dehydrogenase
The method is exactly the same as for lactate dehydrogenase, except that a solution of 18
mM oxaloacetate is used instead of the pyruvate.
c) Glutamate dehydrogenase
The method is similar to those above, but a different cocktail is used. The "GDH
cocktail" contains 0.05 M Tris buffer pH 8.2, 0.1 mM NADH, 0.05 M ammonium acetate and
1 mM ADP (which activates the enzyme). Following the outlines of the lactate
dehydrogenase instructions (above) use 3.0 ml of "GDH cocktail", then a sample of a
fraction (try 20 µl), then the substrate, this time 0.05 ml of 0.4 M 2-oxoglutarate. Don’t
worry if the absorbance jumps up when you add the oxoglutarate – it absorbs at 340 nm to
a small extent.
N.B. For the testis extracts (less active) use 50 µl for all enzyme assays.
3. Calculation of enzyme content per organ from assay data

The slope obtained from the Excel file has units of A sec-1. You know the volume
of the sample (mitochondria or supernatant) that you added to the cuvette (typically 10 µl),
and also the total volume of the fraction (typically 40 ml). The task is to use the raw data to
calculate the total amount of the three enzymes in the tissue that you used. Use the rates
measured in the presence of Triton. We can split the calculation up into logical steps.
(a) Convert from rate of change of Absorbance to rate of change of

concentration of NADH.
Refer to Appendix 3 on “Spectrophotometry” and recollect that Absorbance (A) is
proportional to concentration (c), with the molar absorption coefficient ( εM) as the
constant of proportionality. (The Beer-Lambert law.)
A = εM·c·l

Divide ∆A by the molar absorption coefficient of NADH (εM = 6220 M-1 for a 1 cm cell, so
l = 1) to turn absorbance change into change in substrate concentration.
Rate of change of [NADH] in the cuvette =
(b) Convert from rate of change of concentration to rate of change of amount

of NADH.
To go from concentration (moles per litre) to amount (moles), we need to multiply by the
volume of the assay mixture, v (typically 3.06 ml i.e. 3.06x10¯3 l).
Rate of change of NADH in the cuvette =
The amount of enzyme that converts 1 mole of substrate per second corresponds to a unit
called the katal. Thus we now have the number of katals in the cuvette as a measure of
enzyme activity.
(c) Scale up from the volume of fraction used for the assay to the total volume
of the fraction.
Suppose that sample of fraction, volume y ml, was added to the cuvette in the assay
(typically 0.01 ml = 10 µl). Thus we can calculate the activity of the enzyme in katals per ml
of fraction by dividing by y.
Activity of enzyme per ml of fraction =
Finally, we multiply by the total volume of the fraction, z ml (typically 40 ml).
Record this value in µkatal in the results table (for Q1 in the blue sheets).
(d) Estimate the recovery of Enzyme

Use the data from the LDH measurement obtained from the 1 ml sample that you kept
back from the initial homogenate to calculate the total amount of LDH in the initial
homogenate. Then compare that with the value calculated in step (d) above to estimate the
percent recovery. In other words, what proportion of enzyme activity in the initial
homogenate was still present in the fractions that you prepared?
activity in mitochondria + supernatant fraction

Percent recovery = x 100%
activity in initial homogenate
Record this value in the results table (for Q1 in the blue sheets).

4. Calculate percent distribution of enzyme between mitochondrial

and supernatant fractions prepared from an organ
By comparing the amount of the enzyme in the mitochondrial fraction with that in the
supernatant, you can discover the percentage in each compartment.
Supernatant % =
Amount in supernatant fraction
x100%
Amount in (supernatant fraction + mitochondrial fraction )
and similarly for the mitochondrial fraction.
Record these values in the second results table (For Q3 in the blue sheets).
Look back at your results to see what effects Triton had. What information does this give
you about the latency of the enzymes (latent: from the Latin for hidden)?
5. Electrophoresis of LDH isoenzymes
Safety Note: Acrylamide is toxic. Wear disposable gloves when handling gels.
Make a polyacrylamide separating gel with a final acrylamide concentration of 7.5%. Each
bench is provided with 10 ml of a "monomer mixture" in a conical flask made from 25 parts
of 30% acrylamide + 0.3% bisacrylamide, 25 parts of 1.5 M Tris-HCl buffer pH 8.8, 47.9 parts
of water and 0.1 parts of TEMED. This is enough for one gel. If you are not pouring the gel
yourself, you should watch the pouring procedure. Add, when ready, all of the 10%
ammonium persulphate (the polymerising agent) provided in the tube labelled AMPS (it
holds 0.2 ml) and mix. Pour the mixture into the gel-holding frame; insert the plastic "comb"
to form the sample “wells” and allow the whole to set (about 30 minutes). Put the flask
containing remaining monomer onto the tray for toxic wastes (ask the demonstrator).
After the gels have set in the frame, carefully remove the tubing around the edge of the
frame (it is to be saved for re-use).
Half fill the bottom tank with tank buffer (0.05 M Tris/0.38 M glycine). Put the gel in its
frame into the running apparatus. Place the gels by sliding them in at a left to right angle to
minimise the trapping of air bubbles at the bottom of the gel. Make sure you put the gel
frame into the tank the correct way round, with the shorter glass plate facing the top tank.
Fill up the top tank with more tank buffer before removing the comb and after clamping the
gel frame into place. Make sure the clamping is firm (otherwise the top tank will "leak").
Two benches (4 groups) can share the use of one gel.
Extracts of various rat organs will be provided (as well as liver, kidney and brain there will
be heart and skeletal muscle); these extracts will have been made denser by being premixed
with half their volume of a saturated sucrose solution containing bromophenol blue. Load
samples (5 µl) into the slots ("wells") in the gel left by removal of the comb. You have to
add the samples through the tank buffer: they will be sufficiently coloured to watch for

successful loading. Load a couple of samples of "bromophenol blue + sucrose alone" into
two vacant slots to use as "front markers". You may wish to run some of your own extracts
if there is room on the gel; the “low-speed supernatant”, the high-speed supernatant”, and
the mitochondria. Think about what you might expect. You will need to premix your
material with and equal volume of sucrose + bromophenol blue (ask a Demonstrator). Run
the electrophoresis at a current of 30 mA per gel. This should correspond to a PD of about
100 – 150 V. Watch for the movement of the bromophenol blue "front", and continue the
electrophoresis until the dye gets near the bottom of the gel.
The electrophoresis takes about 1-1.5 hours.
Stop the electrophoresis, remove the gel (remember gloves) and stain it with an LDH
assaying mixture, made of the following: 2.0 ml lactate (1.0 M); 2.0 ml NAD (5 mM); 4.0 ml
Tris-HCl, pH 8.5; 4.0 ml nitroblue tetrazolium (2 mg/ml); 0.1 ml phenazine methosulphate
(10 mg/ml); 13 ml water. Use the small square plastic trays for staining the gels. Place the
enamel beaker over these trays to keep the incubation dark (phenazine methosulphate is
light sensitive), but look at regular intervals for the precipitation of purple formazan bands
at the sites of LDH activity. When ready wash away the staining solution into the “waste
Stain” container. Then photograph the gel. The reason for the appearance of bands is that
the following reaction pathway exists in the presence of LDH:

Notes

Subcellular Fractionation – Results and Assessment
Subcellular Fractionation
When you have completed (your part of) the Subcellular Fractionation practical, try to
obtain other relevant data from the members of your team, and complete as much as you
can of this sheet. Discuss the results with a demonstrator.
Q1. Total dehydrogenase activity in rat tissues
Total µkatal
µkatals of
mitochondrial supernatant
enzyme whole % Recovery
fraction fraction
organ sample
LIVER (HALF)
Lactate DH
Malate DH
Glutamate DH
2 KIDNEYS
Lactate DH
Malate DH
Glutamate DH
BRAIN
Lactate DH
Malate DH
Glutamate DH
2 TESTES
Lactate DH
Malate DH
Glutamate DH

Q2. What is the effect of adding the detergent Triton to the assays?
Q3. Percent distribution of enzyme activity in rat organs

Using the optimum experiments, and assuming that the sum of the activity in the
mitochondrial and supernatant fractions equal 100%, complete the following table:
Percent of recovered enzyme activity

mitochondrial supernatant
fraction fraction
LIVER
Lactate DH
Malate DH
Glutamate DH
2 KIDNEYS
Lactate DH
Malate DH
Glutamate DH
BRAIN
Lactate DH
Malate DH
Glutamate DH
2 TESTES
Lactate DH
Malate DH
Glutamate DH
Q4. What can you conclude about these enzyme distributions?
Q5. What do you conclude from the spacing of the bands? What would have
been the result if the polyacrylamide gel electrophoresis had been carried out in
the presence of SDS?

Lent Week 2: Enzyme Kinetics – Chymotrypsin NST 2018-19
Week 2 (Lent) Portrait of an Enzyme: Chymotrypsin –

A Model Serine Protease
Objectives
In this practical we want you to obtain hands-on experience of setting up and carrying out
experiments to explore enzyme behaviour and of alternative ways of analysing the results.
The experimental manipulations themselves are simple but you will need to work out how
to construct the reaction mixtures and allow for possible limitations imposed by the nature
of the enzyme substrate.
• Plan and execute protocols for measuring reaction velocity over a range of substrate
concentrations using spectrophotometer and recorder.
• Devise and carry out an experiment to investigate the potency of an irreversible
inhibitor.
• Analyse kinetic data using the direct linear plot and linear transformations of the
Michaelis-Menton equation.
Background (Berg, J. M., Tymoczko, J. L. and Stryer, L. Biochemistry. Freeman,

7th edn, 2012 pp. 237-260, 263-274)
The serine proteases play greatly diverse roles in a broad range of biological processes,
ranging from dietary protein turnover to coagulation, fibrinolysis, complement-mediated cell
killing, fertilisation and embryonic development. Their inappropriate activation can
exacerbate, if not cause, many pathophysiological effects in diseases such as metastasis in
cancer, myocardial infarction and cystic fibrosis. Chymotrypsin (CT), because of its ease of
isolation and paradigmatic kinetic behaviour, has been extensively studied and thus provides
a good model of this class of protease.
Chymotrypsin is a ~25 kDa enzyme consisting of 3 polypeptide chains linked together

through interchain S-S bonds. As a digestive enzyme, it is selective for cleavage of peptide
bonds on the carboxyl side of aromatic amino acids and large hydrophobic residues. The
reaction will look something like this:
peptide O R' O
H H H
N N N R'
N O-
H H
N
O +H3N
:
O + O
H H
water
In vitro it can also cleave ester bonds and there is some evidence that this may be the major
physiological role of many serine "proteases".

For kinetic analysis, it is most common to use substrates that yield coloured products, but
for very sensitive detection one can also use fluorogenic substrates. (What property makes
the fluorogenic compounds more sensitive than the chromogenic ones?).
In this practical you will be using the amide N-benzoyltyrosyl p-nitroanilide (BTNA) as the
substrate for CT:
The products are thus:
(You will be detecting the increase in [p-nitroaniline] spectrophotometrically. Why would you expect
the U.V.-visible spectrum of p-nitroaniline to differ from that of BTNA?)
Kinetic studies have established that there are two stages to the mechanism of amide (or
ester) substrate hydrolysis catalysed by CT:
• Stage 1: acylation, where an acyl-enzyme intermediate is produced, releasing

stoichiometric amounts of amine (or alcohol).
• Stage 2: deacylation, where the enzyme-substrate intermediate is hydrolysed to yield
free carboxylate under regeneration of the free enzyme.
The second step closes the enzymatic cycle. Only after the second stage is complete can the
enzyme bind another substrate molecule.

Formally therefore, the reaction can be written as:
The residues involved in catalysis form the famous "catalytic triad" in which Ser-195
provides the attacking nucleophilic oxygen and becomes acylated during catalysis; His-57
accepts the proton from the -OH of Ser-195 and donates it to the nitrogen atom of the
susceptible peptide bond which is thus cleaved. (Its role was demonstrated using an affinity
label: tosyl-L-phenylalanyl-chloromethylketone (TPCK)). This compound alkylates one of the
ring nitrogens of His-57 blocking the ability for His to act as a base.
[Proof: (i) its reactivity can be protected against by adding a substrate and (ii) the rate of
inactivation by TPCK is dependent on pH in nearly the same way that catalysis rate varies
with pH].
The Asp-102 orients the imidazole ring of His-57 and helps neutralise the charge that
develops on His-57 during the transition state. The essence of catalysis by CT is the relative
stabilisation of the tetrahedral transition-state over the substrate and product.

Simple Kinetics
Why study enzyme kinetics?
Enzymes are amazingly efficient catalysts but all enzymes are not equal: the rate of an
enzyme-catalysed reaction varies over several orders of magnitude, depending on enzyme,
substrate and cellular or tissue location. Moreover, to avoid chemical chaos, all enzyme-
catalysed reactions must be under tight control for a cell to operate effectively. This means
that the catalytic competence of an individual enzyme molecule may also vary. Obviously,
for a good understanding of how enzymes co-operate within cellular mechanisms, we need
to be able to analyse and discuss their kinetic properties. In the particular case of
Chymotrypsin, kinetic analyses also helped establish an understanding of its mechanism of
action (see the discussion in Stryer).
The simplest enzyme system we could analyse is one with a single substrate and the
simplest analysis we could do is to look at the dependence of the rate of reaction on the
concentration of that substrate. Although many (if not most) enzymes are considerably
more complicated, knowledge of this simple system enables us to describe the more general
case in a straightforward manner.
Derivation of Michaelis-Menten equation

At a fixed enzyme concentration, the initial rate of substrate hydrolysis will increase
hyperbolically with substrate concentration, displaying a nearly linear rise at low enzyme
occupancy but falling off as a higher proportion of the enzyme molecules in solution become
occupied by the substrate. At maximal occupancy, the rate is governed by how fast the
enzyme can cleave the bound substrate (hence zero-order kinetics – i.e. rate independent of
substrate concentration).
These properties were formally defined by Leonor Michaelis and Maud Menten, back in
1913 deriving what is known as the Michaelis-Menten (MM) equation. Let's go briefly
through the derivation:
Take the following mechanism (S is substrate, E is enzyme and P is product):
The rate v of the production of product P is:
v = kcat[ ES] (II)
How can we determine the concentration of [ES]? To solve this in terms of known
quantities, we need to define [ES] in terms of the rate constants k1 k2 and kcat and the
concentrations [S] and [E].
From first principles we can express the rate of change of [ES], d[ES]/dt, as the difference
between the rates of the reactions that produce and remove it.
d[ES ]
= k1 [E ] ⋅ [S ] − ( k2 + k cat ) ⋅ [ES ] (III)
dt

If we work under conditions where the substrate is at a much higher concentration than
enzyme ([S] >> [E] and [S] >>[ES]) we obtain conditions of steady state where the
formation of ES is balanced by its breakdown so that [ES] remains constant:
d[ES ]
=0
dt
By substituting in Equation (III), we reduce the problem to simple algebra
hence k1 [E ] ⋅ [S ] = ( k2 + k cat ) ⋅ [ES ]
solving for [ES]
k1[ E] ⋅ [S ] [ E] ⋅ [ S] [E] ⋅[ S]
[ ES ] = = = (IV)]
( k2 + k cat ) (k 2 + kcat ) Km
k1
The expression
(k 2 + kcat ) is known as the Michaelis constant, or Km.
k1
Now we’ve replaced one unknown ([ES]) by two unknowns. So, what are [E] and [S]?
We are restricting ourselves to conditions where [S] >> [E] and [S] >>[ES]. Therefore [S],
the free substrate concentration approximates to [S]T, the total concentration of S that is
added to start the reaction.
But [E], the concentration of free enzyme is not >> [ES] so we need to define it in terms of
[E]T and [ES]:
Since [ E]T = [E] + [ES ] then [ E] = [ E]T − [ES ]

Now we can rewrite equation (IV) as
([E ]T − [ES ]) ⋅ [ S] = [E]T[ S] − [ES ] ⋅ [S ] (V)

[ ES ] =
Km Km Km
A little algebra and – voilà –
[ E]T[S]
[ ES] = (VI)
K m + [S ]
Inserting expression (VI) into equation (II) we get:
kcat [E]T [ S] Vmax[S]

v= or v= (VII)
Km + [ S] K m + [S ]
Note that V max = kcat ⋅ [ E]T

Equation (VII) is the Michaelis-Menten equation, derived using the steady state
approximation (which was not what Leonor and Maud did – they made the assumption that
k
E and S are in equilibrium with ES (k2 >> kcat , in which case K m = 2 so that Km is an
k1
indication of affinity of the ES complex.)
Properties of Michaelis Menten equation

The values of Vmax and Km can be determined by varying the substrate concentration and
by measuring the velocity.
Does Equation VII predict the observations of Leonor and Maud? YES. How so?
Look at the graph below, and think how it is described by Equation (VII)
The MM equation describes a rectangular hyperbola.
It’s helpful to look at two zones:
(i) When [S] << Km
Vmax[S] #k &
v= !!→ v = % cat ( [S][E]T
K m + [S ] $ Km '
so the reaction is first-order in [S] and the rate will increase linearly with an increase in [S].
k
The term cat is the corresponding second-order rate constant (in M-1s-1) and is a measure
Km
of catalytic efficiency. The preference of an enzyme for different substrates is expressed via
the kcat/Km value.
Since K m =
( k2 + k cat ) we can rewrite this expression as:
k1
k cat k ⋅k
= cat 1
Km (k2 + kcat )

so the maximal rate of reaction will be ultimately limited by the value of k1, which at its
fastest is restricted by the diffusion encounter between [S] and [E] just like in any ordinary
bimolecular interaction between two molecules.
(ii) When [S] >> Km
Vmax[S]
v= !!→ v = Vmax
K m + [S ]
so the reaction is zero-order in [S], and the rate is independent of [S].
All of the enzyme molecules are loaded by substrate so the rate of the reaction will be
maximal, governed simply by the rate constant of catalysis kcat and the enzyme
concentration [E]T. This provides the means of deriving the turnover number, the
number of catalytic cycles the enzyme can perform per unit time.
If not all enzyme is occupied then the fraction of active sites filled can be defined as
v [S]
!"# = = [S] ( K
Vmax m
Methods for estimating Km and Vmax

By fitting data to a hyperbola
Computer iteration techniques can be used to fit data directly to the MM hyperbola
(Equation VII), using non-linear regression. This is probably the best method, as
measurement errors can be taken into account properly and estimates of the errors in the
results can be derived without difficulties. While producing good results, such approaches
might be little intuitive. Historically, a great deal of effort has gone into devising manual,
paper plot methods, which are easy to use and give reliable values. These are still worth
knowing about – if you want to illustrate the effects of some experimental variable on Vmax
and Km, then a plot of a linear transformation of the MM equation is much easier to
understand at a glance and therefore in this practical you will be concentrating on manual
methods.
By re-arranging the MM equation into linear forms.

Km and Vmax can be estimated from straight line plots after converting Equation VII to a
variety of linear forms. Remember that the general equation for a straight line is:
y = slope × x + intercept
or y = mx + c

The common analysis methods based on x/y-axis plots are summarised below:
1/v vs 1/[S] Lineweaver-Burk or double reciprocal plot
[S]/v vs [S] Hanes-Woolf plot
v vs v/[S] Eadie-Hofstee plot
v/[S] vs v Scatchard plot
All of these plots have a statistical bias (for example because of the use of reciprocals of
velocity), which should be allowed for by appropriate " weighting" of a linear regression.
This is an important (but understandably) often rather neglected topic amongst non-
specialists. The most widely-used plot, the Lineweaver-Burk, gives the worst distortions of
experimental error but often the best-looking straight line when fitted by eye.
1 Km 1 1
= ⋅ + Equation for Lineweaver-Burk plot
v Vmax [ S] Vmax
The safest plot to use is [S]/v vs [S] – the Hanes-Woolf plot – which gives the least distortion
of error. The corresponding equation is:
[S] 1 K
= ⋅ [S ] + m Equation for Hanes-Woolf plot
v V max Vmax
Fig2. Plot of 1/v against 1/s from Eqn (2), showing the effect of Fig 3. Plot of s/v against s from Eqn (3) with error bars
of
the reciprocals on errors of ±0.05 V/max (Cornish-Bowden 1976) ± 0.05 Vmax (Cornish-Bowden. 1976)
The Direct Linear plot

A rather different approach is the "Direct Linear" or Cornish-Bowden plot.
This re-arranges the MM equation so the Vmax becomes the "y" variable and Km the "x"
variable. This takes a little thinking about to get used to but is very powerful:
! v$
V max = # & ⋅ K m + v Equation for Direct Linear plot
" [S ]%

So for any ([S],v) pair, we can plot Vmax against Km as a straight line linking an intercept of
-[S] on the Km axis and of v on the Vmax axis. This line gives all values of Vmax and Km that
satisfy the particular ([S],v) pair. If straight lines are drawn like this for several observations,
they should intersect at a common point – the unique values of Vmax and Km that satisfy all
of the observations. With real data you get a mesh of intersections, each of which is an
estimate of Vmax and Km. The median values provide an estimate of the true magnitudes.
The "mesh" may be rather "loosely woven", as compared to the tight clustering of points
when the same data set is subjected to the double reciprocal plot. What are the
implications of this, do you think?
Computers can help

We want you to manually plot your own data during the practical class to compare all these
different ways of estimating Vmax and Km. If you would like you can fit your data at home on a
computer using excel spreadsheet, and compare with the manual analysis.
Depending on substrate type, the initial kinetics of chymotrypsin can deviate

from steady state.
Note that the mechanism for which we solved the MM equation is simpler than that written
for the chymotrypsin reaction. If the rate constant for the release of P1 (the alcohol or
amine product is slow compared to that for hydrolysis of the acyl-intermediate to release
P2, then MM laws apply right from the start). (Why?) If the release of P1 is faster than the
release of P2 (k3>k4) then we have an awkward situation where there might be a burst of
product in the first round of catalysis before the steady state is entered, something looking
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Many irreversible and reversible inhibitors of chymotrypsin have been synthesised. Two
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ions. In TPCK, specificity is conferred by the phenyl ring (a substrate analog) that binds in
the active site and there is a Cl¯ ion as the leaving group.
currentpoint
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CH2 C C-CH2Cl
NH
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O
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O currentpoint 192837465 CH3 currentpoint 192837465
Phenylmethylsulphonylfluoride Tosyl-L-phenylalanylchloromethyl
(PMSF) ketone (TPCK)
Safety Note: These should be treated with respect! After all, they
are irreversible and PMSF is not very specific!
An irreversible inhibitor reacts with an enzyme in two stages. First it binds reversibly to
form a non-covalent complex (EI), and then a covalent reaction occurs to give a modified
form of enzyme (EI*). Aficionados are welcome to develop the rate equation from the
following mechanistic scheme.
We can define a constant (kass) that gives an estimate of the efficacy of the inhibitor, taking
into account both the strength of its reversible association with the enzyme and the rate of
inactivation. The units of kass are M-1s-1 – it is a second order rate constant.
k i1 ⋅k i3
k ass =
k i2
How can we measure the “strength” of interaction of an irreversible inhibitor with the
enzyme? If it produces a coloured intermediate, or is labelled, then it’s easy – you simply
mix it with the enzyme and follow the fate of the label (direct route).
When the reaction of the inhibitor with the enzyme does not itself yield a detectable
product, one must study the rate at which it inhibits the ability of the substrate to produce

product (indirect route). Deriving the overall rate constant for the interaction between I
and E is now tricky. One approach is to incubate the enzyme with an excess concentration
of the inhibitor, and follow how the activity of the enzyme falls with time by assaying
samples at intervals. The initial rate of product formation is measured over as short a time
as possible so that [S], [ES] and [EI] all remain roughly constant. Using an excess of [I]
forces the above reaction to become pseudo 1st order. Therefore, assuming that [S]>>[E]
and [I]>>[E] one can derive the expression:
ln(residual enzyme activity) = −k obs ∙ t

from which one can approximate the rate of inhibition kass from the relationship:
k obs
k ass =
[I ]
pH Dependence of Chymotrypsin Catalysis

Some catalytic groups on amino acid side chains are ionisable and exist in different ionic
forms at different pH values, where they may or may not contribute to catalysis. All this
shows up in effects of pH on rates or binding. Dependencies on pH are important pieces of
information to understand enzyme mechanism as well as the physiological context of
enzyme action.
The starting point for discussion of the pH dependence of an enzymatic reaction is the pH-
rate profile of the background reaction, amide hydrolysis.
Catalysis is defined as transition state stabilisation. Thus neutralising the energetically costly
build-up of charges in the transition state and making the reacting groups more powerful
will accelerate the reaction. This is achieved for the non-enzymatic reaction at the extremes
of pH:

o At low pH: protonation of the carbonyl group and amide leaving group helps to
offset the build up of negative charge
o At high pH: making more OH- (a better nucleophile than H2O) available speeds up
the reaction.
When we look at pH-rate profiles for enzymatic reaction, we have to distinguish what
parameter is plotted against pH.
This is the pH rate profile for the kcat/KM parameter (rate at low [S]), with an uncharged
peptide as a substrate:
This kcat/KM pH rate profile turns out to be the aggregate of two effects. You will measure
part of the kcat vs pH profile in this practical.
pH dependence for 1/KM :

In the first approximation we interpret KM as a binding constant (which is strictly only

correct for k2 >> kcat). The pH dependence of KM would then reflect differential interactions
at various pHs resulting from ionisations in free enzyme and free substrate. A molecular
explanation for this observation was only possible after the structure of chymotrypsin was
elucidated:
Conformational Changes
Figure 5-4
At high pH the B-carboxylate group of aspartate-194 blocks the specificity pocket of

chymotrypsin. At neutral pH, the protonated isoleucine amino group forms an ion pair with
the aspartate carboxylate group, which opens the pocket and allows the hydrophobic side
chains of specific substrates to bind. The diagrams also show the carboxylate group of
aspartate-102 which holds the imidazole group of histidine-57 in place. The imidazole can
then accept a proton from the hydroxyl group of serine-195 when this hydroxyl group
attacks the substrate.

Enzyme Kinetics – Experimental
The substrate concentrations and assay procedure

In order to obtain an accurate estimate of Km and Vmax it is necessary to use a range of
substrate concentrations that straddle Km. You will be given the chromogenic amide
substrate N-benzoyltyrosyl p-nitroanilide (BTNA). The enzyme-catalysed reaction can be
followed spectrophotometrically by changes in absorbance at 400 nm. The molar absorption
coefficient of the product, p-nitroanilide, is 10,000 M¯1cm¯1 (or 10 mM¯1cm¯1).
It is very important NOT TO CROSS CONTAMINATE YOUR REACTIONS so

PLEASE take care when constructing different mixtures. The water solubility of
the reagents is limited: just before inserting the cuvette in the
spectrophotometer, cover it with parafilm and once more invert it several
times to achieve good mixing (don’t shake as this will generate bubbles).
Stock solutions are:

• BTNA, 5 mM in DMSO
• Buffer, 100 mM Tris/Tris HC1, adjusted to pH7.4 at room temperature containing
20% ethanol
• a- Chymotrypsin, 4 mg/ml in 1 mM HC1
• PMSF (10 mM) in isopropanol
• TPCK (30 mM) in DMSO
Safety note: The inhibitors should be treated with respect! After all,
they are irreversible and PMSF is rather non-specific!
1. For spectrophotometer and computer operation see instructions on next page.
2. Pre-prepare cuvettes containing mixtures of the substrate BTNA + buffer to obtain

a range of concentrations (we suggest 7 or 8) between 25-600 µM, in a final volume
of 1 ml. Think carefully about the distribution of concentrations you use (why?).
It is vital to make sure that the substrate and buffer are mixed thoroughly
at this stage. If you hold the cuvette up to the light you will see that the
BTNA will fall to the bottom. Mix properly by covering the cuvette with
parafilm and invert several times (don’t shake).
3. Insert a cuvette into the photometer for blanking (see spectrometer instructions).
Once the blanking is complete, add 10 µl of µ- chymotrypsin solution to one of your
pre-prepared cuvettes to start the reaction, mix and place the cuvette into the
spectrometer. Again, you need to mix thoroughly, but this time you also
need to be quick! Teamwork is the best approach here. Each cuvette
needs blanking!

A trial run or two is sensible to make sure the enzyme is alive and to establish a
concentration range where the enzyme is not saturated and the substrate remains soluble.
Remember also to cater for possible non-enzymatic hydrolysis of the substrate (how?)
4. Keep each reaction going for a few minutes so that you can measure bona fide linear
rates from the slope of the curve.
5. Alter one of the following parameters and investigate its effects on the reaction
kinetics. It is best again to use a set of at least 5 substrate concentrations for each
new value of your chosen parameter. You will have a good idea of the range from
first experiment. This should give information on how Vmax and Km are altered.
Different groups should try different conditions, so that a range is covered. Ask our
demonstrators which one you should do.
• pH (either 6.5 or 8.0).

• Enzyme concentration (say 5 µl or 20 µl of stock solution).
In order to get things done in time, do only one alternative set of conditions.
Come to some agreement with groups on the benches around you so that you
can get results from other sets of conditions. In any case put your values on the
whiteboard.
6. For the inhibitor experiments, choose either (a) PMSF or (b) TPCK.
Start the reaction with 150-200 µM substrate (choose a concentration where the reaction
is not too fast to allow convenient monitoring) and establish the initial rate in the absence of
inhibitor (record for a maximum of ~30 sec).
Then add PMSF so that the final [PMSF] is ~0.4-0.5 mM (or TPCK to a final concentration of
~0.5 mM) and observe the reduction of the rate as time proceeds. As the inhibitor binds,
less and less active enzyme will be available and the cleavage rate of BTNA will go down.
Determine initial rates for a number of time points from the tangents on the time course.
Compare the slopes of the tangents against the initial rate in the absence of the inhibitor
and express these in %-activity of the initial rate (100% activity).
without inhibitor
(=100% activity)
tangent at t1
tangent at t2
tangent at t3
[product]
addition of
inhibitor
t3
to t2
t1
time

Plot the residual %-activity against time – you will see an exponential decay in activity from
the original 100% without inhibitor. You can obtain the pseudo 1st order rate constant kobs
for the reaction of enzyme and inhibitor by linearising the exponential plot in a semi
logarithmic fashion [ln(residual %-activity) vs time] – the slope will be the observed rate kobs
measured in the presence of a large excess of inhibitor. You can convert this into the rate
of inhibition kassoz by dividing kobs by the inhibitor concentration [I] in your solution (watch
the units !!!).
If it's all over too quickly or there is little response, adjust the inhibitor concentration
accordingly.
7. Turn to your assessment sheet for calculations and write your obtained values on
the whiteboard for comparison and to discuss the effects of varying the reaction
conditions.
Enzyme Kinetics – Use of the Computer

Control of spectrophotometer via computer using ‘Resolution software’
1. Open ‘Resolution software’; this will take control of the spectrophotometer.
There is no need to press any buttons on the spectrophotometer.
2. Open the ‘Application’ tab. Click on ‘Kinetics’ – this will open a new measurement
window.
3. Go to the ‘Acquire’ menu – set wavelength to 400 nm; select absorbance mode; set
the pathlength to 10 mm.
4. Press the ‘Timing’ button. Here you can change the maximum recording time, and
also the step size – set this to 1 s.
5. To take a measurement, first insert a blank (buffer + BTNA) and click ‘Take
reference’. After this, the start button is highlighted. Add the enzyme and press
‘Start’. (NB: this will have to be done for each concentration). The measurement will
begin and the results will be displayed in the measurement window. If the vertical y-
axis is too short, the data will not be displayed, but will still be saved, and displayed
in the result window. If the x-axis is too short, the data recording will stop at the end of
the x-axis. Therefore, it is better to choose a longer time and stop the recording early.
6. Once the measurement is finished, choose ‘Yes’ to save the data. This will transfer
the data to a result window.
7. In the result window, it is possible to add a title to label the graph (either ‘Design’ or
‘Layout’ tab). The result window will also add two vertical lines, with readings for
the x and y values. These can be used to calculate the gradient. (NB: need to x 60 to
get into min). Alternatively, choose ‘Add linear section’ and ‘linear’ to calculate the
gradient. (Chords and tangents can be chosen, useful for the inhibitor data). The
results from this are displayed below the graph window; click on the linear sections
tab and use the value in the ‘Slope’ column.
8. During the inhibitor measurements, there is no way to pause the run – remove the
sample quickly, add the inhibitor, and return to the spectrophotometer. For these
measurements, it will be necessary to increase the length of the acquisition to 5000
s.
9. Data from the result window can be exported to an excel file. Click on the circle
(top left) and choose export.

Enzyme Kinetics – Question and Assessment Sheet

Preliminary questions: It will help you if you think about these before the practical
starts!
(A) What property makes fluorogenic compounds more sensitive than the chromogenic ones?
(B) Why would you expect the U.V.- visible spectrum of p-nitroaniline to differ from that of BTNA?
(C) If the rate constant for the release of P1 (the alcohol or amine product) is slow compared to that
for hydrolysis of the acyl-intermediate to release P2, why will MM laws apply immediately?
(D) What values for [S] have you chosen? Why?
(E) How can you cater for possible non-enzymatic hydrolysis of the substrate?

Experiment 1: Establishing the kinetic properties of Chymotrypsin

10 µl of 4 mg/ml Chymotrypsin, pH 7.4
Calculate the volumes of 5 mM BTNA and buffer required to make up reactions with your
chosen [BTNA]:
Vol. Chymotrypsin Total Vol.

[BTNA] (µM) Vol. BTNA (µl) Vol. Buffer (µl)
(µl) (µl)
10 1000
10 1000
10 1000
10 1000
10 1000
10 1000
10 1000
10 1000
Record the initial rate of reaction at each substrate concentration in Abs s-1 and convert the
rates to Abs min-1:
[BTNA] (µM) Rate (Abs s-1) Rate (Abs min-1)
Plot your data manually using different linearisations methods as listed in the sections below.

Q1. Estimates from raw plot of v vs [S]

Plot the values of the initial rates for Experiment 1 (pH 7.4, 10 µl E) against substrate
concentration. (Direct v vs [S] plot). Estimate Km and Vmax from this plot.
BTNA, pH 7.4, 10 µl Chymotrypsin Km (µM) Vmax (Abs min-1)
Measured values
Q2. Estimates from linearisations of the Michaelis-Menten equation

Plot the data from Experiment 1 for yourselves using one (or more) of the linearisations of
MM equation and derive Km and Vmax values for BTNA (enter the method used). Write your
Km and Vmax values on the whiteboard.
Plotting method Km (µM) Vmax (Abs min-1)
Q3. Estimates from direct linear plot

Replot the data using a direct linear plot (Cornish-Bowden). It's quite difficult to get all the
intersections on a single graph and if your data are scattered you will struggle to manage.
BTNA Km (µM) Vmax (Abs min-1)

Median values from direct linear plot
Q4. Comment on how the Km and Vmax estimates vary between different plot
modes. Does any method seem preferable and why?

Experiment 2: Comparing the kinetic properties of chymotrypsin

under different conditions
Study the effects of one of the following experimental parameters:

• pH (either 6.5 or 8.5).
• Enzyme concentration (5 µl or 20 µl of 4 mg/ml Chymotrypsin).
Different groups should try different conditions, so that a range is covered. Ask the
demonstrators which one you should do.
Experimental condition chosen:

Vol. Chymotrypsin: µl Buffer pH:
Choose at least 5 substrate concentrations and record the rates of reaction.
[BTNA] (µM) Rate (Abs s-1) Rate (Abs min-1)
Plot your data from Experiment 2 using the Michaelis-Menten plot and your preferred
method of linerisation of the Michaelis-Menten equation. This would give information on
how Vmax and Km are altered.
Q5. Compare your estimates of the kinetic properties of Chymotrypsin at pH

7.4 with the other conditions at which you and your colleagues on other benches
have assayed:
Vol.
pH Km Vmax
Chymotrypsin (µl)
7.4 10
8.0 10
6.0 10
7.4 5
7.4 20

Q6. Is the behaviour of the enzyme affected by pH? From your knowledge of the
mechanism of the reaction, would you expect a pH dependence?
Q7. How about the volume of enzyme used? Would you expect this to have an
effect?
Experiment 3: Enzyme inhibition

Inhibitor (Choose one): PMSF / TPCK
Calculate the volume of inhibitor stock solution required:
Substrate concentration ([BTNA]) used:
% Enzyme
Time (s) Rate (Abs s-1) ln (% Enzyme Activity)
Activity
0
100
(no inhibitor)
Q8. Plot the data obtained for the loss of enzyme activity with time in the
presence of your inhibitor.
Plot [ln (value of residual enzyme activity) vs time] and derive kobs and kass (see Green Sheets).
kobs (S-1) kass (M-1 S-1)

PMSF
TPCK

Q9. Why might the values for the two inhibitors be different?
Q10. Calculate the turnover number for chymotrypsin action on BTNA.

The Mr of chymotrypsin is ~25,000 Da and you may assume that it is pure. To convert
velocity from absorbance units per time, use the molar absorbance coefficient of the
product, p-nitroanilide. Its value is 10,000 M¯1cm¯1 (or 10 mM¯1cm¯1 ). Use this to
calculate the kcat for BTNA as a substrate in units of s-1.
Q11. How would you distinguish between the possibility that the inhibitor really
inactivated the enzyme of that something toxic was added (like isopropanol in
the case of PMSF) that "killed" the enzyme.

Lent Week 3: Metabolic Control Analysis NST 2018-19
Week 3: Metabolic Control Analysis Practical
Metabolic Control Analysis in a Nutshell:
Metabolic engineering is generally referred to as the targeted and purposeful alteration of

metabolic pathways found in an organism in order to better understand and utilize cellular
pathways for chemical transformation, energy transduction, and supra-molecular assembly
(Lessard, 1996). Metabolic Control Analysis (MCA) is a theoretical framework for
investigating and understanding the control and regulation of metabolic pathways. The aim
of this practical is to study the control of a metabolic pathway quantitatively focusing
on how different steps in the pathway exert different amounts of control over the flux
through the pathway.
It is an oversimplification to identify a single rate-limiting step imposing control over the

whole pathway. More realistically, control is often shared, though unequally, amongst all
the steps in a pathway, although the extent of control exerted by many of these steps
may be negligible. The flux control coefficient (CJVi) of a particular enzyme quantifies how
much control the enzyme exerts over the pathway flux at steady state. CJVi is the fractional
change in the overall pathway flux (∆J/J) brought about by a fractional change in the activity
or concentration of the enzyme per unit time (i.e. velocity of the reaction step catalysed
by that enzyme) (∆Vi/Vi) for a particular reaction step i.
The sum of all the flux control coefficients for a simple linear unbranched pathway is
1.0 indicating that the total control exerted over the flux through the pathway is shared
among the enzymes acting along the pathway. This is defined as the Flux Control
Summation Theorem.
Thus, if physiological circumstances change, and a particular step becomes a more important
control point i.e. its flux control coefficient is increased, the other steps in the pathway are
bound to become less important control points.
The elasticity coefficient (e) describes the fractional change in the activity of an isolated
enzyme per unit time (∆Vi/Vi) in response to a fractional change in the concentration of a
substrate (∆S/S) or an effector (∆X/X) with all the other substrates and effectors of
that enzyme being held constant at the values that they have in the cell. The
assumption here is that these values (the concentrations of all substrates and effectors of
an enzyme) are known exactly. Unlike the flux control coefficient, which is a system
property, the elasticity coefficient is a property of an individual enzyme and is defined as:

The induction or repression of the synthesis of an enzyme, for catalytically changing the
amount of active enzyme and its activation or deactivation by covalent modification, are
ways of controlling pathways by altering the availability of catalytically active enzyme. These
mechanisms will produce alterations in the flux control coefficients of pathway enzymes.
The kinetic characteristics of enzymes may also be modified via routes such as the
introduction of allosteric effectors, which change an enzyme’s affinity for its substrate.
Response coefficients are used to explain such control mechanisms over pathways. A
response coefficient (R) is defined as the response of the metabolic system to an effector
(X):
Getting to Know the Kinetic Model Used in the Practical:

A recent comprehensive kinetic model of yeast glycolysis revealed the existence of widely
distributed control over the part of the metabolic network, which was most directly
involved in glycolysis in yeast (Smallbone et al., 2013). In this practical, you will be using
a simpler kinetic model of the glycolytic pathway (Teusink et al., 2000) (Figure 1). The
substrate and the effector concentrations, as well as the kinetic constants of the enzymes
in this model, were experimentally determined in yeast cells. The model was developed to
answer the question of whether the behaviour of a complex metabolic pathway could be
calculated from the known kinetic properties of its constituent enzymes. The answer to
this question, as the research article suggests, is yes, but only to a limited extent.
The following set of ordinary differential equations may be used to describe the time
dependence of the metabolite concentrations:
d[Glcin]/dt = vGLT - vHK
d[G6P]/dt = vGLK - vPGI - 2vtrehalose - vglycogen
d[F6P]/dt = vPGI - vPFK
d[F1,6bP2]/dt = vPFK - vALD
d[DHAP+GAP]/dt = 2vALD - vGAPDH - vG3PDH
d[BPG]/dt = vGAPDH - vPGK
d[3PGA]/dt = vPGK - vPGM
d[2PGA]/dt = vPGM - vENO
d[PEP]/dt = vENO - vPYK

d[PYR]/dt = vPYK - vPDC
d[AcAld]/dt = vPDC - vADH - 2vsuccinate
d[ATP]/dt = -vGLK - vPFK + vPGK + vPYK - vATPase

-vtrehalose - vglycogen - 4vsuccinate
d[NADH]/dt = vGAPDH - vADH -vglycerol + 3vsuccinate
d[NAD]/dt = - d[NADH]/dt
A unique rate equation representing the velocity of the reaction step is described in the
model. In the case of PGI, PGM or ENO, one substrate-one product reversible Michaelis-
Menten kinetics were used, such that:
where a and p represent the concentrations of the corresponding substrate and product,
respectively. G is the mass action ratio (p/a) and Keq is the equilibrium constant
(peq/aeq). Ka and Kp are the Michaelis-Menten constants for a and p, respectively.

Figure 1. A schematic representation of the glycolytic pathway. The alternative names

for metabolites and enzymes given in "Red" indicate the corresponding nomenclature in
COPASI.

COPASI as a Tool for Building and Investigating Kinetic Models:

COPASI (COmplex PAthway SImulator), is a user-friendly software environment that
allows the construction, simulation and analysis of biochemical networks and their
dynamics (www.copasi.org, Hoops et al., 2006). Any metabolic model, including the
kinetic model presented above, can be integrated numerically into the software in
order to be analysed.
COPASI can handle several analyses on metabolic pathways including, but not limited
to: Metabolic Control Analysis, sensitivity analysis, elementary mode analysis, mass
conservation analysis and time-scale separation analysis. To illustrate its use, we will run a
Metabolic Control Analysis of the kinetic model of glycolysis in yeast in this practical
session.
The Teusink model will be used to run some in silico experiments in COPASI. The effect of
varying the enzyme concentration per unit time on the flux through the metabolic pathway
will be investigated in the first instance. The concentration of 6-phosphofructo-1-kinase
(PFK) per unit time will be varied and its effect on the glycolytic flux will be monitored by
following the flux through the glucose transporter (GLT). This will then be used to estimate
the flux control coefficient of that enzyme for glycolysis. The effect of varying the
enzyme activity on metabolite levels will also be investigated in the glycolytic pathway. The
activity of triosephosphate isomerase (TPI) will be varied and its effect on the
concentration of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate
(GAP) will be monitored. Finally, the metabolic regulation of a pathway via an external
allosteric effector will be investigated. The concentration of fructose-2,6-bisphosphate
(F26bP) will be varied and its effect on the flux through the glucose transporter (GLT)
and on the concentration of fructose-6-phosphate (F6P) and fructose-1,6- bisphosphate
(F16bP) per unit time will be investigated.
In the following practical, we will present the biological question that is being addressed in
each step of the process. We will then introduce the mathematical formulation of this
biological question within the framework of Metabolic Control Analysis. We then show
how COPASI can be used to handle this question and finally, we will provide some tips
on how to use COPASI more efficiently. For each question, you will be expected to run a
set of simulations and record your results in a MS Excel Worksheet, which is provided
along with other documentation in Course Share on the PC Desktops in the folder called
‘Metabolic Control in silico’. The cells you will be expected to fill in are highlighted in
“Yellow” in the worksheet tabs. You will also be expected to interpret your results and
use them to answer the biological question we have posed for each step of the analysis.

Getting to know the model: (20 min)

The kinetic model of yeast glycolysis has already been implemented in COPASI and is ready
to be used. Double clicking ‘Glycolysis model wt.cps’ in the Folder called ‘Metabolic
Control in silico’ under Course Share on the PC Desktop will open the model in
COPASI. On the left hand menu in the dialog, you will come across a tree for browsing
and changing the model as well as running the simulations and saving the results from each
successive step of the analysis. Once a model is loaded, the default node that is
selected is ‘Model’.
The ‘Biochemical’ and the ‘Mathematical’ structure of the model can be retrieved from the
‘Model’ node. The information on the different metabolites that appear in the model
(‘Species’), the biochemical reactions constituting the model (‘Reactions’), the sub-cellular
compartments in which these reactions take place (‘Compartments’) and the numerical
values of the parameters (initial concentrations of the metabolites as well as the kinetic
and the thermodynamic parameters of the enzymes catalysing each reaction step) that are
supplied in the model (‘Parameter Overview’) are provided under the ‘Biochemical’ sub-
node.
Please note that the present model involves 19 reactions and some 25 unique
metabolites and all reactions take place in a single compartment, the cytosol. Please click
on any random metabolite under the ‘Species’ element and observe that the initial
concentration and the reactions in which the metabolite is either produced or consumed
are displayed on the right-hand side; the input to the model may be changed as needed.
Similarly, clicking on any randomly selected enzyme under the ‘Reactions’ element will
open up the details for that individual reaction on the right-hand side. Please observe that
the rate law for the reaction, details of which can be accessed by clicking ‘Edit Rate Law’
in the right-hand window, along with the corresponding parameter values as well as the
substrates and the products of the reaction are provided individually. Any parameter
belonging to a species or a reaction may be modified by clicking on its name and making
the required changes in the window on the right- hand side. Please make sure to click
on the COMMIT button after making the changes to ensure that the changes are saved in
the model.
The structure of the mathematical model for the pathway under investigation is provided in
the ‘Mathematical’ node in the tree on the left-hand menu. The set of ‘Differential
Equations’ constituting the kinetic model of the glycolytic pathway is automatically
generated once parameter inputs are provided in the ‘Biochemical’ node. The ‘Matrices’
node provides the stoichiometric matrix of the pathway, which is mainly required for
metabolic analyses that are conducted at steady state. The reaction stoichiometries are
used to generate this matrix automatically.
Important things to note regarding the model:

• vmax is denoted as V in COPASI terminology.
• In figure 1, 3PGA is denoted as P3G and 2PGA as P2G in the COPASI model.
• COPASI assumes that the model simulations take place in a unit compartment
volume (1L) and uses mmol/min as the unit of concentration per time. These
values could safely be used to make comparisons with the values given in mM/min
in this text.

• ‘Transport’ and ‘HK’ reactions in Figure 1 are denoted as ‘GLT’ and ‘GLK’ flux,
respectively, in the COPASI model.
Getting ready to run simulations: (10 min)

This is a very straightforward procedure; therefore, we only provide details on how to
set things up in COPASI.
• General information
• The ‘Tasks’ node provides options for running the simulations. ‘Time Course’
simulations will be carried out during this practical using the initial conditions
and kinetic parameters provided in the model.
• The reaction rates and the metabolite concentrations will be calculated at every
time step throughout the dynamic simulation. This will allow us to monitor how
the concentrations of the species and the fluxes of the reactions change over time
until they reach a steady state.
• The output plots and reports need to be set up prior to running any simulations.
• Setting up the output formats
• Any existing plots and reports first need to be cleared. To do this, click on:
• ‘Output Specifications’ (on the left- hand menu) → ‘Plots’ (on the left-hand
menu) → ‘Delete All’ (bottom of the right-hand window) will appear executable
(not as shadow) if there are any plots that need to be deleted. Clicking this
button will clear out any existing plots.
• ‘Output Specifications’ (on the left- hand menu) → ‘Reports’ (on the left- hand
menu) → ‘Delete All’ (bottom of the right-hand window) will appear executable
(not as shadow) if there are any reports that need to be deleted. Clicking this
button will clear out any existing reports.
• For setting up a new set of plots and reports in which the new simulation results
will be recorded, follow the steps:
• To generate a plot of the metabolite concentrations click on the following:
• ‘Tasks’ (on the left-hand menu) → ‘Time Course’ (on the left-hand menu) →
‘Output Assistant’ (right hand window) → ‘Concentrations, Volumes, Global
Quantities’ (under Plots section) → ‘Create’
• To generate a plot of the reaction fluxes click on the following:
‘Output Assistant’ (right hand window) → ‘Reaction Fluxes’ (under Plots
section) → ‘Create’
• To generate a report of the metabolite concentrations click on the following:
‘Output Assistant’ (right hand window) → ‘Time, Concentrations, Volumes,
Global Quantities’ (under Reports section) → ‘Create’

• Report (right hand window still at ‘Time Course’) → ‘Open folder icon’ →
Choose where to save the data file → Name the file using an .xls extension →
Select Any File(*) as the Save As Type → Save → Unclick Append → Confirm
Important:
• Reports and plots are created after the simulation has run.
• You only need to set up the plots and reports once; they will then be produced
every time you click “run”.
• It is up to you what combination of plots and reports you use to collect data,
but the most straightforward method is to create a report for metabolite
concentrations and then record VGLT flux from a plot of fluxes. If you need to know
the flux with greater accuracy (as is required for Q11), use the ‘Save data’ command
in the plot of the reaction fluxes.
• Report files need to be closed before each simulation in order for the new
results to be overwritten on the same file.
o Throughout this practical, whenever a parameter is changed in the
simulations, all output results should change. Obtaining the exact same
result as that of the previously run simulation would indicate either: Report
files were left open prior to running the simulation and they could not
be overwritten.
OR
o A bug has occurred in Report files. A quick solution to this would be
repeating the report saving procedure provided above or saving the results
from the Plot windows by overwriting those reports manually by:
§Save Data (in Plot window) → name/rename/select the existing
report file → Save
• The time interval of the model should be set such that 1000 iterations would be run
(number of intervals) for intervals of 2 minutes, thus 2000 minutes will be the total
time to be dynamically simulated in the in silico experiments.
• Before proceeding any further, check that your plots and reports are set up
correctly by running the simulation (clicking ‘Run’ in the ‘Time Course’ window on
the right-hand side) – check that two different plots (only 2) and a single report are
being generated in each run.
• It is useful to jot in as many significant digits as possible. Some perturbations
may result in very subtle changes.
• The concentration and flux values may be read from the plots generated in
COPASI or, alternatively, or by selecting ‘Time Course’ → ‘Result’ on the left hand
menu and looking for the steady state values presented in the Table on the main
window.

Running simulations using the model: (15 min)

For the first simulation, you should run the glycolysis model (Glycolysis model
wt.cps) without performing any modifications. In fact, you have already done so as the test-
step in the previous task. When you run the model, the plot for ‘Concentrations, Volumes
and Global Quantity Values’ will display how the dynamic profiles of the concentrations
of all metabolites vary from their initial values until they attain a steady state. The
plot for
‘Reaction Fluxes’ shows the transient fluxes of all reactions. The profiles for any of the
metabolites can be hidden on the plot by clicking on their label at the bottom of the figure.
Two very short questions to answer before you proceed with the actual
practical:
Q1. What is the steady state concentration of F6P?
Q2. The steady state flux through both the glucose transporter; VGLT and the glucokinase;
VGLK are both 85.45614 mM/min. What is VPGI? Why is it lower? Can we use ‘V’ or ‘J’
interchangeably for either one of the three?
Hint: The schematic representation of glycolysis (Figure 1) may be helpful.
The effect of PFK concentration per unit time on the glycolytic flux –
Determination of a flux control coefficient at steady state: (40 min)
This exercise aims to investigate how the glycolytic flux (J) represented by the glucose
uptake rate (VGLT) and the concentrations of the following glycolytic intermediates; G6P,
F6P, F16BP and P3G are affected by variations in the velocity of the reaction step
catalysed by PFK. For this purpose, you will vary the maximum rate of PFK (Vmax –
denoted as V in COPASI) to change the velocity of the individual reaction step. To vary
the Vmax for PFK, click on the following nodes in the left hand menu: ‘Model’ →
‘Biochemical’ → ‘Reactions’ → ‘PFK’. Please make sure that you click on the COMMIT
button in order for the changes to take place. For different Vmax values of PFK, you will
record the steady-state concentrations of G6P, F6P, F16BP and P3G and the steady-state
flux VGLT. The data should be recorded in Tab A of the MS Excel Worksheet provided.
Please make sure that you change the name of the file to your name as you will be
handing that document in at the end of the practical. You will be simulating the model
for 3 different values of VmaxPFK = 50, 50.1, and 60 mM/min. The simulation results for
other values of VmaxPFK (up to 200 mM/min) are already provided in the worksheet for
the sake of convenience.
Q3. Two different scales are used in the ‘Metabolite Concentrations’ plot provided in the
MS Excel worksheet Tab A. Why do you think we need two different scales? Hint: Check
how the metabolites are positioned in the glycolytic pathway in Figure 1.
Now try simulating the model at VmaxPFK = 40 mM/min.
Q4. What happens if Vmax = 40 mM/min? Why do you think this happens? Hint:
Answer to Q3 might be helpful in explaining this phenomenon.

Now you will try to investigate how much control the PFK enzyme exerts over the
glycolytic flux. The flux control coefficient of PFK for glycolysis (CJVPFK) is given by:
where ∆X/X=(Xfinal-Xinitial)/Xinitial
Hint: making use of all the flux and V data recorded on the MS Excel worksheet Tab A, calculate
∆J, ∆J/J, ∆V, ∆V/V and finally C values for each simulation. It is important to note that the final and
the initial values should preferable be consecutive ones.
Q5. What is the effect of changing the VmaxPFK on glycolytic flux? Is the effect more
evident at higher or lower values of VmaxPFK?
The Flux Control Coefficient is the fractional change in flux following an infinitesimally small
fractional change in the concentration or activity of an enzyme. Experimentally, this value is
impossible to achieve since an infinitesimally small increase or decrease in flux cannot be
measured. However, it is possible to conduct such an experiment in silico by decreasing the
step size. This would be very similar to measuring the tangent to the slope of the graph JGLT
vs VPFK at a specific point VPFK = 80 mM/min. For the actual VmaxPFK = 80 mM/min, the true
value of the Flux Control Coefficient is 0.646.
Q6. Conduct one in silico experiment to get a more realistic value for the Flux
Control Coefficient.
Q7. How does the glycolytic flux and the control of PFK over the glycolytic flux
change when the velocity of the reaction step catalysed by PFK is varied? A major
portion of the remaining control exerted on this pathway would be shared between the
glucose transport (GLT) and hexokinase (GLK) activities. What do you think happens as
the control of PFK over the glycolytic flux changes?
Q8. The Flux Control Coefficient of PFK at low PFK concentrations (e.g. PFK VmaxPFK =
50) is greater than 1.0. Why is this value so high? Why is it greater than 1.0?
The effect of TPI enzyme activity on the glycolytic flux –

Determination of a Flux Control Coefficient at steady state: (25 min)
In the next exercise, you will work with an enzyme that is conventionally thought to
have a very low Flux Control Coefficient for glycolysis, triosephosphate isomerase (TPI)
catalysing the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde
3-phosphate (GAP). You will investigate how varying the activity of TPI affects its control
over the glycolytic flux. The Vmax for PFK should first be set back to 80 mM/min (‘Model’
→ ‘Biochemical’ → ‘Reactions’ → PFK’ (all in the left hand menu)). Re-running the
simulation, note down the values for the steady state concentrations of GAP, DHAP

as well as VGLT in Tab B of your MS Excel worksheet. The next step would be to vary
the kinetic constants for TPI in the cell. This enzyme catalyses a reversible reaction
occurring in the cell. The kinetic constants need to be varied together in order to
maintain the thermodynamic equilibrium constant (K = (k1/k2) = 22.2) as constant at all
times. In order to achieve a considerable change in enzyme activity, both kinetic constants
should be increased or decreased by several orders of magnitude (keeping K constant at
all times). The worksheet Tab B suggests a reduction of 105 in magnitude. After
committing this change and re- running the simulation, you should record the GAP and
DHAP concentrations as well as VGLT.
Q9. What happens to the glycolytic flux in response to changing the enzyme activity of TPI?
What happens to DHAP/GAP concentration ratio? Is the reaction catalysed by TPI still at
equilibrium in the cell? What does this mean thermodynamically? Why do you think we
encounter such an outcome?
Now you will see the effect of this change that you have artificially created on the control of
TPI over the glycolytic flux. For each case, we suggest making a small change of 1% in k1,
and calculating the change that needs to be made in k2 accordingly, keeping K = 22.2. This
time, only VGLT needs to be recorded in the simulations. Either k1 or k2 can be used to
calculate the Flux Control Coefficient for TPI over the glycolytic flux following a similar
procedure as the one you have carried out for PFK.
Q10. Does TPI have high flux control? Explain why the control of TPI over the glycolytic
flux is affected by the displacement of the reaction catalysed by TPI from equilibrium.
F26bP as a regulator of the glycolytic flux: (35 min)

Fructose-2,6-bisphosphate (F26bP) is perhaps the most important regulator of the glycolytic
flux. F26bP affects the reaction step catalysed by PFK in glycolysis. This exercise is designed
to explore this relationship, which is described by the elasticity coefficient of PFK for F26bP:
As elasticity coefficients are challenging to obtain directly, you will make use of the concept
of response coefficient, which establishes the relationship between the glycolytic flux and
both the enzyme’s elasticity and its Flux Control Coefficient, as previously introduced. The
response of the glycolytic pathway to F26bP could be formulated as:
You will also investigate how the upstream substrate, fructose-6-phosphate (F6P), and the
downstream product, fructose-1,6-biphosphate (F16bP), of the reaction step catalysed

by PFK is affected by varying the concentration of the effector metabolite, F26bP. This
analysis will further be extended to monitor the same effect at different reaction velocities
of PFK (i.e. for different values of Vmax for PFK).
The previous exercises demonstrated that PFK would have a relatively high Flux Control
Coefficient for glycolysis in yeast at low concentrations. However, the true Vmax for PFK
is close to 200 mM/min producing a very low flux coefficient for the enzyme over the
glycolytic flux ( ) (Davies & Brindle, 1992).
As an initial step, the k1 and k2 settings for TPI need to be reverted back to the original
values from the previous exercise. To access the relevant window on the right-hand side,
click on the nodes in the left-hand menu in the dialog in the following order: ‘Model’
→ ‘Biochemical’ → ‘Reactions’ → ‘TPI’ and set k1 = 2.2222e+07 and k2 = 1e+06 on the
right-hand window. In order to implement the current exercise in COPASI, the Vmax for
PFK (represented as V in COPASI) needs to be varied. To access the relevant window on
the right-hand side, click on the nodes in the left-hand menu in the dialog in the
following order: ‘Model’ → ‘Biochemical’ → ‘Reactions’ → ‘PFK’. The required settings are
provided in Tab C of the MS Excel Worksheet. Please note that as you have already
simulated the model for V=80 and V=200 for PFK (as given in COPASI), in the first
exercise, these values are already included in the worksheet. The glucose uptake rate VGLT
needs to be recorded with a precision of 5 decimal places as well as the concentrations of
F6P and F16bP.
To determine the effect of F26bP on flux, the simulations need to be carried out at a
different concentration of F26bP = 0.2 mM. To make this change, please follow the
nodes in the given order on the left-hand menu: ‘Model’ → ‘Biochemical’ → ‘Species’ →
‘F26bP’. The glucose uptake rate VGLT as well as the concentrations of F6P and F16bP
again need to be recorded with a precision of 5 decimal places. These values will then be
J
used to calculate the response coefficient (R F26bP), which is the fractional change in VGLT,
and to determine the elasticity coefficient of PFK for F26bP using the relationship provided
above.
IMPORTANT: Please note that incremental changes created in F26bP concentration will
be used to calculate the response coefficient for a fixed value of VmaxPFK. The
incremental changes created in VmaxPFK will be used to calculate FCC for a fixed value of
F26bP concentration.
Q11. What does the elasticity tell us about the response of PFK to F26bP? How is
substrate F6P and product F16bP concentrations are affected by changes in F26bP
concentration at a constant reaction velocity for PFK? How do these results relate to PFK
having high or low control over the glycolytic flux at Vmax values of 80 or 200 mM/min?
Congratulations, you have completed the MCA practical!
BONUS for Math Gurus: If you are interested in the topic and would like to
construct your own kinetic model from scratch, please inquire !!

Metabolic Control Analysis – Questions Addressed in the Practical
Q1. What is the steady state concentration of F6P?
Q2. What is J(PGI)? Why is it lower? Hint: The schematic representation of glycolysis on the
second page of the green sheets may be helpful.
Q3. What happens if the Vmax for PFK is lowered below 50?
Q4. What is the effect of changing the PFK Vmax on glycolytic flux? Is the effect more
evident at higher or lower values of Vmax?
Q5. Is there a cross-over in metabolite concentrations as the PFK activity is changed?
Q6. What is the range of values of the PFK flux control coefficient? How does that compare
with the effect of enzyme concentration on the glycolytic flux (as in Q4)?
Q7. Why is flux control coefficient of PFK very high and why is it greater than 1.0 at low
PFK concentrations (e.g. PFK Vmax = 50)

Q8. What happens to the flux in response to the changing the rate constants of TPI? Is the
reaction catalysed by TPI still at equilibrium in the cell? Does it have a high flux control
coefficient? How does this relate to any simplistic notions we might have regarding control
– would you expect an enzyme catalysing a reaction that is displaced from equilibrium to
have a high flux control coefficient?
Q9. What does the elasticity tell us about the response of PFK to F26P? Is this consistent
with the changes in F6P and F16P concentrations?
Q10. If we calculated the elasticity coefficient of PFK for F26P with the Vmax of PFK set to
80, we would find it is very low, around 3.08e-03. Why might this be? Remember that PFK
has a relatively high flux control coefficient under these conditions.

Lent Week 4: Oxidative phosphorylation NST 2018-19
Week 4 (Lent) – Mitochondrial Oxidative Phosphorylation
Objectives
This practical is intended to complement the lectures on Bioenergetics (Energy transduction
in bacteria, mitochondria and chloroplasts). You will use mitochondria to assess the
coupling of respiration to ATP synthesis, by measuring the P/O ratio and the respiratory
control ratio. You will explore the characteristics of chemiosmotic energy coupling using
uncouplers and inhibitors, and do a variety of calculations based on the results.
• Use an oxygen electrode to assess how well respiration is coupled to
phosphorylation in a preparation of mitochondria by measuring the respiratory
control ratio.
• Measure P/O ratios for respiratory substrates.
• Follow and devise protocols to study the action of inhibitors and uncouplers on
mitochondrial energetics.
Introduction and Background

In this practical you will use the oxygen electrode to demonstrate the effects of ADP and a
selection of inhibitors (oligomycin, antimycin) and uncoupler (FCCP) on oxygen
consumption by mitochondria isolated from rat liver. This part of the practical will be
structured and is designed to illustrate the basic properties of isolated mitochondria.
To get the most out of the practical you should try to predict what each addition will do to
the rate you are measuring, and then (if your predictions fail) try to explain the rate changes
that you see as the experiment is running (or at least, before you run the next incubation).
If you can't, talk to a demonstrator before proceeding.
Oxygen uptake by mitochondria can be studied using an oxygen electrode. The electrode
(located at the bottom of the incubation vessel) is covered by a Teflon membrane
(separating it from the incubation medium), which is permeable to gases only. As soon as
oxygen diffuses across this membrane from the medium it is reduced at the surface of the
platinum electrode (held at –0.6 volts), causing a current to flow that is proportional to the
oxygen concentration in the medium.
Mitochondria contain the enzymes necessary to oxidise substrates, particularly those of the
citric acid cycle, and to generate ATP from ADP and phosphate. The two processes of
oxidation and phosphorylation are fairly tightly coupled together.
The phosphate:oxygen (P:O) ratio is defined as the number of molecules of phosphate
incorporated into ATP per atom of oxygen consumed by a mitochondrial preparation
oxidizing a substrate. Maximum values for the P:O ratio will be given by the number of H+
ejected per oxygen consumed during electron transport divided by the number of H+ used
to synthesize each ATP (P:O ratio ≤ H+:O ratio / H+:P ratio). If it is assumed that all the
ADP utilized by mitochondria is converted to ATP and there is a negligible rate of ATP
hydrolysis, then the P:O ratio may be accurately calculated from the amount of oxygen
consumed by mitochondria supplied with a limiting amount of ADP in the presence of an
excess of the substrate and Pi.
The respiratory control ratio is the rate of respiration of mitochondria in the presence of
substrate, Pi and ADP (state 3) divided by the rate of respiration of the mitochondria in the

presence of substrate, and ATP (state 4). This indicates how ‘tightly-coupled’ or intact the
mitochondrial preparation is.
The experiments to be carried out include a study of the P:O ratios achieved by
mitochondria in oxidising various substrates, and an investigation of the effects of some
typical inhibitors on mitochondrial respiration.
MITOCHONDRIA
How many protons? OUT IN OUT
NADH2
The number of protons transported by the -
mitochondrial respiratory chain is indicated I 2e NAD + 2H+
4H+ 4H+
on the left PER 2 ELECTRONS.
2H+ 2H+
Complex I transports 4 H+.
QH2 Q
Complex II does not transport any protons.
Complex III transports 2 H+ across the membrane 2H+
plus releases 2 extra H+ outside. 2e-
2H+ 2H+
Complex IV transports 2 H+ across the membrane, 2H+
III
moves 2 extra – charges inside,
and takes up 2 extra H+ inside.
c
So for NADH-linked substrates (e.g. glutamate
and malate) the total number of protons transported 2e-
is 10 H+ per 2 e- moving from NADH to oxygen, and NADP + 2H+
2H+ 2H+
for succinate is 6 H+ per 2 e-. IV O + 2H+ NADPH
2
H2O
ATP synthesis requires 8 H+ per 3 ATP made, but PO4H2-
transport of ATP, ADP and Pi requires H+
the equivalent of another proton per ATP.
ADP3-
The P/O ratio = H+/O ratio / H+/P ratio ATP4-
Thus for NADH linked substrates: 3ADP3-
P/O = 10/3.67 ≈ 2.7 + 3PO4H2-
For succinate: 8H+
P/O = 6/3.67 ≈ 1.6 3ATP4-

Oxidative Phosphorylation – Experimental
1 Mitochondrial respiration and coupling to ATP production

The mitochondria provided will have been isolated earlier in the day from rat liver. For
maximum coupling and respiratory rates, the mitochondrial suspensions must be kept cold
and must not be contaminated by ice or by pipettes that might have come into contact with
inhibitors or uncouplers.
A. Rates of respiration and P:O ratios with succinate

This experiment is designed to measure the respiration rates and P:O ratio of mitochondria
respiring on succinate.
• Shake your flask of ‘medium A’ (110 mM KCl, 10 mM KH2PO4, 5 mM NTA

[nitrilotriacetic acid], 2 mM MgCl2, pH 7.4) to ensure air saturation.
• Remove the lid from the oxygen electrode assembly and suck out the contents using
the suction tube attached to the vacuum line. Wash with water, then use a P5000
Gilson pipette to add 1.9 ml of medium into the electrode vessel. Make sure that the
stirrer is on. Check that the recorder pen is roughly 90% over to the right of the
paper. If unsure ask a demonstrator – don’t fiddle with the knobs.
• Check that the chart motor is on and the pen is making a mark on the paper. Make
sure the chart is moving at 10 mm/min.
• Now add 30 µl (or other recommended volume) of mitochondrial suspension (using
Gilson P200 pipette and yellow tip).
• Place the lid on the electrode, and make sure no air bubbles are trapped. Do
not switch off the stirrer while you do this. NOTE: It is ESSENTIAL that no
air bubbles are trapped. To remove air bubbles, push the lid steadily down
so that the air exits through the capillary “chimney” in the lid and a bubble-
free column of liquid enters the chimney. Don’t push so far that too much
liquid is pushed up and forms a pond in the top part of the lid. Don’t push
down on the lid with your finger over the top of the lid (that will prevent air
leaving). Check with a demonstrator if in doubt.
• Check that a reasonably steady trace is obtained on the recorder (1 minute will tell).
There may now be a slow rate of oxygen consumption (why?).
• Add 50 µl of 0.5 M sodium succinate, as respiratory substrate, through the “chimney”
in the lid using a white Gel-tip with capillary extension attached to a Gilson P200
(check that the end of the tip is in the vessel). You can insert the Gel-tip so that
the shank rests on the lip of the lid, but don’t press so hard that you make an air-tight
seal. Press the plunger of the Gilson as far as the resistance point (and no further) to
expel the liquid and then remove Gilson (with tip attached) before releasing the
plunger. Press the “Event Marker” on the top panel of the recorder to mark the
addition on the chart paper, for this and subsequent events, and write what you added
on the chart paper.
• Watch for an increased rate of oxygen consumption (give it 1 – 2 minutes) and then:
• Add exactly 20 µl 10 mM ADP using a white Gel-tip attached to a P20 Gilson. This
should take the mitochondria into state 3 with a faster rate of respiration, but after a

minute or two (when all the ADP added has been converted to ATP) the rate should
slow down again (this is state 4). When the mitochondria have been in state 4 for
about 2 minutes:
• Followed by 5 µl of FCCP.
Preferably allow your experiment to reach anaerobiosis; this should be close to zero on the
chart (consult a demonstrator if in doubt). However, these may be a slow process: if so,
you can establish the zero by pressing the “Zero Button” on the recorder and noting the
position of the pen. When finished, suck out the contents of the vessel and wash it and its
top thoroughly with ethanol (supplied in a wash bottle) and then with distilled water. This is
to remove all traces of the FCCP before the next experiment. If you are not going to do
the next run immediately, put about 2 ml of distilled water in the vessel to stop the
membrane drying out.
You can convert recorder readings into values of oxygen concentration because the
concentration of oxygen in air-saturated medium at 22 °C is known to be 500 nmol O
atoms per ml (0.5 mM O atoms). Thus, the reaction vessel (containing say 2.0 ml liquid) will
contain a total of 2.0 x 500 = 1000 nmol O at the beginning of the experiment, and this
amount of oxygen would be represented by the usable span across the recorder chart (e.g.
170 small scale divisions if the starting reading was 180 and ‘zero’ reading was 10). Note
that oxygen concentrations have been expressed here in terms of O (atoms); in reality of
course the oxygen is present as O2 (at a concentration of 0.25 mM, or 250 nmol O2 per
ml), but, since each O atom accounts for a pair of electrons, it is convenient to use O
concentrations.
If this experiment has worked well, you should be able to determine the respiratory control
ratio, the P:O ratio and the state 4 rate for succinate. If not, repeat the experiment in order
to determine these values.
The respiratory control ratio (RCR) is calculated by dividing the state 3 rate of respiration by
the state 4 rate, and this indicates how intact the mitochondria are. The P:O ratio is calculated
by dividing the amount of ADP (nmol ADP) added by the amount of oxygen (nmol O)
consumed in state 3. This indicates the ratio of ATP produced to oxygen consumed by the
mitochondria, and varies with different substrates. Put your calculated P:O ratio and RCR
values on the board.
B. Substrates and inhibitors of the mitochondrial respiratory chain

The aim of the following experiment is to illustrate the sites of action of various substrate
and inhibitors of the mitochondrial respiratory chain.
If you have not already done so, wash out the vessel and rinse its top thoroughly with
ethanol and then with distilled water to remove all traces of FCCP. Make additions to the
reaction vessel in the following sequence (and wait a minute or two between additions to
see the effects):
• 1.90 ml medium A.
• 0.05 ml (50 µl) mitochondrial suspension (now put the top on)
• 50 µl glutamate + malate substrates (produce NADH for oxidation by Complex I)
• 100 µl of 10 mM ADP (this quantity will keep the mitochondria in state 3); then
add

• 10 µl rotenone (an inhibitor of Complex I)

• 50 µl succinate (a substrate for Complex II)
• 10 µl antimycin (an inhibitor of Complex III)
• 50 µl ascorbate + TMPD (non-physiological substrate for Complex IV)
• 10 µl cyanide (an inhibitor of Complex IV).
Wash out the vessel at the end of the experiment and leave with water in it.
Note. Rotenone is dissolved in DMSO and antimycin is dissolved in ethanol.
The concentrations of inhibitors provided are rotenone, 500 µg/ml; antimycin, 500 µg/ml;
cyanide, 0.1 M; oligomycin, 1.25 mg/ml.
Safety Note: These poisons are poisonous!

The concentrations of substrates provided are: 0.16 M glutamate and 40 mM malate; 0.5 M
succinate; 0.4 M ascorbate and 20 mM TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine).
C. Control of respiration by ATP production and consumption

This experiment is designed to examine how the rate of mitochondrial respiration is
controlled by ATP production and consumption. Hexokinase is an enzyme of glycolysis that
catalyses the following reaction:
glucose + ATP à glucose-6-P + ADP
If you have not already done so, wash out the vessel and rinse its top thoroughly with ethanol
and then with distilled water to remove all traces of inhibitors. Make additions to the reaction
vessel in the following sequence (and wait a minute or two between additions to see the
effects):
• 1.90 ml medium A.
• 0.05 ml (50 µl) mitochondrial suspension (now put the top on)
• 50 µl glutamate + malate substrates (produce NADH for oxidation by Complex I)
• 20 µl of 10 mM ADP (and wait until rate has slowed down to state 4)
• 10 µl glucose (hexokinase substrate)
• 10 µl hexokinase (and wait 1 min)
• 10 µl of oligomycin (an inhibitor of the ATP synthase, wait 1 min)
• 5 µl of FCCP (and wait 1 min).
Wash out the vessel at the end of the experiment and leave with water in it.
Try to think why hexokinase stimulates oxygen consumption, and what oligomycin is doing.
Calculate the RCR and P:O ratio on glutamate and malate. Put the values on the board.
Calculate the rate of respiration (in arbitary units) after each addition of hexokinase. Plot the
rate of oxygen consumption (J, in arbitrary units) against the amount of hexokinase ([H] in

arbitrary units) on a piece of graph paper (with origin 0 & 0). Fit a curve to these points by
eye. Try to think why the graph has the shape it does.
The flux control coefficient (C) of hexokinase over the oxygen consumption rate at any
particular value of H can be calculated as:
C = (dJ/dH) . (H/J)
i.e. the gradient of your curve multiplied by H/J at that point. This is the extent to which
hexokinase controls the rate of oxygen consumption (see Introduction to next practical).
Calculate C at your lowest and highest values of H.
D. Design an experiment to test how a toxin works on mitochondria

This section is intended to complement the theoretical “experimental design” sessions you
will be attending, and challenges you to play the detective to discover how a mystery poison
works. We have provided a sample of a toxin (labelled T). The toxin interferes with
oxidative phosphorylation, and we want you to narrow down its site of action. Design one
or more experiments to answer the following question, making selective use of the
materials provided for experiments A, B & C.
You may wish to discuss your experiment with a demonstrator before you start.
Is substance T (a) an uncoupler, (b) an inhibitor of the electron transport chain or (c) an
inhibitor that affects ATP formation by mitochondria?
Start with an incubation mixture like this, as a basis for further additions:
1.90 ml Medium A
50 µl mitochondria
50 µl glutamate + malate
Use 10 µl of substance T when you want to add it to the incubations.
NB. The rate of oxygen consumption in the absence of ADP varies with different substrates,
so if you wish to study the effects of T on the different complexes of the electron transport
chain (option (b) above) it is best to rule out options (a) and (c) first, and add excess ADP.
Summarise your design, and note your conclusion in the blue sheets. You may like to
suggest further steps that would be needed to get more exact information about the site of
action of substance T.

Oxidative Phosphorylation – Results

A. Rates of respiration and P:O ratio with succinate
a. What was the value of the P:O ratio?

Is this the expected ratio?
b. What was the value of the Respiratory Control Ratio?

c. What was the value of the state 4 respiration rate?
d. Why, in a sentence, did ADP stimulate the rate?
e. Why did FCCP stimulate the rate?
B. Substrates and inhibitors of the mitochondrial respiratory chain
a. Why did rotenone have the effect they did?
b. Why did antimycin have the effect they did?
c. Why did cyanide have the effect they did?
d. How did glutamate and malate supply electrons to the respiratory chain?
e. How did succinate supply electrons to the respiratory chain?
f. How did ascorbate and TMPD supply electrons to the respiratory chain?

C. Control of respiration by ATP production and consumption
a) What was your value of the P:O ratio on glutamate and malate?
b) Open the internet browser on your phone or computer, go to:

www.graphpad.com/quickcalcs/ttest1.cfm, and enter the class results for the P/O ratio
on succinate and glutamate/malate into the data table, and do an unpaired t-test. What
were the means and standard deviations of the P:O ratios on succinate and
glutamate+malate?
Why might different students get different results for the same experiment?
c) What was the two-tailed P value of the t-test?

What is the P value the probability of?
Was the difference between the P:O ratios significant?
Does the significance tell you whether there are real differences between the P/O ratios?
d) Why did hexokinase + glucose stimulate the rate?
e) Why did further hexokinase stimulate the rate less?
f) What were the values of the flux control coefficient of hexokinase over respiration rate
at the lowest and highest amounts of hexokinase?
g) Why did oligomycin inhibit the rate of oxygen consumption?

D. Design an experiment to test how a toxin works on mitochondria
a. Which experiments did you do to test how toxin T acts on oxidative phosphorylation?
b. How does toxin T act on oxidative phosphorylation?
c. Did you use the optimum protocol to test how toxin T acts on oxidative
phosphorylation, and if not, what would have been the best protocol to test this?

Lent Weeks 5 & 6: Cell Signalling I: Tyrosine Phosphorylation in Platelets NST 2018-19
Cell Signalling 1. Tyrosine Phosphorylation in Platelets

There are two Cell Signalling practicals using human platelets that (including analysis and
discussion) will occupy the next four weeks. This first practical takes two weeks and
investigates the action of various agonists and antagonists to modify tyrosine
phosphorylation of platelet proteins. Tyrosine phosphorylation of a range of proteins is
intimately involved in platelet activation and subsequent aggregation.
Objectives
This practical is intended to give you experience in the technique of Western blotting
(immuno-blotting) in the context of mechanisms of transmembrane signalling. You will
examine the response of platelets to physiologically important agonists and explore
the effects of antagonists.
After this practical you should be able to carry out the following procedures:
• A timed protocol for stimulation of cells with different agonists.
• Set up, load and run SDS polyacrylamide gels.
• Transfer proteins from a gel to a nitrocellulose membrane by semi-dry blotting.
• Detect specific proteins (epitopes) using antibodies (Western blotting).
Introduction
Platelets
Human platelets are a useful experimental system because they express a variety of signal
pathways and are very robust in comparison to other cell types. They are easily
available from the National Blood Transfusion Service. Platelets are small, membrane-
bound structures (not cells, but fragments of much larger precursors, megakaryocytes) that
are responsible for haemostasis (prevention of blood loss) after injury.
Agonists and Antagonists

An agonist is a substance which initiates a physiological response when it binds to a
receptor, while an antagonist blocks the action of an agonist. Platelets may be activated by a
variety of both physiological and non-physiological agonists and antagonists. Those used in
this practical are briefly described below.
Agonists
Collagen
Collagen is the most abundant human protein. One location of collagen is below the
endothelial cells that line blood vessels, which can become exposed as a result of injury.
Collagen binds to specific receptors on platelets, causing them to adhere to sites of vascular
damage. The signalling pathways activated by these receptors may then promote platelet
aggregation, thereby plugging leaks in the vasculature. The amino acid sequence in collagen
is generally a repeating tripeptide unit, Gly-Pro-4-hydroxyproline. Synthetic peptides
comprising repeats of this sequence also activate platelets via the glycoprotein VI receptor
(GpVI). This practical uses one such peptide, (G-P-O)10, known as collagen-related
peptide (CRP).

Thrombin
Thrombin, the proteolytic product of the zymogen prothrombin, functions in blood
clotting to promote the proteolysis of fibrinogen to form fibrin, the insoluble fibrous
protein that holds blood clots together. However, thrombin also activates platelets and
promotes their aggregation by interacting with protease activated receptors (PARs).
Human platelets express PAR1 and PAR4, both of which are G-protein coupled
receptors activated when thrombin cleaves a fragment from the N-terminus permitting the
new N-terminus to bind to the receptor of which it is a part and activate intracellular
signalling. Thus the agonist is an integral part of the receptor and is referred to as a
tethered ligand.
A23187
A23187 is an ionophore that forms stable complexes with divalent cations. It is produced at
fermentation by Streptomyces chartreusensis. Its lipid solubility permits the equilibration of
ions across membranes. A23187 will rapidly increase the intracellular concentration of
Ca2+ as a consequence of the high concentration gradient of this ion across the plasma
membrane (~10,000-fold).
Antagonists
BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’tetraacetic acid tetrakis(acetomethoxyl ester)
BAPTA is derived from EGTA and is a strong Ca2+ chelator. In the acetomethoxyl ester
form the four carboxylate groups of EGTA are esterified, permitting diffusion of these
otherwise negatively charged groups across the plasma membrane. Once inside the cell the
ester bonds are hydrolysed by endogenous esterases to generate the chelator BAPTA.
BAPTA is therefore used to chelate Ca2+, which increases in concentration during platelet
activation.
PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine)

PP1 is a cell permeable drug and a potent inhibitor of tyrosine kinases (e.g. Src, Lyn, Fyn,
etc.). It acts as a competitive inhibitor of ATP binding.
Phenylarsine oxide (PAO: C6H5AsO)

PAO is a tyrosine phosphatase inhibitor.
Dimethyl sulfoxide (DMSO: (CH3) 2SO)

DMSO is a colourless, dipolar, aprotic solvent. It is readily miscible with a wide range
of organic solvents as well as water and is frequently used as solvent. DMSO is widely used
as a cryoprotectant, for example, in the preservation of organs, tissues and cell suspensions.
It has the distinctive property of readily penetrating the skin. The antagonists used in this
practical are dissolved in DMSO as they are insoluble in water.

Summary of procedure: Week 1

The practical takes place over two weeks. In week 1 platelets are incubated with an agonist
and an antagonist. The cells are lysed in Laemmli buffer (containing SDS and
b-mercaptoethanol) and the proteins separated by SDS polyacrylamide gel electrophoresis
(PAGE). You will prepare an 8% cross-linked gel that separates proteins over a wide range
of molecular weights, up to about 200 kDa, but with relatively poor resolution. This degree
of cross-linking is close to the limit at which the gels become physically difficult to
handle. In other words, if we use much more open gels, they become very sloppy. Higher
levels of resolution would be achieved for smaller proteins (15-60 kDa) by increasing the
cross-linking of the gel but large proteins (>150 kDa) would then not be resolved since
they could not enter the gel from the loading wells. To increase the resolution you will
also prepare a stacking gel.
After running, the gels are transferred to a semi-dry blotting apparatus. We will do this
for you as a demonstration. The blotting apparatus transfers the proteins to a PVDF
(polyvinylidene fluoride) membrane by applying an electric current at right angles to the gel.
We will remove the blots (i.e. the PVDF membranes) from the apparatus and store them
for you until week 2.
Summary of procedure: Week 2

Ponceau staining. Ponceau is a diazo dye (general formula R2C=N2) that provides a relatively
crude method of detecting protein. It is widely used as it is quick, easy and readily
reversible. A successful stain confirms transfer of protein from the gel to the PVDF
membrane, and will indicate if the amounts of protein loaded into each well were broadly
similar (which is important when comparing the densities of the specific proteins identified
by Western blotting).
Western blotting. Western blotting often uses antisera raised against pure protein or
against peptides synthesised to correspond with cDNA sequence data. This practical uses a
commercially available mouse monoclonal antibody (PY20) that detects
phosphotyrosine. The antibody is then recognized by a secondary antibody raised against
mouse IgG. This secondary antibody is conjugated to HRP (horse radish peroxidase)
permitting direct detection of binding. Use of the secondary antibody allows amplification of
the signal and increases the sensitivity of the method. Bound antibody is detected by the
HRP peroxidase activity using a dye plus hydrogen peroxide. The oxidation product is
coloured.
Safety Notes
1. The platelets are supplied by the National Blood Transfusion Service and have been
screened for hepatitis and HIV. Nevertheless, human blood products represent a risk, so
follow good practice: wear gloves when handling the platelet preparation; wear a lab
coat at all times. Many of the chemicals involved in setting up the gel contain strong
detergents and cross-linking reagents that are powerful irritants. Acrylamide is a
neurotoxin: wear gloves and take care when handling these materials.
2. Perform all manipulations of materials over the trays provided. After use gloves and
contaminated tissues should be removed carefully and discarded in the bins provided and
small items such as Gilson tips placed in the waste containers on the bench.

Some background about antibodies

Antibody proteins contain immunoglobulin (Ig) domains and are produced by B
lymphocytes (B cells). They are Y-shaped molecules comprising four chains, two light and
two heavy. The light chain contains one pair of Ig domains and the heavy chain
contains two pairs of Ig domains connected by a flexible linker or hinge. The chains are
linked by disulphide bonds, as illustrated for the IgG class of immunoglobulin (the most
common class).
The binding sites for antigens are at the tips of the arm (hypervariable regions) and in a
single antibody both arms will bind to the same antigenic component. All of the antibody
molecules produced by a single B cell have the same antigen binding site. Since the immune
system can respond to very many antigens, the organism must be able to produce a
corresponding diversity of B cells that make antibodies. The antigen binding sites are
produced during B cell maturation. An array of different V (variable), D (diversity) and J
(joining) genes exists in the heavy chain locus (in the light chain locus only V and J are used)
and from these a single V-D-J (or V-J) DNA sequence is created by a process called somatic
recombination. Expression of these sequences forms the variable region on the arms of the
Y, which are combined with the stalk of the Y (the constant region) as a result of RNA
splicing. The enormous number of possible V, D and J DNA sequence combinations, along
with different light and heavy chain combinations means that antibody diversity in terms
of specific, high affinity binding to the antigen is almost limitless.
Antibodies are produced for research and diagnostics by injecting an antigen into an
animal, often a mouse or a rabbit. An antigenic molecule, such as a protein, will contain
many antigenic determinants, known as epitopes. Individual B cells display a surface
immunoglobulin that can recognise the antigen and go on to differentiate and multiply (with
the help of other cells), producing and secreting antibodies. Each B cell progenitor produces
a different type of antibody with specificity for one epitope on the antigen. Thus the blood
from the immunized animal will contain a mixture of antibodies to the antigen and this
mixture is known as a polyclonal antibody.

To produce a monoclonal antibody (meaning a unique antibody made by a population of

identical B cells derived from a single progenitor, in other words a clone), B cells
are removed from the spleen of an immunized animal and are fused with cells from a
myeloma (a B cell tumour) to produce hybrid cells that grow indefinitely like cancer
cells but produce large amounts of antibody. The antibodies produced by single cells are
tested for antigen binding. The cell that produces the tightest binding antibody can then be
selected and used to generate a cell-line that manufactures large amounts of the antibody.
Antibodies can be used as molecular probes for the presence of antigen but this requires a
system to detect the presence of antibody. One method is to use a secondary antibody (an
“antibody to an antibody”). Secondary antibodies are raised against the constant region
of the primary antibody and so recognise all IgG from an organism (e.g. mouse). For
example, if the primary antibody is from mouse, the secondary antibody may be from rabbit.
Use of secondary antibodies amplifies the signal from the primary antibody, due to several
secondary antibody molecules binding to a single primary antibody molecule. Secondary
antibodies can be conjugated to an enzyme, such as horseradish peroxidase, that is used for
production of a coloured or luminescent product, or the secondary antibodies can be
directly attached to a fluorescent dye.
Some examples of uses of antibodies that you may encounter:
• Detection of antigen in a lysate by western blotting.

• Detection of modified antigen by using an antibody raised against (say) a
phosphorylated peptide.
• Quantitation of antigen in a lysate/serum/urine sample by ELISA. Identification of the
localisation of antigen in fixed cells by immunostaining. Purification of antigen from a
lysate by immunoprecipitation.
• Identification of protein ligands of an antigen by co-immunopurification.
• Exploiting bivalency of IgG in cross-linking studies (e.g. tyrosine kinase receptors).
• ‘Panning” in phage display.
• Neutralisation (i.e. inhibition of) protein function.
• Medical applications: “humanised” antibodies. Studies of transition state stabilisation
in enzymes. Tethering of flagellar protein components.

Cell Signalling I – Experimental procedure – Week 1
1. Prepare the polyacrylamide gel

The gel apparatus has been assembled for you. It consists of two glass plates, one with
raised pieces on each side, like ears. Plastic tubing prevent leaks while the gel sets. Before
pouring your gel make sure the apparatus is level. There are two stages: a “separating gel”
is poured first and allowed to set, then a smaller “stacking gel” is poured on top of the
separating gel. A plastic comb is used to create wells in the stacking gel into which the
samples will be loaded.
Materials
These are coded as follows:
A Acrylamide (4.2 M); N, N´-Methylene bisacrylamide (52 mM)
B1 Tris-HCl buffer, pH 8.8 (1.5 M)
B2 Tris-HCl buffer, pH 6.8 (0.5 M)
C SDS (sodium dodecyl sulphate, 10% weight for volume)
AMPS Ammonium persulphate (polymerising agent, 10% weight for volume)
T N,N,N´,N´-tetramethylenediamine (TEMED: cross-linking reagent)
Preparation of the separating gel (8%)

Mix as follows, in the order below. Be careful to measure accurate volumes:
A 5.4 ml
B1 5.0 ml
C 0.2 ml
AMPS 170 µl
Water 9.2 ml
T (TEMED) 30 µl
Make sure that the materials are well mixed by swirling; do not shake as that may
introduce air into the mixture that will slow down the setting process. Using a 5 ml
Gilson, carefully pipette the gel mix between the plates to fill the space up to 1 cm below
the bottom of the teeth of the comb. Then add 1 ml ethanol on top to ensure a flat level
surface for the next gel. Leave it to set (about 30 minutes). Tip off the ethanol and rinse
between the plates with distilled water.
Preparation of the stacking gel (4%)

Mix as follows, in the order below. Be careful to measure accurate volumes:
A 0.65 ml
B2 1.25 ml
C 0.1 ml
AMPS 85 µl
Water 2.9 ml
T (TEMED) 15 µl

Pipette the stacking gel mix on top of the separating gel. Then insert the comb making sure
that there are no tiny air bubbles trapped between the comb and the solution. Leave to set
(about 15 minutes).
2. Platelet assays
You are provided with:
1. A platelet preparation at 5 x 108/ml.
IMPORTANT: Do not put this on ice. Leave it at room temperature.
2. Laemmli buffer (0.26 M Tris-HCl, pH 6.8, 10% SDS, 40% glycerol, 5% b-
mercaptoethanol, 0.05% bromophenol blue). Glycerol increases the density of
the solution so that it collects at the bottom of the well when applied to the gel.
Bromophenol blue is a dye that runs at the “front” of the gel.
3. Molecular weight markers dissolved in Laemmli buffer.
Each pair will set up FOUR pre-incubations in screw-cap, plastic microfuge tubes using one
of the antagonists: EITHER BAPTA or PP1 or PAO: these are dissolved in DMSO, so
DMSO as a control must be added to the other tubes.
Start the incubations by adding 40 µl of platelet suspension to each of the four tubes at
30 second intervals. After 10 minutes, add the agonist at intervals of 30 seconds as
indicated in the table.
Replace the tubes in the 30 °C heat block and stop each incubation after 5 minutes,
at staggered 30 second intervals, by adding 50 µl of Laemmli buffer. Tighten the lids and put
the tubes in the 95 oC heat block for 5 minutes. The molecular weight markers do not
need to be heated. Place the tubes on ice for 2 minutes to cool.
Experimental design for platelet assays:

TUBE No. (screw-cap)
STEP Agent added Vol. 1 2 3 4
BAPTA or BAPTA or
1 Antagonist 5 µl DMSO DMSO PP1 or PP1 or
(1%) (1%)
PAO PAO
2 Platelets 40 µl Add to each tube
~10 min @ 30 oC (heating block)
Thrombin or Thrombin or
3 Agonist 5 µl H2O CRP or H 2 O CRP or
A23187 A23187
5 min @ 30 oC
Laemmli
4 50 µl Add to each tube
buffer
5 Transfer to heating block (5 min)
6 Transfer to ice (2 min)
7 Load gel
8 Run @ 60 mA/gel (~1 hr)
9 Remove gel: place in blotting buffer
10 Transfer gel to blotter
Final concentrations: Thrombin 10 U/ml, CRP 250µg/ml, A23187 10 µM.

3. Gel loading
Carefully remove the bulldog clips and then the plastic tubing from around the sides of the
plates. Mount the plates in the apparatus, making sure the plate with ‘ears’ is to the
rear, adjacent to the upper reservoir. Secure them with the larger bulldog clip on the left
and the smaller on the right. Fill the top and bottom tanks of the gel apparatus with
running buffer (25 mM Tris-HCl, pH 8.3, 200 mM glycine, 0.5% SDS) making sure that
the level in the top tank flows onto the top of the gel to establish electrical contact.
Mark the positions of the wells with your marker pen and then remove the comb.
Using a P20 Gilson load the molecular weight markers (20 µl). Load 20 µl from each
incubation tube into other wells, going from left to right. Use adjacent central wells,
unless you have bubbles that need avoiding. Plug in to the power pack, taking care to get
the positive (RED) and negative (BLACK) terminals correct. Switch on and set the current
to 60 mA. The gel will take about an hour to run.
4. Setting up the Western blot

After the gel has run switch off the power pack, remove the leads and carefully pour away
the running buffer. Take the plates out of the apparatus and place in your tray on top of
some paper towel. Use a large spatula gently to lever the top plate off. Apply the leverage
BETWEEN the top and bottom ends of the plate so you do not break the ‘ears’ or the
corners. Carefully remove the gel and place it in blotting buffer (25 mM Tris-HCl, 150
mM glycine, 5% v/v methanol, pH 8.8) in the square plastic box. Take it to the blotting
apparatus where we will assemble the blot. This involves laying the gel over a piece of
PVDF membrane and sandwiching it between layers of filter paper. Application of a
current at right angles to the gel transfers the proteins from the gel to the nitrocellulose
membrane. Assembly of the blot completes the first week’s work.
We will take the blots off and store them ready for the detection step the following
week.

Cell Signalling I – Experimental procedure – Week 2
You will stain your blot for protein and then use a monoclonal antibody to detect
phosphorylated tyrosines. Remember that the platelets were stimulated with various
agonists, and you want to see if changes can be detected in the level of tyrosine
phosphorylation.
These should reflect the level of platelet activation.
Ponceau staining
Collect your PVDF blot and pour off the blotting buffer. Wash the blot with distilled water.
Then pour in enough Ponceau stain to just cover the blot. Leave for 1 minute ONLY. A
longer exposure makes it difficult to remove the stain. Pour off the stain into the small
beaker provided, and wash 2 – 3 times with distilled water. Dispose of these washes into
the larger beaker. Add distilled water to cover the blot; do not let it dry out. You should be
able to see some of the proteins that have been transferred from the gel to the PVDF
membrane, and be able to check relative amounts of protein in each sample you loaded
onto the gel.
Draw a rough sketch. The most important point to note is the intensities of the bands in
each lane. In an ideal experiment they would all be the same. If they don’t look equal then
note this down. It will help with your final analysis.
Probing with Antibodies
1. Pour off the water and add the solution containing antibody PY20.
2. Incubate for 45 minutes in the shaker.
3. Tip off the PY20 solution and wash the membrane with PBS for 2 x 10 seconds.
4. Add the secondary antibody and incubate for 15 minutes on the shaker.
5. Tip off the secondary antibody solution
6. Wash the membrane for 2 x 10 seconds with PBS.
Detection
1. Prepare detection buffer (collect the detection reagents to add to the detection
buffer on your bench from a demonstrator)
2. Add the detection reagent to your blot and allow the bands to develop (don’t
shake), this should take 5-10 minutes.
3. Try and identify any differences in the densities of similarly sized bands and see if
they relate to your treatment of the sample. You can stop the blot darkening
further if you need to by gently tipping off the solution and adding water
4. Photograph the result.

Cell Signalling I – Question Sheet
1. Sketch or attach the photo of your Ponceau stained membrane and your
Western blot
2. Indicate by a simple drawing how the immunodetection procedure works.

3. Compared to the control (DMSO alone) what were the levels of changes
to tyrosine phosphorylation elicited by
(a) thrombin?
(b) collagen?
(c) A23187?
4. What effects did the antagonists have on the levels of tyrosine

phosphorylation?
i) BAPTA
ii) PP1
iii) PAO
5. What do you deduce about the effects of the agonists and

antagonists on platelet activation?
6. What further experiments might you perform to test your theories?

Lent Weeks 7 & 8: Cell Signalling 2: The production of Thromboxane by Human Platelets NST 2018-19
Cell Signalling 2: The production of Thromboxane by human

platelets
This, the second of the Cell Signalling practicals, will correlate with the appropriate
signalling lectures. You will investigate the ability of platelets to synthesise thromboxane
A2, (TxA2), which is unstable, so levels of its stable metabolite, thromboxane B2 (TxB2) are
measured. As you will recall from the first Cell Signalling practical, platelets bind to
collagens via specific receptors, such as integrin a2b1 and glycoprotein VI (GpVI). Human
platelets are stimulated by binding to collagen, which promotes arachidonic acid release
from membrane phospholipids. Free arachidonic acid is then converted to thromboxanes.
The experimental work will be completed in Week 1, and data will be analysed and
discussed in Week 2.
Objectives
You will learn how to perform an ELISA (Enzyme-Linked Immunosorbant Assay), a
technique that is used to measure a huge number of agents and metabolites in many
areas of biochemistry.
The strategy is to use collagen or other known activators to stimulate human platelets
and measure the production of thromboxane. You will also investigate the action of
aspirin, which blocks this pathway, and examine the role of intracellular calcium in
mediating the collagen response.
At the end of this practical you should be able to carry out the following tasks:
• Carry out timed protocols on activated platelets.
• Prepare samples for ELISA and use the 96-well plate reader.
• Prepare a standard curve and extract and analyse data.
• Plot concentration-response curves and determine the EC50 for the
activator
Introduction and Background

Phospholipids in many different cell membranes are enriched at the R2 position (see figure
below) in arachidonic acid. The enzyme that releases arachidonic acid from membrane
phospholipids is known as cytoplasmic phospholipase A2 (cPLA2). It is regulated either
by ligands that bind to some G protein-linked receptors or by some tyrosine kinase-
mediated signals, as you saw in the Cell Signalling I practical (Western blotting) last week.
The regulation of cPLA2 is not understood in detail, but several second messenger pathways
interact to stimulate the activity of the enzyme.
Arachidonic acid is a C20 fatty acid with 4 double bonds (formally, 5:6, 8:9, 11:12, 14:15
eicosatetraenoic acid). It is the precursor of prostaglandins and some related paracrine or
autocrine agents collectively known as eicosanoids. The eicosanoids are important
mediators of inflammation and pain. In blood platelets, production of the thromboxane A2
(TxA2, a prostaglandin) amplifies the activation process via the paracrine activation of a 7-
TM-R, TP2. This leads to platelet aggregation, an event vitally important to thrombosis and
haemostasis (a topic included in the Cardiovascular Biology option of the Biochemistry Part
II and Part III courses).

Platelet Thromboxane Production

You will be provided with suspensions of washed human platelets, obtained from National
Blood Service. You will use the platelets untreated (the control) or treated with aspirin
(acetyl salicylic acid) or BAPTA-AM to investigate the effects of aspirin and the role of
intracellular calcium in the synthesis of TxA2. TxA2 is unstable and rapidly converts to the
stable TxB2, which is what you will measure in the Practical.

Collagens of the blood vessel wall become exposed after injury to the endothelial cell
layer and are major physiological activators of circulating platelets. You will investigate
thromboxane synthesis stimulated by collagen itself or one of the other known platelet
activators.
You will produce concentration–response curves for novel synthetic collagens and you will
discover whether you can stimulate TxB2 production in aspirin-treated platelets. You will
investigate the role of intracellular Ca2+ using BAPTA-AM, an intracellular Ca2+ chelator.
The functions of these modulators are summarised below.
Platelet Thromboxane Production – additional notes:

Modulators of thromboxane synthesis
Aspirin is a specific inhibitor of the cyclo-oxygenases, COX1 and COX2, which convert
arachidonic acid into prostaglandin H2, from which prostanoids including TxA2 are formed.
It acts by acetylating the active site of the enzyme, leading to irreversible inhibition.
BAPTA (1,2bis[2-aminophenoxy]ethaneN,N,N',N'-tetraacetic acid) is a Ca2+ chelator, not
unlike EGTA or EDTA, which blocks the Ca2+ rise that would otherwise occur as Ca2+ is
mobilised from internal stores in response to collagen. (BAPTA itself is not membrane-
permeant, so instead we use the acetoxymethyl ester, BAPTA-AM, which can readily pass
through membranes. Intracellular esterases hydrolyse the AM group, trapping the active
chelator in the cytoplasm. This trick is quite often used to load membrane-impermeant
organic anions into cells.)
DMSO (dimethylsulphoxide) is the solvent used for all inhibitors. The final DMSO
concentration in platelet suspensions with inhibitors is 0.1% (v/v) or 14 mM; after water and
NaCl, it is the most abundant species in the pre-incubations. Therefore it is imperative that
we add DMSO to the untreated platelet suspension as well.
Table 1. Concentration of TxA2 synthase modulators
Concentration in platelet suspension

MODULATOR
provided
None 0.1% (v/v) DMSO
Aspirin 100 µM, with 0.1% (v/v) DMSO
BAPTA-AM 20 µM, with 0.1% (v/v) DMSO
Technique
Thromboxane production assay
The platelets will be incubated with activator at 37 °C. The platelets will be centrifuged and
the supernatant removed and diluted so that the [TxB2] is within range for measurement by
ELISA.
96-well plate competitive ELISA
Each well is coated with anti-rabbit Ig antibody (raised in goat). The principle of the assay is
to mix TxB2 from your samples with a fixed amount of TxB2 to which alkaline phosphatase
has been conjugated, in the presence of an anti-TxB2 antibody (a rabbit polyclonal). The two
forms (unconjugated and phosphatase-conjugated) of TxB2 compete for binding to the

antibody, and the whole complex is captured on the base of the well by the immobilised
anti-rabbit Ig.
After washing off unbound proteins, a chromogenic substrate is added, which is converted
by the phosphatase to a yellow-coloured product that can be detected
spectrophotometrically.
The colour is then measured in a plate-reader at 405 nm. To compensate for scattering in
the sample, the plate reader will also take a measurement at 595 nm (blue absorption)
where the product will not absorb, but any scattering by particles in the sample or
scratches or stains at the bottom of the plate are taken into account and subtracted from
the reading at 405 nm. This is done automatically and you do not need to worry about this
– you will receive the corrected absorbance readings. A standard curve will also be
constructed by adding known quantities of TxB2 to the conjugated TxB2 and developing
these samples in the same plate in which your samples are. You will use these data to draw
a standard curve and use it to determine the TxB2 concentrations in your samples during
the data analysis session the week later.

Safety Notes
Human blood products

Platelets must be regarded as potentially pathogenic, though these platelets have been
screened for viral contamination.
Thus the platelet suspensions present a relatively minor risk, but it is essential that you
wear lab coats and gloves throughout, and perform all manipulations over the
trays provided. This includes sampling of platelet supernatants, dilution, pipetting etc.
Gloves should be discarded into the (floor) bins provided, and tips, tubes etc into the
plastic- lined waste buckets on the benches.

Week 7 – Cell Signaling: The production of Thromboxane by Human

Platelets – Experimental
Platelet Stimulation with Collagens (synthetic or native):
Each group of students will test one ligand only (i.e. native collagen fibre preparation or
synthetic collagen peptide or thrombin or ionomycin) to produce a 7-point concentration-
response curve, as well as examining the effects of aspirin and BAPTA-AM on stimulation by
that ligand.
The assays are performed by incubating platelets with the activating ligand for a defined
period of time, stopping the reaction, centrifuging the platelets, removing a supernatant
sample and then diluting it to follow the synthesis of thromboxanes, measured as the
amount of stable metabolite, TxB2, described above.
Using the data generated in this experiment, during the data analysis session next week, we
will produce a standard curve using authentic standards from the ELISA kit, prepare pooled
dose–response curves for each ligand, compare the relative potencies of different ligands
and discover whether TxA2 synthesis requires an elevation in intracellular calcium. We will
also study the effect of aspirin in this process.
Materials
You should have on your own bench (or obtain from the demonstrator as needed):
1. Platelet suspension at 2 x 108 platelets/ml
Platelets have already been isolated from plasma by centrifugation and resuspended in an
appropriate isotonic buffer (PB). Ensure that you have 2 ml of untreated platelets (PT)
and 0.5 ml each of aspirin (ASP) – and BAPTA (BAP) – treated platelets.
2. Platelet Termination Reagent (TR)
This contains EDTA and indomethacin, which very quickly stops the synthesis of TxA2,
acting like aspirin to block COX1 and COX2. The reagent will be ready on your bench.
3. Activators
You are provided with 60 µl of a stock solution of activator ligand in buffer (HEPES-buffered
isotonic saline + 0.5 mM EGTA), ready to use at the highest concentration condition A7).
A1 is a control containing the buffer alone. You are provided with 20 ml of this buffer,
labeled PB (platelet buffer). The lower concentrations of activator, A2 to A6, are made by
diluting a small volume of the higher concentrations – see the dilution scheme below. For
A2-A5, you need to prepare sufficient for 2 x 10 µl aliquots for the incubations. A6 is also
used for the BAPTA- and aspirin-treated platelets.
Details of the activators to be used will be provided by the demonstrators. Make a
note which activator you are using and its original concentration:
4. Snap-top microfuge tubes for incubations and dilutions
These are available on the bench or from the demonstrators.
5. ELISA reagents
Each group will use 22 wells of a 96-well plate, 11 wells in each of two rows. The
last position in each row will be used for TxB2 concentration standards, added by the
demonstrators when the rest of the plate has been filled. Thus each plate will be shared
between four groups.
Make sure to write your name in the form next to the plates to indicate which
rows contain your samples!

Cell Signalling 2: Experimental Procedure

1. Dilution of the ligand (activator)
Your first task is to dilute the ligand to produce 6 concentrations (the 7th is the negative
control, with no ligand) that should cover its active range. The final concentrations in both
stocks and the assay will depend on which ligand you are using, so make sure you
understand how this will be done, and which ligand you are testing.
Begin by labelling snap-top Eppendorf tubes for the ligand dilutions i.e. A1-A7 and add to
these tubes PB buffer as shown in Table 2 (below). Starting with the activator stock
solution, dilute the activator by adding the indicated volume of the sample into the next
tube. Mix the new sample well with whirlimixer. Using a new tip, dilute this new solution
further, until all samples are prepared (i.e. A7 to A2). Condition A1 is your negative
control with no activator, just PB buffer.
Tick the box when done:

Table 2. Dilution of activators
Stock Add to PB [Stock] [Assay]
Condition Final volume
volume volume µg/ml µg/ml
A7 60 µl A7 0 60 µl (less 30 µl) 1000 100
A6 30 µl A7 70 µl PB 100 µl (less 30 µl) 300 30
A2 30 µl A3 70 µl PB 100 µl 3 0.3
A1 (use PB as is) 0 70 µl PB 70 µl 0 0
These same dilutions apply to all activators. It is important that, as you prepare each
dilution, the tube is thoroughly whirlimixed to ensure the correct amount of activator is
transferred to the next dilution.
Done:
2. Dispensing ligand and setting up assay tubes

You will use 1.5 ml snap-cap Eppendorf tubes for your assays. Set up the assay tubes in
duplicate, labelling them 1a-11a and 1b-11b (22 tubes in total), with the conditions as in
Table 3.
Tubes 1-7 will include 10 µl from one of A1-A7. Tubes 8 and 9 will have 10 µl of A1 and A6
but the platelets used will contain aspirin; tubes 10 and 11 will also have 10 µl of A1 and A6
but the platelets used will contain BAPTA-AM.
Done:
You will need to label a second set of tubes (D1a-D11a and D1b-D11b) which you will use
to dilute the supernatant from the experiment before pipetting the sample onto the ELISA
plate.
You should thus have a total of 44 labelled tubes. You could use a different colour marker
for the different sets, to ensure they do not get mixed.
Done:

Take the diluted activators (A1-A7) and dispense them as appropriate in 10 µl aliquots to
your 22 assay tubes (Table 3) and transfer the rack of tubes to the heat block. To ensure you
can easily lift the tubes from the block during the experiment, place them staggered in the
block, leaving an empty place between the tubes.
Done:
Table 3. Assay conditions for tubes Ia-IIa and Ib-11b.
1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a
Tube IDs
1b 2b 3b 4b 5b 6b 7b 8b 9b 10b 11b
Agonist A1 A2 A3 A4 A5 A6 A7 A1 A6 A1 A6
dilution 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
Do not add the platelets into the tubes yet!!!
3. Pre-incubation
You should have platelets in your rack, ready for use, in total 2 ml of untreated platelets (at
2 x 108 platelets/ml, containing 0.1% DMSO) and 0.5 ml platelets to which modulators
(aspirin or BAPTA, with 0.1% DMSO as solvent) have already been added. The modulators
require some time to equilibrate with their sites of action, so the platelet suspension plus
modulators have been incubated at room temperature for at least 25 min before you
receive them.
Pre-incubate all the platelet tubes (untreated, aspirin- and BAPTA- treated) for 5 min in the
37 °C heat block before use. Do not mix the platelets and activators yet!
Done:
4. Incubation with activator
The assays will be started by transferring appropriate 90 µl of platelets (untreated, aspirin-
or BAPTA-treated) into the pre-warmed tubes (1-11) in the heat block containing the
activator (10 µl) at seven concentrations, each in duplicate, as per table 4 below. For all
of the incubations, after 10 min exactly, the reaction will be stopped by the addition of
termination reagent (TR).
It is important to time these 10 min incubations with activator precisely so that the results
between the different groups will be comparable. Timings are shown in Table 5 below. In
each case, 90 µl platelets will be added to the tubes. All assays will be performed in
duplicate. Working in pairs, one student should call the times and the other one do all the
pipetting. The better organized you are, the more punctual you can be with the timings and
the better your duplicate samples will match each other. Make sure tips are close by, you
know the order of the tubes, the waste container is close to your pipette hand etc. If you
have a problem in the middle of the experiment, do not panic, but make a note when the
next experiment starts and simply shift the schedule to ensure all samples are in the
experiment for 10 minutes exactly.
Before starting the experiment, invert the three platelet tubes once, to ensure you have an
even suspension of platelets, as they could have settled towards the bottom of the tube.

Table 4.
1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a
Tube IDs
1b 2b 3b 4b 5b 6b 7b 8b 9b 10b 11b
Platelet PT PT PT PT PT PT PT ASP ASP BAP BAP
condition 90 µl 90 µl 90 µl 90 µl 90 µl 90 µl 90 µl 90 µl 90 µl 90 µl 90 µl
Table 5. TIMINGS of Additions
Start Incubation.
Stop Incubation.
Add 90 µl platelets to Tick box
Platelets Tube ID Add 100 µl
duplicate tubes Mark the box
termination reagent
containing 10 µl ligand when started
1a, 1b 0.00 min, 0.15 min 10.00 min, 10.15 min 1a 1b
Untreated 3a, 3b 1.00 min, 1.15 min 11.00 min, 11.15 min 3a 3b
platelets (PT) 4a, 4b 1.30 min, 1.45 min 11.30 min, 11.45 min 4a 4b
7a, 7b 3.00 min, 3.15min 13.00 min, 13.15 min 7a 7b
Aspirin-treated 8a, 8b 3.30 min, 3.45 min 13.30 min, 13.45 min 8a 8b
(ASP) 9a, 9b 4.00 min, 4.15 min 14.00 min, 14.15 min 9a 9b
BAPTA-treated 10a, 10b 4.30 min, 4.45 min 14.30 min, 14.45 min 10a 10b
(BAP) 11a, 11b 5.00 min, 5.15 min 15.00 min, 15.15 min 11a 11b
Start the assay by adding 90 µl of control platelet suspension (containing 0.1% DMSO) to
your assay tube labelled 1a and then15 sec later to tube 1b,15 sec later to 2a and so on.
Whirlimix or flick the tube gently and return them to the heat block. Change the pipette
tip before starting assays with aspirin-treated platelets and again before starting assays with
BAPTA-treated platelets. Try to keep the timing between duplicates constant.
Done:
After exactly 10 min incubation, add 100 µl of termination reagent (TR), cap and
whirlimix, maintaining the timing intervals as in Table 5. Transfer the stopped tubes to ice.
Done:
When all the incubations are complete, microfuge the tubes for 1 min at full speed. After
centrifuging, handle the tubes gently to avoid disturbing the platelet pellet, and carefully
remove a 20 µl sample of the supernatant (avoiding the pellet) and transfer it to the
second set of labelled tubes (D1-D11, in duplicate). Add 580 µl of Assay buffer (from the
classroom staff) to each of these dilution tubes. Cap and whirlimix the tubes well.
Done:

5. ELISA
To your two rows of the 96-well ELISA plate, add 100 µl of sample from the dilution tubes,
D1-D11, in duplicate (see Table 6). It is important that exactly the same volume is added to
each well, and therefore it is advisable that only one person does the pipetting and uses the
same pipette without adjusting the volumes, to minimize problems with variable calibration
between pipettors. Make note of the plate number and the rows you are using and write
your names and the agonist you used on the sheet by the ELISA plates.
Done:
A range of seven standards (10,000 to 1 pg TxB2/ml) is added to the 12th column of the plate by
the classroom staff. The last well, H12, is a background or “zero” well without TxB2.
Once all the samples are on the plate 50 µl of alkaline phosphatase-conjugated TxB2 (blue
solution) will be added to each well, followed by 50 µl of anti-TxB2 antibody solution (yellow).
The classroom staff will complete this step, using a multichannel pipettor.
Table 6: ELISA layout.

Each group uses two rows, columns 1-11, inside dark lines.
1 2 3 4 5 6 7 8 9 10 11 12
A D1a D2a D3a D4a D5a D6a D7a D8a D9a D10a D11a Std 1
B D1b D2b D3b D4b D5b D6b D7b D8b D9b D10b D11b Std 2
C Same for Second group Std 3
D Std 4
E Same for Third group Std 5
F Std 6
G Same for Fourth group Std 7
H zero
My plate number: My rows:

Once the ELISA plate is full, it is incubated for 2 hours at room temperature on the shaker.
**Break for lunch (plan for discussion at approx. 2pm)**

Wash each well three times with 400 µl washing buffer (Tris-buffered saline plus detergents,
provided by the kit manufacturer). Volunteers from the class will do this with guidance from
the classroom staff using multichannel pipettes.
Add 200 µl of the phosphatase substrate (provided by the kit manufacturer) to each well.
Incubate 45 min, room temperature, without shaking.
Finally, add 50 µl of tri-sodium phosphate to terminate the reaction. (This reagent is
provided by the kit manufacturer.)
Read absorbance at 405 nm (ABS405) using the plate-reader. This is usually done by
demonstrators after the practical, but the use of plate reader will be demonstrated during
the class.

Data Handling and Interpretation – Week 2
Drawing the standard curve

Use the absorbance results from the standards on the plate your samples were to plot a
standard curve for the TxB2 assay on four-cycle semilog paper with standard
concentration on the X-axis (logarithmic) and absorbance values (ABS405) on the Y-axis. The
standard wells contained: 0, 1, 10, 30, 100, 300, 1000 and 10000 pg/ml of TxB2.
For this assay the standard curve is not linear but takes the form of an inverse
rectangular hyperbola. On a logarithmic scale, this appears as an upside-down sigmoidal
curve (as in the computer derived example below). At high and low TxB2 concentrations
the curve tends towards an asymptote. This means that the assay is most accurate for those
concentrations of TxB2 in the middle of the standard curve, i.e., 10 – 1000 pg/ml.
Remember that there is no zero on logarithmic scale, so place the 0 ug TxB2 standard outside
the graph, assuming it is in infinity.
If data analysis is done computationally, the standard concentration data can be fitted to a four
parameter logistic equation (see below). Once the parameters are known, it is possible to
calculate TxB2 levels from individual absorbance values.
Variables: ABS405 = Measured absorbance value (dependent variable)

TxB2 = Concentration of TxB2 standard
Parameters: Max = ABS405 when [TxB2] = ¥

Min = ABS405 when [TxB2] = 0
T50 = [TxB2] which gives an ABS405 value of (Max + Min)/2
nH = Hill coefficient (relates to steepness of slope)

Determining the amount of TxB2 in your samples

Standard curve can be used to determine the amount of TxB2 in each of your sample
wells. Use the table on the next page to first calculate average absorbance for your
duplicate samples (D1-D11) and use this average value to read the corresponding TxB2
concentration from the standard curve you have drawn.
As the curve becomes flat at both ends, the reliable part of the curve is in the middle,
where there is a significant slope and differences in the absorbance can be correlated with
differences in TxB2 concentrations.
Dose response curve

To evaluate the potency of your activator, you need to construct a concentration-response
curve from your data and estimate the EC50 value (concentration at which the ligand
induces half maximal, 50%, response in the cells) of the activating ligand that you used. This
is done by plotting the amount of TxB2 as a function of agonist concentration using four-
cycle semi-log paper and determining the agonist concentration from the point in the X-
axis where the effect is half of the maximum. Enter your EC50 value into the table at the
front of the class room, and an average value will be determined from all the
experiments over the week of practicals.
Converting the concentrations to biologically meaningful values

The values obtained from the standard curve are expressed as pg/ml and so is your
EC50 value. But in order for this to be useful and comparable with data obtained with
different amounts of platelets (or cell types), this value needs to be converted to a scale
that reflects the experimental conditions, in this case the number of cells used in the
experiments. This ensures that data done on different dates, using different concentrations
of cells can be compared directly.
Taking into account all the steps in the experiment (various sample volumes, additions of
reagents and dilutions), you need to derive a conversion factor to change the TxB2
concentrations (pg/ml) to pg of TxB2 / 108 platelets. There is only one conversion factor to
be determined as all the samples were treated the same way. Check from the
demonstrators that the conversion factor is correct before doing the conversions in the
table below.

Cell Signalling 2 – Results and Questions

Name: Day & Date: Activator tested:
TxB2 Stds (pg/ml) 0 1 10 30 100 300 1000 10000

ABS405
Samples D1 D2 D3 D4 D5 D6 D7
ABS405 (a)
ABS405 (b)
Mean ABS405
[TxB2] (pg/ml)
TxB2 (pg/108 platelets)

Samples Aspirin samples: D8 D9 BAPTA samples: D10 D11
ABS405 (1)
ABS405 (2)
Mean ABS405
[TxB2] (pg/ml)
TxB2 (pg/108 platelets)
What is the EC50 of the ligand? (Assume the maximum dose that you have
used causes maximal TxB2 production, in the case your curve does not
reach plateau.)
Compare the TxB2 production of the aspirin- and BAPTA-treated samples with
corresponding non-treated platelets. Plot this as a bar chart showing the data for all
relevant samples. What are the effects of aspirin and BAPTA?

Easter Weeks 1 & 2: Protein targeting and “global” gene regulation in bacteria NST 2018-19
Protein targeting and “global” gene regulation in bacteria.

Background to the practical
Bacteria are faced with identical protein targeting problems to those of eukaryotes i.e. how
to direct proteins to cellular locations appropriate to their biological functions. Bacteria can
be classified into Gram-positive and Gram-negative bacteria based on their reaction to
staining with particular dyes. Gram-negative bacteria have a more complex envelope
structure than Gram-positive bacteria, in that the former have two membranes – the
cytoplasmic (inner) membrane (as in Gram-positives) and an outer membrane. The region
between the inner and outer membranes is the periplasm. Consequently, proteins can be
targeted to one of five cellular locations in a Gram-negative bacterium: retained in the
cytoplasm, inserted into the inner membrane, exported to the periplasm, inserted into the
outer membrane or completely secreted to the environment. There are transposons
(mobile genetic elements or ‘jumping genes’) that have been engineered for use specifically
in tagging genes encoding proteins that are targeted to the inner membrane or beyond i.e.
for identifying and tagging genes encoding non-cytoplasmic proteins. You will exploit one
such transposon in this practical.
Another problem faced by bacteria is the co-ordinate control of gene expression. The
simultaneous activation or repression of multiple genes is often essential for bacterial
survival in response to changing environmental conditions e.g. during temperature shifts, pH
shifts, osmolarity changes etc. when moving between niches. It has been long known that
bacteria co-ordinate expression of multiple genes via operonic organisation of contiguous
genes. However, in addition to operons, bacteria have higher order gene control systems
called regulons in which many, genetically-unlinked genes can be co-ordinately switched on
or off by virtue of common regulator proteins acting as activators or repressors of the
genes in the regulon. Mutants defective in such activators or repressors are often
pleiotropic in that they display multiple phenotypes, and occasionally in apparently unrelated
physiological processes. So, one simple way of trying to define “global” regulation mutants is
to search for mutants that simultaneously display two or more phenotypes due to a single
mutation.
The aims of this practical are to investigate aspects of both the protein targeting and
“global” regulation features of bacteria. Both will be approached by the use of transposons
as random mutagenic agents and as agents for the enrichment of particular types of mutants.
A. Protein targeting and use of TnblaM positive selection.

One engineered transposon that is useful for bacterial protein targeting studies is TnblaM.
This transposon carries a gene encoding resistance to the antibiotic, spectinomycin, but,
more importantly, it also carries the “mature” (hence blaM) coding sequence of b-lactamase
– an enzyme capable of degrading b-lactam antibiotics, such as ampicillin. The blaM gene in
this transposon lies at the extreme left hand end of the mobile element but it has no
promoter, ribosome binding site or 5’ end of the blaM gene (encoding the signal
sequence of the b-lactamase enzyme); all of these sequences have been deleted.
When a copy of TnblaM hops into a gene it may, under specific conditions, generate a
hybrid gene that encodes a protein comprising an N-terminal part of the protein encoded
by the target gene fused to b-lactamase. If the target gene encodes a protein that is
normally targeted into or across the membrane then the b-lactamase moiety may be
directed to the periplasm. b-lactam antibiotics such as ampicillin kill bacterial cells by
inhibiting peptidoglycan synthesis. Peptidoglycan is the complex molecule that determines

bacterial shape and confers structural rigidity and it is found between the inner and outer
membranes in Gram-negative bacteria. Inhibition of peptidoglycan synthesis leads eventually
to cell lysis. For a b-lactamase enzyme to inactivate ampicillin it has to be exported to the
periplasm so that it can meet and inactivate the antibiotic before it can exert its lethal
effects. So, strains carrying exported Bla+ fusion proteins will have the ability to grow in the
presence of ampicillin, but only if the fusion proteins have access to the periplasm.
Consequently, only a very specific subset of TnblaM fusions in specific genes will confer the
ability to grow in the presence of ampicillin. Of course, it is even more likely that a random
TnblaM insertion event will not produce an in-frame transcriptional or translation fusion.
A key feature of transposons is that they are capable of “hopping” randomly in a genome.
However, on occasion, a transposon (or a copy of it) from one site may hop to another,
thereby inactivating another gene. Such “secondary” hops with TnblaM may or may not
generate a Bla+ fusion. Clearly, the phenotypes induced by secondary hops will depend on
the ultimate target site of insertion.
B. “Global” regulatory mutations in bacteria.

Serratia strain ATCC39006 is a Gram-negative bacterium that makes a red pigmented
antibiotic (prodigiosin). This strain is also able to make a b-lactam antibiotic (a carbapenem;
1-carbapen-2-em-3-carboxylic acid). Finally, this strain also makes a freely diffusible signal
molecule that can be detected by its effect in another pigment bioassay. In this assay, the
signal molecule from Serratia can be detected by its ability to switch on production of a
violet pigment (violacein) in a particular white mutant of the bacterium, Chromobacterium
violaceum (carrying a mutation in a gene required for production of its own signalling
molecule).
The aim of this part of the practical is to isolate Serratia mutants (defective in any of the
above phenotypes) and then consider the possible molecular bases of such mutants.
C. Environmental bacteria that make signalling molecules or antibiotics.

Bacteria isolated from local sites such as river, soils and ponds may have the ability to make
N-acyl homoserine lactones and antibiotics. You should pick a random selection of the
colonies provided (say 20-50) and replicate them into Serratia and Chromobacterium signalling
molecule bioassay plates to screen for environmental isolates that make detectable quorum
sensing molecules (or antibiotics that kill the indicator strain).
References:
• Broome-Smith, J.K et al., (1990) b-lactamase as a probe of membrane protein assembly and
protein export. Molecular Microbiology, 4, 1637-1644.
• Tadayyon, M and Broome-Smith, J.K. (1992) TnblaM – a transposon for directly tagging genes
encoding cell envelope and secreted proteins. Gene, 111, 21-26.
• McClean, K.H. et al., (1997) Quorum sensing and Chromobacterium violaceum: exploitation of
violaceum production and inhibition for the detection of N-acylhomoserine lactones.
Microbiology, 143, 3703-3711.
Background reviews for general interest:

• Coulthurst, S et al., (2005) Regulation and biosynthesis of carbapenem antibiotics in bacteria.
Nature Reviews Microbiology, 3, 295-306.
• Williamson, NR et al., (2006) The biosynthesis and regulation of bacterial prodiginines. Nature
Reviews Microbiology, 4, 887-899.

Day 1 – Experimental Procedure

Note: Safety and aseptic technique
In experiments with bacteria, the possibility of contamination is high and such contamination
with other bacteria will generate results that are of little value. You must pay particular
attention to aseptic technique to avoid contamination. This will be demonstrated to you. In
addition, as you are working with microbes, it is essential that you pay attention to good
microbiological practice. You must wear a buttoned lab coat at all times and it is
essential that you wash your hands – with soap and water – before leaving the
laboratory.
All contaminated materials must be disposed of appropriately in the plastic bins provided on
your bench – for sterilisation by autoclave.
You should also pay attention to the fact that there will be Bunsen burners used in this lab
and so you should be very careful that you do not lean over the flame. Of course, this is
particularly important if you have long hair!
A. Secondary transposition, “high hopping” and tagging targeted

proteins
You will be given an overnight culture of a Serratia strain Cam1. This strain carries a TnblaM
insertion in an unknown genomic location. However, this insertion is in a “non-
fusion” site i.e. it does not generate a Bla+ fusion as determined by the isolated
colony test of Tadayyon and Broome-Smith (1992) or Broome-Smith et al.,
(1990). Nevertheless, it does give rise to some isolated ampicillin resistant (Apr) colonies if
plated (patched) at high cell density. It is thought that this insertion is therefore an example
of a “high hopper” that can produce Apr fusions through secondary hops.
1. You should set up sterile tubes for making serial dilutions. Add 900 µL of sterile diluent
to each then take the bacterial culture and make serial dilutions (1 in10; 100 µL: 900
µL) in the sterile diluent (using sterile tips and pipettes). Make sure you mix each tube
thoroughly (e.g. a few seconds on a whirlimixer) before sampling it. Make these serial
dilutions from 10-1 down to 10-7. It is essential that you take care with this
aseptic technique, as contamination will almost certainly prevent you from
getting meaningful data in this experiment.
2. Take the nutrient agar plates, and the nutrient agar plates containing various
concentrations of ampicillin. Make sure you mark them with your initials, group number
and day on the back of the plate (not on the lid!). On the back of the plate draw out
quarters and mark them first with the corresponding dilution that you will plate out.
For the nutrient agar “control” plate (with no ampicillin) you should plate out a 20 µL
sample (in a single spot) of each of the10- 4,10- 5,10- 6 and 10- 7 dilutions only.
3. For the nutrient agar plates containing ampicillin, take one plate each of each ampicillin
concentration and mark them with your initials, group number and day. Again mark
quarters on the plate, but this time only for dilutions, ‘Neat,’ (undiluted), 10- 1, 10- 2 and
10- 3. Again, plate out a 20 µL spot for each dilution of the bacterial culture.

4. When the spots have dried into the agar (taking care to avoid the spots running
together) the plates will be incubated at 30 °C for 2-3 days, and then stored until
examination next week.
B. Mutants, pleiotropy and “global” gene regulation in bacteria

You will be provided with colonies of Serratia strain ATCC39006 that have survived a
mutagenesis programme and so some of these colonies will be of bacteria carrying random
mutations.
1. Examine the plates carefully to see if you can identify any affected in red pigment
(prodigiosin) production. Any colonies affected in any way (increased or decreased) in
pigment production are, by definition, mutant. Quantify the frequency of pigment
mutants in the population.
2. Using sterile toothpicks, pick a selection of these mutants, plus a selection of colonies
that still make the pigment at wild type levels (around 200 colonies in total is preferred)
and stab inoculate each into the carbapenem assay plate then stab inoculate into the
Chromobacterium signal molecule assay plate (this will be demonstrated to you). Also,
make sure you have labelled and numbered your plates appropriately. These plates will
then be incubated at 30 °C for a few days and then stored prior to inspection in the
next lab.
C. Environmental bacteria that make signalling molecules or

antibiotics.
You will be provided with a series of agar plates containing bacterial colonies that have
grown from local environmental samples, such as soil, pond water or river water. You
should screen a selection of the colonies (say, around 20-30) to score for the ability to
make quorum sensing molecules (using the Chromobacterium and Serratia mutants that can
detect the presence of such molecules and then switch on pigment production – pigment
bioassays). Also consider the possibility that environmental isolates may make antibiotics
that inhibit or kill the indicator strains.

Day 2 – Experimental Procedure
A. Secondary transposition, “high hopping” and tagging targeted

proteins.
1. Examine the nutrient agar plates and the ampicillin plates. Count the colonies and
calculate the number of viable cells mL-1 in the original culture that grew on the
corresponding plates.
2. Plot a simple graph (log10 survivors against concentration of ampicillin). This may be
easier as a class exercise, depending on the results obtained.
Answer the questions on the blue sheets.
B. Mutants, pleiotropy and “global” gene regulation in bacteria

1. Examine the Serratia plates from last week. Record the frequencies and phenotypes
observed with regard to pigment, carbapenem and signal molecule production. Pay
particular attention to the possibility that a mutant may have more than one phenotype
(pleiotropic). (This may be easier as a class exercise as larger numbers will have been
sampled).
Answer the questions on the blue forms.
C. Environmental bacteria that make signalling molecules or

antibiotics.
Examine your bioassay plates detecting production of quorum sensing molecules and
antibiotics.
Answer the questions on the blue pages.

Day 2 – Questions:
1. Secondary transposition and tagging targeted proteins.
a) What molecular events can you envisage that could give rise to Apr cells in the original
Cam1 culture?
b) From the graphical representation of your results, what is the explanation for the
changing pattern of ampicillin resistant survivors versus ampicillin concentration?
c) How might you explain colony formation at very high concentrations of ampicillin?
2. Mutants, pleiotropy and “global” gene regulation in bacteria.
a) Why did you have to replicate the colonies from the carbapenem assay first, before
replicating to the Chromobacterium assay?
b) Are there any differences in frequency of the three phenotypes being assayed?
c) If so, how do you explain such differences?
d) Are there any mutants with two or more phenotypes? If so, what are their phenotypes
and frequencies?
e) What are the possible molecular bases of the mutants described in d)?
3. Environmental bacteria that make signalling molecules or antibiotics.
a) Did any colonies make putative signalling (quorum sensing) molecules?
b) If so, were these detected in one, or both, bioassays? How do you interpret these
results?
c) Did any isolates show antibiotic production activity?
d) Are any isolates positive for both signalling molecule production and antibiotic activity? If
so, how might you explain your results?
  
  
  
  
  



  

  
  
  
  
  
Periodic Table of the Elements
IA VIIIA
1 +1 2 -
-1
H He
Hydrogen Helium
1.0079 IIA IIIA IVA VA VIA VIIA 4.0026
3 +1 4 +2 5 +3 6 +2 7 -3 8 -1 9 -1 10 -
+4 +2 -2
Li Be B C N +3 O F Ne
Lithium Beryllium Boron Carbon Nitrogen+4 Oxygen Fluorine Neon
6.941 9.0122 10.811 12.011 14.007 +5 15.999 18.998 20.180
11 +1 12 +2 13 +3 14 +4 15 -3 16 -2 17 -1 18 -
+3 +2 +1
Na Mg Al Si P +5 S +4 Cl +3 Ar
Sodium Magnesium Aluminium Silicon Phosphorus Sulfur +6 Chlorine +5 Argon
22.990 24.305 IIIB IVB VB VIB VIIB VIIB VIIB VIII IB IIB 26.982 28.086 30.974 32.066 35.453 +7 39.948
19 +1 20 +2 21 +3 22 +3 23 +2 24 +2 25 +2 26 +2 27 +2 28 +2 29 +1 30 +2 31 +3 32 +2 33 -3 34 -2 35 -1 36 +2
+4 +3 +3 +3 +4 +3 +4 +1
K Ca Sc Ti V+4 +6Cr +4
Mn +3 Fe +3
+6 Co +3 Ni Cu +2
+3 Zn Ga Ge As +5 Se +6 Br +3 Kr
Potassium Calcium Scandium Titanium Vanadium +5 Cromium +5
Manganese+6 Iron Cobalt Nickel Copper Zinc Gallium Germanium Arsenic Selenium Bromine +5 Krypton
39.098 40.078 44.956 47.897 50.942 51.996 54.938 +7 55.847 58.933 58.693 63.546 65.39 69.723 72.61 74.922 78.96 79.904 83.80
37 +1 38 +2 39 +3 40 +4 41 +3 42 +2 43 +2 44 +2 45 +2 46 +2 47 +1 48 +2 49 +1 50 +2 51 -3 52 -2 53 -1 54 +2
+5 +3 +4 +3 +4 +3 +4 +1
Rb Sr Y Zr Nb +4Mo Tc +7 Ru +3
+4 Rh +3
+4 Pd +4 Ag +2
+3 Cd In Sn Sb +5 Te +6 I +5 Xe +4
+6
Rubidium Strontium Yttrium Zirconium Niobium Molybdenum+5 Technetium Ruthenium +6 Rhodium +5 Palladium Silver Cadmium Indium Tin Antimony Tellurium Iodine +7 Xenon +8
85.468 87.62 88.906 91.224 92.906 95.94 +6 [98] 101.07 +8 102.91 105.42 107.87 112.41 114.82 118.71 121.75 127.60 126.90 131.29
55 +1 56 +2 72 +4 73 +3 74 +2 75 +2 76 +3 77 +2 78 +2 79 +1 80 +1 81 +1 82 +2 83 +3 84 +2 85 -1 86 +2
57-71 +4 +3 +3 +4 +3 +4 +3 +2 +3 +4 +5 +4 +1
Cs Ba Hf Ta+5 +4W Re +4 Os +6 Ir
+4 +6 Pt Au Hg Tl Pb Bi Po +6 At +3 Rn
Cesium Barium * Hafnium Tantalum Tungsten +5 Rhenium +6 Osmium +8 Iridium +6 Platinum Gold Mercury Thalium Lead Bismuth Polonium Astatine +5 Radon
132.91 137.33 178.49 180.95 183.85 +6 186.21 +7 190.23 192.22 195.08 196.97 200.59 204.38 207.2 208.98 [209] [210] +7 [222]
87 +1 88 +2 104 +4 105 +5 106 107 108 109 110
89-103
Fr Ra Rf Db Sg Bh Hs Mt Uun
Francium Radium ** Rutherfordium Dubnium Seaborgium Bohrium Hassium Meitnerium Ununnilium
[223] [226.03] [261] [262] [263] [264] [269] [268] [271]
57 +3 58 +3 59 +3 60 +3 61 +3 62 +2 63 +2 64 +3 65 +3 66 +3 67 +3 68 +3 69 +3 70 +2 71 +3
+4
* Lanthanide series
La Ce +4 Pr Nd Pm Sm +3 Eu +3 Gd Tb +4 Dy Ho Er Tm Yb +3 Lu
Lanthanum Cerium Praseodymium Neodymium Promethium Samarium Europium Gadolinium Terbium Dysprosium Holmium Erbium Thulium Ytterbium Lutetium
138.91 140.12 140.91 144.24 [145] 150.36 151.97 157.25 158.93 162.50 164.93 167.26 168.93 173.04 174.97
89 +3 90 +4 91 +4 92 +3 93 +3 94 +3 95 +3 96 +3 97 +2 98 +2 99 +2 100 +2 101 +2 102 +2 103 +3
+5 +4 +4 +4 +4 +4 +3 +3 +3 +3 +3 +3
** Actinide series
Ac Th Pa +5 U +5 Np +5 Pu+5 Am +4 Cm +4 Bk Cf Es Fm Md No Lr
Actinium Thorium Protactinium Uranium +6 Neptunium +6 Plutonium +6 Americium +6 Curium Berkelium Californium Einsteinium Fermium Mendelevium Nobelium Lawrencium
[227] 232.04 231.04 238.03 237.05 [244] [243] [247] [247] [251] [252] [257] [258] [259] [262]
Alkali metals Nonmetals C Solid

IVA Group classification
Atomic number 6 +2 Oxidation degree Alkaline earth metals Noble gases Br Liquid
Symbol +4
C Transition metals Lanthanide series H Gas
Carbon Name
Atomic weight 12.011 Poor metals Actinide series Tc Synthetic
é



 



 

 

 


 
 
  
  
 
 
 







 


 






   

   
   


   
   
   


   

   
   
   


   
   
   
   

   

   
   
   


   
   
   
 

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 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  
 
 

 
 

 
  

  
 
 
 

 
  
  


 

  

Genome Sizes
Virus Length, nt Approx. MW, Da Length of
Eukaryotes Approx. MW, Da
Bacteriophage ΦX174 5,380 3.5 x 106 DNA, nt
Bacteriophage Lambda 48,502 3.1 x 107 Anopheles gambiae PEST
2.78×108 1.80×1011
(malaria mosquito)
Human Immunodeficiency Virus 1 9,181 3.1 x 106
Arabidopsis thaliana (flowering plant) 1.15×108 7.47×1010
Rous Sarcoma Virus 9,392 3.2 x 106
Caenorhabditis briggsae
SARS Coronavirus 29,751 1.0 x 107 1.04×108 6.76×1010
(soil-dwelling nematode)
Simian Virus 40 (SV 40) 5,224 3.4 x 106 Caenorhabditis elegans (round worm) 1.21×107 7.86×109
Vaccinia Virus 191,737 1.2 x 108 Ciona intestinalis (ascidian tadpole) 1.16×108 7.54×1010
Variolla (Small pox) Virus 185,578 1.2 x 108 Drosophila melanogaster (fruit fly) 1.37×108 8.90×1010
Gallus gallus (chicken) 1.05×109 6.82×1011
Length of
Bacteria Approx. MW, Da Guillardia theta (chromophyte algae) 5.51×105 3.58×108
DNA, nt
Agrobacterium tumefaciens C58- Homo sapiens (human) 3.15×109 2.07×1012
4.91×106 3.19×109
Cereon Mus musculus (mouse) ~3.00×109 ~1.95×1012
Aquifex aeolicus VF5 1.55×106 1.00×109 Neurospora crassa OR74A
4.30×107 2.80×1010
Bacillus subtilis 168 4.20×106 2.73×109 (filamentous fungus)
Bacillus halodurans 4.20×106 2.73×109 Saccharomyces cerevisiae S288C
1.21×107 7.86×109
(budding yeast)
Bordetella pertussis Tohama I
4.07×106 2.65×109 Schizosaccharomyces pombe
NCTC-13251 1.40×107 9.10×109
(fission yeast)
Chlamydophila pneumoniae CWL029 1.23×106 8.00×108
Pan troglodytes (chimpanzee) 3.02×109 1.96×1012
Deinococcus radiodurans R1 3.28×106 2.13×109
Plasmodium falciparum 3D7
Escherichia coli K12 4.64×106 3.02×109 2.29×107 1.49×1010
(human malaria parasite)
Escherichia coli 0157:H7 EDL933 4.10×10 6
2.66×10 9
Oryza sativa japonica (rice) 4.20×108 2.73×1011
Haemophilus influenzae KW20 1.83×106 1.19×109
Helicobacter pylori 26695 1.66×106 1.08×109 Length of
Archaea Approx. MW, Da
Lactobacillus acidophilus NCFM 1.99×10 6
1.29×109 DNA, nt
Lactococcus lactis IL1403 2.36×106 1.53×109 Halobacterium sp. NRC-1 2.01×106 1.31×109
Mycobacterium leprae TN 3.27×106 2.12×109 Methanosarcina acetivorans C2A 5.75×10 6
3.74×109
Mycobacterium tuberculosis H37Rv 4.45×106 2.89×109 Methanococcus jannaschii DSM2661 1.66×10 6
1.08×109
Mycoplasma genitalium G037 5.80×105 3.77×108 Methanosarcina mazei Go1 4.10×10 6
2.66×109
Mycoplasma pneumoniae M129 8.16×105 5.30×108 Methanobacterium
1.75×106 1.14×109
thermoautotrophicum delta H
Neisseria meningitidis Z2491 2.18×10 6
1.42×109
Pyrococcus horikoshii OT3 1.74×106 1.13×109
Salmonella typhimurium LT2 SGSC1412 4.86×106 3.16×109
Pyrococcus abysii GE5 1.76×106 1.14×109
Staphylococcus aureus MW2 2.82×106 1.83×109
Pyrococcus furiosus DSM3638 1.91×10 6
1.24×109
Streptomyces coelicolor A3(2) 8.67×106 5.64×109
Sulfolobus acidocaldarius DSM639 2.23×10 6
1.45×109
Streptococcus pneumoniae R6 2.04×106 1.33×109
Sulfolobus solfataricus P2 2.99×10 6
1.94×109
Streptococcus pyogenes SF370 (M1) 1.85×106 1.20×109
Thermoplasma acidophilum 1.56×10 6
1.01×109
Pseudomonas aeruginosa 6.26×106 4.07×109
Pseudomonas fluorescens Pt-5 7.07×106 4.60×109
Calculation the number of copies of a template
Thermus thermophilus HB8 2.12×10 6
1.38×109
amount of genome DNA (µg) × Avogadro constant (mol-1)
Vibrio cholerae N16961 4.00×106 2.60×109 Genome copy number =
length of DNA (bp) × 106 × 650
This calculation is based on the assumption that the average weight of a base pair (bp) is 650 Daltons.
Example: Human genome copy number in 1 µg of genome DNA.
1 (µg) × 6.022 × 1023 (mol-1)

Genome copy number = = 2.94 × 105
3.15 × 109 (bp) × 106 × 650
References
1. GOLD™ Genomes online Database,
for more complete data and updates, see http://www.genomesonline.org/.
2. Genomes Atlas Database,
for more complete data and updates, see http://www.cbs.dtu.dk/services/GenomeAtlas/.

 

 
DNA Migration in Agarose and Polyacrylamide Gels Physical Constants of the Nucleoside Triphos-
Recommended agarose gels for electrophoretic separation of DNA fragments.
phates and Related Compounds
Range of Approximate positions of tracking dyes, bp* (see section “Nucleotides & Primers” for ordering information)
Agarose
effective Bromophenol blue Xylene cyanol FF
gel, %
separation, bp TBE buffer TAE buffer TBE buffer TAE buffer MW, λmax*, ε,
Compound
Da (acid form) nm M-1 x cm-1
0.5 2000-50000 750 1150 13000 16700
0.6 1000-20000 540 850 8820 11600 ATP 507 259 15400
0.7 800-12000 410 660 6400 8500 CTP 483 271 9000
0.8 800-10000 320 530 4830 6500
GTP 523 253 13700
0.9 600-10000 260 440 3770 5140
1.0 400-8000 220 370 3030 4160 UTP 484 262 10000
1.2 300-7000 160 275 2070 2890 dATP 491 259 15200
1.5 200-3000 110 190 1300 1840
2.0 100-2000 65 120 710 1040 dCTP 467 271 9300
3.0 25-1000 30 60 300 460 dGTP 507 253 13700
4.0 10-500 18 40 170 260
dTTP 482 267 9600
5.0 10-300 12 27 105 165
* Positions of the tracking dyes can be estimated only approximately because the dye front migrates as ddATP 475 261 15200
fuzzy band. To use the data above, the following guidelines are recommended:
• Agarose should be top quality. The Fermentas TopVision™ LE GQ Agarose (#R0491) was used to prepare
ddCTP 451 271 9300
the gels. ddGTP 491 253 13600
• Only freshly prepared electrophoresis buffers should be used. The buffers were prepared from the stocks
supplied by Fermentas (50X TAE Buffer (#B49) and 10X TBE Buffer (#B52), respectively). ddTTP 466 267 9600
dm6ATP 507 265 15400
Recommended polyacrylamide gels for electrophoretic separation of DNA fragments (1).
dm4CTP 481 274 13600
Polyacrylamide Range of Approximate positions of tracking dyes*
gel (with BIS at effective dm5CTP 481 279 8770
1:20), % (w/v) separation* Bromophenol blue Xylene cyanol FF 240 11900
Denaturing gels Aminoallyl-dUTP 523
290 7800
4.0 100-500 nt 50 nt 230 nt
5.0 70-400 nt 35 nt 130 nt 240 10700
Biotin-11-dUTP 862
6.0 40-300 nt 26 nt 105 nt 289 7100
8.0 30-200 nt 19 nt 75 nt NAD 664 260 18000
10.0 20-100 nt 12 nt 55 nt
15.0 10-50 nt 10 nt 40 nt NADH 665 338** 6200
20.0 5-30 nt 8 nt 28 nt NADP 743 260 18000
30.0 1-10 nt 6 nt 20 nt
NADPH 745 260 18000
Non-denaturing gels
ε molar absorption coefficient (absorbance at λmax for 1M solution at pH 7.0)
3.5 100-1000 bp 100 bp 460 bp * determined at pH 7.0
5.0 80-500 bp 65 bp 260 bp ** determined at pH 10.0
8.0 60-400 bp 45 bp 160 bp
12.0 50-200 bp 20 bp 70 bp Conversion Formula
15.0 25-150 bp 15 bp 60 bp
20.0 5-100 bp 12 bp 45 bp C = A / ε x 10 3
* Positions of the tracking dyes can be estimated only approximately because the dye front migrates as C – mM concentration of compounds
fuzzy band.
A – observed absorbance at λmax (nm)
Tracking dye migration in agarose gels. ε – molar absorption coefficient (M-1 x cm-1)
100 kb Bromphenol blue in TBE buffer
Bromphenol blue in TAE buffer Physical Properties of Some Common Radioisotopes

10 kb Xylene cyanol FF in TBE buffer Radioisotope Half-life Specific activity, MBq/mmol
Xylene cyanol FF in TAE buffer
32
P 14.3 days up to 107
33
P 25.4 days up to 107
1 kb 35
S 87.4 days up to 107
131
I 8.04 days up to 105
125
I 60 days up to 107
100 bp 14
C 5730 years up to 103
3
H 12.43 years up to 106
10 bp Summary of Useful Conversion

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 1 Becquerel (Bq) = 1 disintegration per second = 2.7x10-11 Curies (Ci)
Agarose, % 1 Ci = 3.7x1010 Bq = 37 GBq = 2.22x1012 disintegrations per minute (dpm) 1 mCi = 37
MBq = 2.22x109 dpm
Reference 1 µCi = 37 kBq = 2.22x106 dpm
1. Sambrook, J., et al., Molecular Cloning. A Laboratory 1 GBq = 27 mCi
Manual, Cold Spring Harbor Laboratory, Cold Spring 1 MBq = 27 µCi
Harbor, N.Y., 12.89, 5.42, 2001. 1 kBq = 27 nCi
Common Conversions of Nucleic Acids Common Conversions of Oligonucleotides
Estimation of Ends (3’ or 5’) Concentration Common Conversions of Oligonucleotides
Circular DNA Molecular Weight
pmol ends = pmol DNA x number of cuts x 2 MW = 333 x N
Linear DNA Concentration of Oligonucleotides
pmol ends = pmol DNA x (number of cuts x 2 + 2) C (µM or pmol/µl) = A260 / (0.01 x N)
Phage/Plasmid DNA pmol ends (1 µg) C (ng/ml) = (A260 x MW) / (0.01 x N)
1000 bp DNA 3.04 MW – molecular weight
linear pUC18/19 DNA 1.14 A260 – absorbance at 260 nm
linear pBR322 DNA 0.7 N – number of bases
linear SV40 DNA 0.58
linear ΦX174 DNA 0.56
linear M13mp18/19 DNA 0.42 Melting Temperature of Duplex DNA and Oligo-
λ phage DNA 0.06 nucleotides
For Duplex Oligonucleotide shorter than 25 bp,
Spectrophotometric Conversions “The Wallace Rule” (2)
NA A260 µg/ml mM (in nucleotides) Tm (in °C) = 2(A+T) + 4(C+G), where
dsDNA 1 50 0.15 (A+T) – the sum of the A and T residues in the oligonucleotide and
ssDNA 1 33 0.1 (G+C) – the sum of G and C residues in the oligonucleotide.
ssRNA 1 40 0.12 Presence of m5C in oligonucleotide increases melting temperature of
dsDNA 6.7 335 1 duplex. Presence of m4C or m6A decreases melting temperature (3).
ssDNA 10.0 330 1
For Duplex DNA, <100 bp long (4)
ssRNA 8.3 332 1
Tm (in °C) = 81.5°C+16.6(log10 [Na+])+0.41(%[G+C])-675/n-1.0m
The average MW of a deoxyribonucleotide base = 333 Da
n – the number of bases in the oligonucleotide
The average MW of a ribonucleotide base = 340 Da
The average MW of a deoxyribonucleotide base pair = 650 Da m – the percentage of base-pair mismatches
Molar Conversions Temperature Dependence of the pH of 50 mM

Quantity Tris-HCl Solutions
Phage/Plasmid DNA bp
µg pmol
1 1.52 pH of the 50 mM Tris-HCl solutions
DNA 1000 4°C 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4
0.66 1
1 0.57 25°C 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8
pUC18/19 DNA 2686 37°C 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5
1.77 1
1 0.35
pBR322 DNA 4361
2.88 1 General Physical Constants
1 0.29
SV40 DNA 5243 Name Constant
3.46 1
Avogadro’s Number 6.02214 x 1023 mol-1
1 0.28
ΦX174 DNA 5386 Planck’s Constant 6.62608 x 10-34 J s
3.54 1
Boltzmann Constant 1.38066 x 10-23 J K-1
1 0.21
M13mp18/19 DNA 7250 0.0821 L atm/mol K
4.78 1
Ideal Gas Constant 8.314 J/mol K
1 0.03 6.24 x 104 cm3 torr/mol K
λ phage DNA 48502
32.01 11 Speed of Light 2.9979 x 108 m/s
Atomic Mass Unit 1.66054 x 10-27 kg
DNA/Protein Conversions
1 kb of DNA = 333 amino acid ≅ 37 kDa References
10 kDa protein ≅ 0.27 kb DNA 1. Sambrook, J., et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Labora-
tory, Cold Spring Harbor, N.Y., 2001.
30 kDa protein ≅ 0.81 kb DNA 2. Wallace, R.B., et al., Hybridization of synthetic oligodeoxyribonucleotides to phiX174 DNA:
50 kDa protein ≅ 1.32 kb DNA the effect of single base pair mismatch, Nucleic Acids Res., 6, 3543-3557, 1979.
3. Butkus, V., et al., Synthesis and physical characterization of DNA fragments containing
100 kDa protein ≅ 2.70 kb DNA N4-methylcytosine and 5-methylcytosine, Nucleic Acids Res., 20, 8467-8478, 1987.
4. Sambrook, J., et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Labora-
tory, Cold Spring Harbor, N.Y., 10.2-10.4, 2001.
Temperature Dependence of the pH for Commonly Used Buffers
Buffer system Chemical name pKa/20°C ∆pKa/10°C
MES 4-morpholineethanesulfonic acid 6.15 -0.110
ADA [(carbamoylmethyl)imino]diacetic acid 6.60 -0.110 35
S, (half-life – 84.4 days)
Days
PIPES 1,4-piperazinediethanesulfonic acid 6.80 -0.085 Weeks
0 1 2 3 4 5 6
ACES 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid 6.90 -0.200 0 1.000 0.992 0.984 0.976 0.969 0.961 0.954
1 0.946 0.939 0.931 0.924 0.916 0.909 0.902
BES 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid 7.15 -0.160 2 0.895 0.888 0.881 0.874 0.867 0.860 0.853
MOPS 4-morpholinepropanesulfonic acid 7.20 -0.013 3 0.847 0.840 0.833 0.827 0.820 0.814 0.807
4 0.801 0.795 0.788 0.782 0.776 0.770 0.764
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}- 5 0.758 0.752 0.746 0.740 0.734 0.728 0.722
TES 7.50 -0.200
1-propanesulfonic acid 6 0.717 0.711 0.705 0.700 0.694 0.689 0.683
7 0.678 0.673 0.667 0.662 0.657 0.652 0.646
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 7.55 -0.140 8 0.641 0.636 0.631 0.626 0.621 0.616 0.612
TRICINE N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 8.15 -0.210 9 0.607 0.602 0.597 0.592 0.588 0.583 0.579
10 0.574 0.569 0.565 0.560 0.556 0.552 0.547
TRIS 2-amino-2-hydroxymethylpropane-1,3-diol 8.30 -0.310 11 0.543 0.539 0.534 0.530 0.526 0.522 0.518
BICINE N,N-bis(2-hydroxyethyl)glycine 8.35 -0.180 12 0.514 0.510 0.506 0.502 0.498 0.494 0.490
GLYCYLGLYCINE 2-(2-aminoacetyl)aminoacetic acid 8.40 -0.280
Decay Factors for Calculating the Amount of Radioactivity

32
P, (half-life – 14.3 days) 33
P, (half-life – 25.4 days)
Hours Days
Days Days
0 12 24 36 48 60 72 84 0 1 1 3 4 5 6 7 8 8
0 1.000 0.976 0.953 0.930 0.908 0.886 0.865 0.844 0 1.000 0.973 0.947 0.921 0.897 0.872 0.849 0.826 0.804 0.782
4 0.824 0.804 0.785 0.766 0.748 0.730 0.712 0.695 10 0.761 0.741 0.721 0.701 0.683 0.664 0.646 0.629 0.612 0.595
8 0.679 0.662 0.646 0.631 0.616 0.601 0.587 0.573 20 0.579 0.564 0.549 0.534 0.520 0.506 0.492 0.479 0.466 0.453
12 0.559 0.546 0.533 0.520 0.507 0.495 0.483 0.472 30 0.441 0.429 0.418 0.406 0.395 0.385 0.374 0.364 0.355 0.345
16 0.460 0.449 0.439 0.428 0.418 0.408 0.398 0.389 40 0.336 0.327 0.318 0.309 0.301 0.293 0.285 0.277 0.270 0.263
20 0.379 0.370 0.361 0.353 0.344 0.336 0.328 0.320 50 0.256 0.249 0.242 0.236 0.229 0.223 0.217 0.211 0.205 0.200
24 0.312 0.305 0.298 0.291 0.284 0.277 0.270 0.264 60 0.195 0.189 0.184 0.179 0.174 0.170 0.165 0.161 0.156 0.152
28 0.257 0.251 0.245 0.239 0.234 0.228 0.223 0.217 70 0.148 0.144 0.140 0.136 0.133 0.129 0.126 0.122 0.119 0.116
32 0.212 0.207 0.202 0.197 0.192 0.188 0.183 0.179 80 0.113 0.110 0.107 0.104 0.101 0.098 0.096 0.093 0.091 0.088
36 0.175 0.170 0.166 0.162 0.159 0.155 0.151 0.147 90 0.086 0.084 0.081 0.079 0.077 0.075 0.073 0.071 0.069 0.067
40 0.144 0.140 0.137 0.134 0.131 0.127 0.124 0.121 100 0.065 0.064 0.062 0.060 0.059 0.057 0.055 0.054 0.053 0.051
44 0.119 0.116 0.113 0.110 0.108 0.105 0.102 0.100 110 0.050 0.048 0.047 0.046 0.045 0.043 0.042 0.041 0.040 0.039
48 0.098 0.095 0.093 0.091 0.089 0.086 0.084 0.082 120 0.038 0.037 0.036 0.035 0.034 0.033 0.032 0.031 0.030 0.030
52 0.080 0.078 0.077 0.075 0.073 0.071 0.070 0.068
  

   
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   
   
   
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  

  
Commonly Used Media, Stock Solutions and Buffers
Growth Media (1)
LB Medium, per liter: Final 1X concentration: 100X Denhardt Solution: Final concentration:
Tryptone 10 g 1.0% (w/v) Ficoll 400 10 g 0.02% (w/v)
Yeast extract 5g 0.5% (w/v) Polyvinylpyrrolidone 10 g 0.02% (w/v)
NaCl 10 g 1.0% (w/v) Bovine serum albumin 10 g 0.02% (w/v)
H2O to 1 liter H2O to 500 ml
Adjust pH to 7.0. Filter sterilize and store at -20°C in 25 ml aliquots
Low Salt LB Medium, per liter: Final 1X concentration: 1 M Dithiothreitol (DTT):
Tryptone 10 g 1.0% (w/v) DTT 15.45 g
Yeast extract 5g 0.5% (w/v) H2O to100 ml
NaCl 5g 0.5% (w/v) Store at -20°C
H2O to 1 liter 0.5 M Ethylenediamine Tetraacetic Acid (EDTA) (pH 8.0):
Adjust pH to 7.0. Na2EDTAx2H2O 186.1 g
Terrific Broth Medium, per liter: Final 1X concentration: H2O to 700 ml
Tryptone 12 g 1.2% (w/v) Adjust pH to 8.0 with 10 M NaOH (~50 ml)
Yeast extract 24 g 2.4% (w/v) H2O to 1 liter
Glycerol 4 ml 0.4% (w/v) 10 mg/ml Ethidium Bromide:
H2O to 900 ml Ethidium bromide 0.2 g
Autoclave, cool to 60°C or less before adding 100 ml of filter sterilized 10X TB H2O to 20 ml
phosphate (0.17 M KH2PO4, 0.72 M K2HPO4). Mix well and store at 4°C in dark.
SOB Medium, per liter Final 1X concentration: CAUTION: Ethidium bromide is a mutagen. Wear gloves when working with
Tryptone 20 g 2.0% (w/v) solutions and a mask when weighing the powder.
Yeast extract 5g 0.5% (w/v) 1 M KCl:
NaCl 0.5 g 0.05% (w/v) KCl 74.6 g
250 mM KCl 10 ml 2.5 mM H2O to 1 liter
H2O to 900 ml 1 M MgCl2:
Adjust pH to 7.0 and add H2O to 990 ml. MgCl2x6H2O 20.3 g
Autoclave, cool to room temperature and just prior to use add 10 ml H2O to 100 ml
1 M MgCl2 (sterile solution). 10 mM 1 M MgSO4:
SOC Medium, per liter: MgSO4x7H2O 24.6g
SOB Medium (1 liter) with the addition of 20 ml filter sterilized H2O to 100ml
1 M glucose. 5M NaCl:
M9 Minimal Medium, per liter: Final 1X concentration: NaCl 292.2 g
5X M9 salts 200 ml H2O to 1 liter
Sterile H2O to 1 liter 10 M NaOH:
1 M MgSO4 1 ml 1 mM NaOH 400.0 g
20% glucose 10 ml 0.2% (w/v) H2O to 1 liter
1M CaCl2 0.1 ml 0.1 mM 1 M Tris-HCl [tris(hydroxymethyl)aminomethane]:
5X M9 Salts, per liter: Final 1X concentration: Tris base 121.1 g
Na2HPO4x7H2O 64 g 47.8 mM H2O to 800 ml
KH2PO4 15 g 22 mM Adjust to desired pH with concentrated HCl.
NaCl 2.5 g 8.6 mM Mix and add H2O to 1 liter
NH4Cl 5g 18.7 mM 3 M Sodium Acetate (pH 5.2 or 7.0) (2):
Additives Final 1X concentration: Sodium acetate x 3H2O 408.1 g
Antibiotics (if required): H2O to 800 ml
Ampicillin 50 µg/ml Adjust the pH to 7.0 with acetic acid or adjust the pH to 5.2 with glacial acetic acid.
Chloramphenicol 20 µg/ml H2O to 1 liter
Kanamycin 30 µg/ml
Tetracycline 12 µg/ml Buffers (2)
Galactosides (if required): 10X Stock Phosphate-buffered Saline (PBS), per liter: Final 1X concentration:
X-Gal 20 µg/ml NaCl 80.0 g 137 mM
IPTG 0.1 mM KCl 2.0 g 2.7 mM
Media containing agar or agarose, per liter: Na2HPO4 14.4 g 10 mM
Agar (for plates) 15 g 1.5% (w/v) KH2PO4 2.7 g 2 mM
Agar (for top agar) 7g 0.7% (w/v) H2O to 800 ml
Agarose (for plates) 15 g 1.5% (w/v) Adjust to pH 7.4 with concentrated HCl.
Agarose (for top agarose) 7g 0.7% (w/v) H2O to 1 liter
20X SSC, per liter: Final 1X concentration:
Stock Solutions NaCl 175.3 g 150 mM
10 M Ammonium Acetate: Na3citrate x 2H2O 88.2 g 15 mM
Ammonium acetate 385.4 g H2O to 800 ml
H 2O to 500 ml Adjust pH to 7.0 with 1M HCl
1 M CaCl2: H2O to 1 liter
CaCl2x6H2O 219.1 g
H2O to 1 liter
(continued on next page)
20X SSPE, per liter: Final 1X concentration: Commonly Used Abbreviations
NaCl 175.3 g 150 mM
NaH2PO4xH2O 27.6 g 10 mM
A
Na2EDTA 7.4 g 1 mM
A adenine or adenosine; one-letter code for alanine
H2O to 800 ml
A260 absorbance at 260 nm
Adjust pH to 7.4 with 10 M NaOH
ACES N-(2-acetamido)-2-aminoethanesulfonic acid
H2O to 1 liter Ad-2 adenovirus-2
5X Tris-glycine, Native Electrophoresis Buffer, per liter: Final 1X concentration: ADA N-(2-acetamido)-2-iminodiacetic acid
Tris base 15.1 g 25 mM ADP adenosine 5’-diphosphate
Glycine 72.0 g 192 mM AMP adenosine monophosphate
H2O to 1 liter AMV Avian Myeloblastosis Virus
The pH of diluted solution is pH 8.3. AP alkaline phosphatase
5X Tris-glycine-SDS Electrophoresis Buffer, per liter: Final 1X concentration: APS ammonium persulfate
Tris base 15.1 g 25 mM aRNA antisense RNA
Glycine 72.0 g 192 mM ATP adenosine 5’-triphosphate
SDS 5.0 g 0.1% (w/v) B
H2O to 1 liter BCIP 5-bromo-4-chloro-3-indolyl phosphate
The pH of diluted solution is pH 8.3. BES N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid
5X Tris-tricine-SDS Electrophoresis Buffer, per liter: Final 1X concentration: BGG bovine gamma globulin
Tris base 60.6 g 0.1 M BICINE N,N-bis(2-hydroxyethyl)glycine
Tricine 89.6 g 0.1 M Bio-dNTP biotin-deoxynucleoside triphosphate
SDS 10.0 g 0.1% (w/v) BME β-mercaptoethanol; 2-mercaptoethanol
H2O to 1 liter bp base pair
The pH of diluted solution is pH 8.3. Bq Becquerel
50X TAE (Tris-acetate-EDTA) Electrophoresis Buffer, per liter: Final 1X concentration: BSA bovine serum albumin
Tris base 242 g 40 mM B/W blue/white cloning
Glacial acetic acid 57.1 ml 20 mM C
0.5 M EDTA (pH 8.0) 100 ml 1 mM C cytosine or cytidine; one-letter code for cysteine
CA casamino-acids
H2O to 1 liter
CAPS N-cyclohexyl-3-aminopropanesulfonic acid
The pH of diluted solution is ~8.5
cDNA complementary deoxyribonucleic acid
10X TBE (Tris-borate-EDTA) Electrophoresis Buffer, per liter: Final 1X concentration:
CEU cohesive wnd ligation unit
Tris base 108 g 89 mM
Ci Curie
Boric acid 55 g 89 mM CIAP calf intestinal alkaline phosphatase
0.5 M EDTA (pH 8.0) 40 ml 2 mM CMP cytidine monophosphate
H2O to 1 liter cpm counts per minute
10X TPE (Tris-phosphate-EDTA) Electrophoresis Buffer, per liter: Final 1X concentration: CTP cytidine 5’-triphosphate
Tris base 108 g 89 mM D
Phosphoric acid (85%) 15.5 ml 27 mM Da Dalton
0.5 M EDTA (pH 8.0) 40 ml 2 mM dAMP deoxyadenosine monophosphate
H2O to 1 liter dATP deoxyadenosine triphosphate
TE (Tris-EDTA) Buffer, pH 7.4, 7.6 or 8.0, per liter: Final 1X concentration: dCTP deoxycytidine triphosphate
1 M Tris, pH 7.4, 7.6, 8.0 10 ml 10 mM ddATP dideoxyadenosine triphosphate
0.5 M EDTA (pH 8.0) 2 ml 1 mM ddCTP dideoxycytidine triphosphate
H2O to 1 liter ddGTP dideoxyguanosine triphosphate
ddNTP dideoxynucleoside triphosphate
ddTTP dideoxythymidine triphosphate
DE-81 Whatman® diethylaminoethyl cellulose paper
Expressing Concentrations of Reagents
DEPC diethyl pyrocarbonate
Molarity DIG digoxigenin
number of moles solute dITP deoxyinosine triphosphate
M (mol/l) =
number of liters of solution dGTP deoxyguanosine triphosphate
DMSO dimethyl sulfoxide
Percent composition DNA deoxyribonucleic acid
weight of solute (g) DNase deoxyribonuclease
Concentration (%) = x 100
total weight of solution (g) dNMP deoxyribonucleoside monophosphate
dNTP deoxynucleoside triphosphate
Percent composition (weight/volume) dpm disintegrations per minute
weight of solute (g) ds double-stranded
Concentration (% w/v) = x 100 DTT dithiothreitol
total weight of solution (ml)
dTTP deoxythymidine triphosphate
Percent composition (volume/volume) dUTP deoxyuridine triphosphate
weight of solute (ml) E
Concentration (% v/v) = x 100 EDTA ethylenediaminetetraacetic acid
total weight of solution (ml) EGTA ethylene glycol tetraacetic acid
ELISA enzyme-linked immunosorbent assay
References EMBL European Molecular Biology Laboratory
1. Atlas, R.M., Handbook of Microbiological Media, second edition, Ed. Parks, L.C., CRC
EMSA electrophoretic mobility shift assay
Press, N.Y., 1997.
2. Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Endo endodeoxyribonuclease assay
Harbor Laboratory Press, Cold Spring Harbor, NY, A1.2-A2.12, 2001. endo endonuclease
exo exonuclease
Exo 5’ and 3’- exodeoxyribonuclease assay poly(dT) polydeoxythymidylic acid
F PPi inorganic pyrophosphate
FastAP™ FastAP™ thermosensitive alkaline phosphatase PVDF polyvinylidene fluoride
FDU fast didest unit Q
FOA (5-FOA) 5-fluoroorotic acid QCA quality control assay
G qPCR quantitative polymerase chain reaction
G guanine or guanosine; one-letter code for glycine R
Gal D-galactose RACE Rapid Amplification of cDNA Ends
GMP guanosine monophosphate RCA rolling circle amplification
GMPD glycosylase mediated polymorphism detection RDA random displacement amplification
GUS β-D-glucuronidase RE restriction endonuclease
GTP guanosine 5’-triphosphate REase restriction endonuclease
GQ genetic quality RNA ribonucleic acid
H RNase ribonuclease
HC high concentration R-M restriction-modification
HEPES N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) rRNA ribosomal ribonucleic acid
HRP horseradish peroxidase RT reverse transcriptase
HPLC high-performance liquid chromatography RT-PCR reverse transcription polymerase chain reaction
I RT-qPCR reverse transcription – quantitative polymerase chain reaction
IEF isoelectric focussing S
IPTG isopropyl-β-D-thiogalactopyranoside SAM S-adenosylmethionine
K SAP shrimp alkaline phosphatase
kb kilobase SDA strand displacement amplification
kDa kiloDalton SDS sodium dodecyl sulfate
L siRNA small interfering ribonucleic acid
LAMP loop-mediated isothermal amplification SNP single nucleotide polymorphism
LB Luria Bertani media ss single-stranded
LC low concentration SSC sodium chloride/sodium citrate (buffer)
LE low electroendosmosis SSCP single-strand conformation polymorphism
LM low melting point SSPE sodium chloride/sodium phosphate/EDTA (buffer)
LO labeled oligonucleotide STR short tandem repeat
M T
MCS multiple cloning site T thymine or thymidine; one-letter code for threonine
MES 2-(N-morpholino)ethanesulfonic acid TAE Tris/acetate/EDTA (buffer)
MDA multiple displacement amplification TBE Tris/borate/EDTA (buffer)
M-MuLV Moloney Murine Leukemia Virus TdT terminal deoxynucleotidyl transferase
MOPS 3-(N-morpholino)propanesulfonic acid TE Tris/EDTA (buffer)
mRNA messenger ribonucleic acid TEMED N,N,N’,N’-tetramethylethylenediamine
MW molecular weight TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid
N TLC thin layer chromatography
NAD nicotinamide adenine dinucleotide Tm melting temperature
NADH nicotinamide adenine dinucleotide, reduced form TMB 3,3’, 5,5’-tetramethylbenzidine
NADP nicotinamide adenine dinucleotide phosphate TRICINE N-tris(hydroxymethyl)methylglycine
NADPH nicotinamide adenine dinucleotide phosphate, reduced form Tris tris(hydroxymethyl)aminomethane
NBT nitro blue tetrazolium tRNA transfer ribonucleic acid
NDP nucleoside diphosphate TPE Tris/phosphate/EDTA (buffer)
NMP ribonucleoside monophosphate TX-100 triton X-100
NR not recommended T4 T4 polynucleotide kinase
NP-40 Nonidet P-40 (detergent) U
nt nucleotide U uracil or uridine
NTC no-template control u unit
NTP nucleoside triphosphate UDG uracil-DNA glycosylase
O UMP uracil monophosphate
oligo(A) oligoadenylic acid UTP uridine 5’-triphosphate
oligo(dT) oligodeoxythymidylic acid UV ultraviolet
OMP orotidine monophosphate V
P v/v volume/volume
Pi inorganic phosphate W
PAAG polyacrylamide gel WGA whole genome amplification
PAGE polyacrylamide-gel electrophoresis w/v weight/volume
PBS phosphate-buffered saline X
PCR polymerase chain reaction X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
PEI polyethylenimine X-Gluc 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid
PEG polyethylene glycol
PIPES piperazine-N,N’-bis(2-ethanesulfonic acid)
PNK polynucleotide kinase
pNPP 4-nitrophenyl phosphate; para-nitrophenyl phosphate
poly(A) polyadenylic acid
poly(A)+ polyadenylated (mRNA)
poly(dA-dT) poly (deoxyadenylic acid – deoxythymidylic acid)
(continued on next page)
Bulk quantities and custom formulations available upon request
Abbreviation pKa values
Amino acid Mr, Da
pK1 pK2 pKR pI
3-letter 1-letter
(-COOH–) (-NH3+) (R group) SI Unit Prefixes
Nonpolar (hydrophobic)
Prefix Symbol Multiple
Alanine Ala A 89.09 2.34 9.69 6.01
yotta Y 1024
Proline Pro P 115.13 1.99 10.96 6.48
zetta Z 1021
Valine Val V 117.15 2.32 9.62 5.97
Leucine Leu L 131.17 2.36 9.60 5.98
exa E 1018
Isoleucine Ile I 131.17 2.36 9.68 6.02 peta P 1015
Methionine Met M 149.21 2.28 9.21 5.74 tera T 1012
Phenylalanine Phe F 165.19 1.83 9.13 5.48 giga G 109
Tryptophan Trp W 204.23 2.38 9.39 5.89 mega M 106
Polar, uncharged kilo k 103
Glycine Gly G 75.07 2.34 9.60 5.97 hecto h 102
Serine Ser S 105.09 2.21 9.15 5.68 deka da 101
Threonine Thr T 119.12 2.11 9.62 5.87 deci d 10-1
Cysteine Cys C 121.16 1.96 10.28 8.18 5.07 centi c 10-2
Asparagine Asn N 132.12 2.02 8.80 5.41 milli m 10-3
Glutamine Gln Q 146.15 2.17 9.13 5.65
micro µ 10-6
Tyrosine Tyr Y 181.19 2.20 9.11 10.07 5.66
nano n 10-9
Basic
pico p 10-12
Lysine Lys K 146.19 2.18 8.95 10.53 9.74
femto f 10-15
Histidine His H 155.16 1.82 9.17 6.00 7.59
Arginine Arg R 174.20 2.17 9.04 12.48 10.76
atto a 10-18
Acidic zepto z 10-21
Aspartate Asp D 133.10 1.88 9.60 3.65 2.77 yocto y 10-24
Glutamate Glu E 147.13 2.19 9.67 4.25 3.22
Concentrations of Acids and Bases

% Specific ml/liter to prepare
Substance Formula MW Moles/liter* Grams/liter
by weight gravity 1 M solution
Acetic acid, glacial CH3COOH 60.05 17.4 1045 99.5 1.05 57.5
Acetic acid CH3COOH 60.05 6.27 376 36 1.045 159.5
Formic acid HCOOH 46.02 23.4 1080 90 1.20 42.7
Hydrochloric acid HCl 36.5 11.6 424 36 1.18 86.2
Nitric acid HNO3 63.02 15.99 1008 71 1.42 62.5
Perchloric acid HClO4 100.5 11.65 1172 70 1.67 85.8
Phosphoric acid H3PO4 97.9 14.8 1445 85 1.70 67.7
Sulfuric acid H2SO4 98.1 18.0 1766 96 1.84 55.6
Ammonium hydroxide NH4OH 35.0 14.8 251 28 0.898 67.6
Potassium hydroxide KOH 56.1 13.5 757 50 1.52 74.1
Sodium hydroxide NaOH 40.0 19.1 763 50 1.53 52.4
* With some acids and bases, stock solutions of different molarity/normality are in common use.
Amino Acids
Nonpolar (hydrophobic) Polar, uncharged Basic Acidic

Amino acid Structure Amino acid Structure Amino acid Structure Amino acid Structure
Alanine Glycine
Aspartate
Lysine
Proline Serine
Glutamate
Valine Threonine
Histidine
Cysteine
Leucine
Arginine
Asparagine
Isoleucine
Glutamine
Methionine
Tyrosine
Phenylalanine
Tryptophan
Ex. No. 4: Separation of Photosynthetic Pigments by Paper Chromatography.
Chromatography
Aim: To separate photosynthetic pigments by paper chromatography.
Principle: The separation of photosynthetic pigments is based on partition takes place between
the static phase adsorbed to the cellulose matter of the paper and mobile phase.
Materials Required: Mulberry leaves, pestle and mortar, 80% acetone, calcium carbonate, Buchner
filter,, beaker, measuring cylinder, glass jar/test tube with a tight cork,
cork Whatmann No.1 filter paper,
petroleum ether, acetone, hook, micropipette.
Procedure:
1. Take 50 g of fresh leaves in a pestle and mortar. Crush them with 20 ml of 80% acetone. Add a pinch
of calcium carbonate and again crush.
2. Filter the extract on a Buchner filter or with double layered muslin cloth.. The deep green coloured
filtrate containing chlorophylls and carotenoids is obtained. Evaporate the extract to concentrate.
3. Take a glass jar (about 45cm high) with a tight cork fitted in it. The cork should have a hole in the
centre to fix the hook.
4. Now prepare the solvent by mixing 25 ml petroleum ether and 3 ml acetone. Pour the solvent into the
jar and allow the jar to become saturated.
sat
5. Cut a strip of filter paper of the size which can easily be hung on the hook. Apply a circular spot of
pigment extract about 3cm from the base of strip with the help of a micropipette. Now hang the strip
inside the jar to the hook of cork and close the cork. Care should be taken that the spot is not dipped
in the solvent. Make the apparatus air tight and observe.
Observation:
The solvent will run on the filter paper. After few hours, the chloroplast pigments will be separated in the
form of different nt spots on the paper. Take out the paper when the solvent reaches up to the upper level.
After drying the paper, identify the different pigments with the help of their specific colours. Carotene is
yellow, Xanthopyhll is yellow-brown,
brown, Chlorophyll-a
Chlorophyll is bluee green and Chlorophyll-b
Chlorophyll is olive green in
colour.
Experimental set up during running Chromatogram

1. Cork to minimize evaporation of solvent
2. Paper clip hook to hold the paper
3. Filter paper, 4. 4. Spot of pigment,
pigment 5. Solvent
------------
Reprinted from: A Laboratory Manual on Physiology of Mulberry and Silkworm. Ed. Dr.H.B.Mahesha,
Pub. Yuvaraja’s College Cooperative Society, University of Mysore, Mysore, 2014.
Ex. No. 5: Extraction of Photosynthetic Pigments by Solvent Wash method.
Aim: To separate photosynthetic pigments by solvent wash method.
Principle: The separation of photosynthetic pigments is based on solubility of different
pigments with specific solvents.
Materials Required: Mulberry leaves, pestle and mortar, 80% acetone, calcium carbonate,
separating funnel, beaker, measuring cylinder, petroleum ether, acetone, diethyl ether, 30%
methanolic KOH, 95% methanol, distilled water etc.,.
Procedure:
1. Take 10 g of fresh leaves in a pestle and mortar. Crush them with 40 ml of 80% acetone. Add a
pinch of calcium carbonate, crush again and filter the extract on a Buchner filter or double layered
muslin cloth.
2. Fill the homogenate in to separating funnel, add equal amount of petroleum ether, shake the
contents gently and leave it for separation of acetone and petroleum ether layers. The upper
petroleum ether layer contains all the pigments. Care should be taken to release the pressure built
up in the separating funnel by opening the top lid.
3. Discard the lower acetone layer by opening the tap of separating funnel. Wash the petroleum
ether layer with 20-30 ml of distilled water, discard lower water layer.
4. Now to the petroleum ether layer add 20 ml of 95% methanol mix the contents by shaking and
leave it for separation of upper petroleum ether and lower methanol layers.
5. Collect the lower methanol layer and store it separately.
6. To the upper petroleum ether layer add 16 ml of 30 % methanolic KOH and 4 ml of distilled
water shake gently; leave the contents for the separation of two layers. Collect the upper blue
green petroleum ether layer and lower yellowish methanolic KOH layers separately as
chlorophyll a and carotenes.
7. Now take methanol fraction (collected and stored at step 5) in separating funnel and add equal
amount of diethyl ether (16 ml) as well as distilled water (4 ml). Mix the contents and discard
lower methanol layer as it contains no pigments.
8. To the upper diethyl ether fraction add equal amount of methanolic KOH and distilled water (8+8
ml), mix gently and collect upper diethyl ether layer contains greenish chlorophyll b and lower
methanolic KOH layer contains orange red xanthophylls.
Observation: After isolation identify the different pigments with the help of their specific colours as
shown in the previous experiment.
Ex. No 13. Estimation of Amylase Activity
ctivity in Haemolymph of Bivoltine and Multivoltine
ultivoltine races.
Aim: To estimate the amylase activity in haemolymph of bivoltine and multivoltine silkworm strains.strains
Principle: When amylase acts on starch,
starch, it is converted in to glucose units. The resultant glucose units react with 3,
5-dinitrosalicylic
dinitrosalicylic acid (DNS) in alkaline solution to give rise to orange coloured complex, which can be measured
at 540 nm.
Reagents Required:
1. Phosphate buffer 0.1 M pH 7.8:
a. 0.1 M KH2PO4: Dissolve 1.360 g of KH2PO4 in 100 ml of distilled water.
b. 0.1 M K2HPO4: Dissolve 1.7418 g of K2HPO4 in 100 ml of distilled water
Mix the solutions a and b at 1:1 ratio and adjust the pH 7.8.
2. Haemolymph Sample: Dilute 100 µl of silkworm haemolymphhaemo with 1900 µl phosphate buffer congaing 1mM
thiourea.
3. Substrate (1% starch): Dissolve 1 g soluble starch in 90 ml of distilled water and boil to get clear solution.
Make up to 100 ml with water.
4. DNS reagent: Please refer experiment number 11.
Procedure for Standard Curve: For preparation of standard curve please refer experiment number 11 or same
standard curve may used here for the estimation of amylase activity.
Procedure for Experiment:
1. Blank: Take 2 ml of phosphate buffer, 0.5 ml of substrate and 0.5 ml of inactivated haemolymph sample or
distilled water in a clean dry test tube.
2. Test 1: Take 2 ml of phosphate buffer, 0.5 ml of substrate and 0.5 ml of multivoltine haemolymph samples in
a clean dry test tube.
3. Test 2: Take 2 ml of phosphatesphate buffer, 0.5 substrate and 0.5 ml of bivoltine haemolymph samples in a clean
dry test tube.
4. Mix the contents of the tubes by vortexing / shaking the tubes and incubate for 30 min at 37°C.
5. Now add 0.5 ml of DNS to all the test tubes, mix the contents of the tubes by vortexing / shaking the tubes and
incubate for 10 min in a boiling water bath and cool to room temperature.
6. Thenn to the cooled test tubes add 0.5 0.5 ml of distilled water, mix the contents and record the absorbance at 540
nm against blank.
Observations and Calculations
Buffer Substrate Enzyme Mix, DNS Incubate DW A540
(ml) (ml) Sample (ml) incuba (ml) for (ml)
Blank 2 0.5 IE te at 0.5 10 min 0.5 0.00
Test 1 2 0.5 0.5 37°C 0.5 in a boiling 0.5
(MV) for water bath
30’ and cool
Test 2 2 0.5 0.5 0.5 0.5
(BV)
*IE-Inactivated Enzyme
Amylase activity = Standard Curve Value X 60 X Dilution Factor (20)

ml of sample X Time of incubation
= -----µg
µg of glucose released/ml of haemolymph sample/hour at 37°C
Result: The given multivoltine and bivoltine haemolymph

haem samples shown ------ and ----- µg /ml / hour at 37°C
respectively.
-----------
Pub. Yuvaraja’s College Cooperative Society, University
U of Mysore, Mysore, 2014.
Ex. No 14. Estimation of Succinate Dehydrogenase activity in the eggs/ tissue.
Aim: To estimate succinate dehydrogenase activity in silkworm eggs.
Principle: Succinate dehydrogenase is one of the mitochondrial enzymes, which catalyzes the
conversion of succinate to fumerate. In this reaction FAD reduces to FADH2. In vitro, the lemon
yellow colored INT accepts electrons and becomes red coloured farmazan which can be measured at
495 nm.
Reagents Required:
1. Sodium phosphate buffer (0.1M, pH 7.4): Mix 16 ml (0.2 M) of monobasic and 84 ml (0.2 M)
of dibasic and makeup to 200 ml with distilled water.
2. INT [2(4-iodophenyl)-3(4-nitrophenyl)-5-Phenyltetrazolium chloride]: 1mg/ml in distilled water.
3. Sodium succinate: 15mM
4. Glacial acetic acid, Toluene etc.,
5. Sample: Prepare 0.2% egg (5 days old or more) homogenate in cold phosphate buffer/distilled
water using mortar and pestle. Centrifuge the homogenate at 3000 rpm for 10 min, collect the
clear supernatant and use as sample.
Procedure for Standard Curve:
1. Pipette out 0.0, 0.2, 0.4, 0.6, 0.8 and 1 ml of INT in to the series of labeled test tubes.
2. Make up the volume to 1 ml in all the test tubes. A tube with 1 ml of distilled water serves as the
blank.
3. Now add 1 ml of buffer, 1 ml of sodium succinate and 1 ml of sample (egg homogenate) to all the
test tubes including the test tubes labeled 'blank' and 'test'.
4. Mix the contents of the tubes by vortexing / shaking the tubes and incubate at 37°C for 24 h or
until complete reduction of INT.
5. Now add 6 ml of glacial acetic acid to stop the reaction.
6. Then add 6 ml of toluene, mix and keep them in a refrigerator for separation of toluene layer.
7. Now collect the upper red coloured toluene layer containing farmazan in to a tube, cool to room
temperature and record the absorbance at 495 nm against blank.
8. Then plot the standard curve by taking concentration of farmazan (equivalent to INT) along X-
axis and absorbance at 495 nm along Y-axis.
For drawing the standard graph please refer last page figure 1.
Procedure for Test:
1. Blank: Take 1 ml of phosphate buffer, 1 ml of sodium succinate, 1 ml of INT and 1 ml of
inactivated enzyme sample (distilled water may be used) in a clean dry test tube, and incubate at
37°C for one hour.
2. Test: Take 1 ml of phosphate buffer, 1 ml of sodium succinate, 1 ml of INT and 1 ml of enzyme
sample in a clean dry test tube, and incubate at 37°C for one hour.
After incubation add 6 ml of glacial acetic acid to both blank as well as test to stop the
reaction. Then add 6 ml of toluene to each tube, mix and keep them in a refrigerator to separate the
red farmazan. After separation, collect the upper red coloured toluene layer containing farmazan in to
a cuvette and record the absorbance at 495 nm against blank.
Observations and Calculations:
Standard Curve:
INT H 2O Buffe Succin Sam Acetic Tolue Mix & OD
(ml) (ml) r (ml) ate ple Mix, acid ne keep in 495
(ml) (ml) incu (ml) (ml) a fridge
0.0 1.0 1 1 1 bate 6 6 for 24 0.00
h.
0.2 0.8 1 1 1 at 6 6
37° Then
0.4 0.6 1 1 1 6 6 collect
C
0.6 0.4 1 1 1 6 6 the
for
0.8 0.2 1 1 1 6 6 upper
24h. layer
1.0 0.0 1 1 1 6 6
Test:
INT Buffer Succin Sam Mix, Acetic Tolue Colle OD
(ml) (ml) ate ple Incuba acid ne ct 495
(ml) (ml) te at (ml) (ml) farma
B 1 1 1 1 37°C 6 6 zan as 0.00
for 1h. above
T 1 1 1 1 6 6
B - Blank
T - Test
Optical density of the test: ---
SDH activity level = Standard Curve

C Value X 60 .
Tissue taken (mg) X Incubation time (min)
= ----- µg of farmazan formed per hour per mg at 37°C.
Report: The SDH activity in the given sample is ----- µg of farmazan formed per hour per mg tissue
at 37°C.
--------
Pub. Yuvaraja’s College Cooperative Society, University
U of Mysore, Mysore, 2014.
50X TAE Stock Solution
For each litre of solution:
242 g Tris Base (MW=121.1)

57.1 mL Glacial Acetic Acid
100 mL 0.5 M EDTA
mix Tris with stir bar to dissolve in about 600 mL of ddH2O.

add the EDTA and Acetic Acid.
bring final volume to 1 L with ddH2O.
store at room temperature.
Note: Final (1x) working concentration :
0.04 M Tris - Acetate

0.001 M EDTA
50X Modified TAE Stock Solution
For each litre of solution:
242 g Tris Base (MW=121.1)

57.1 mL Glacial Acetic Acid
10 mL 0.5 M EDTA
mix Tris with stir bar to dissolve in about 600 mL of ddH2O.

add the EDTA and Acetic Acid, pH to 8.0.
bring final volume to 1 L with ddH2O.
store at room temperature.
Note: Final (1x) working concentration :
0.04 M Tris - Acetate

0.0001 M EDTA
Andrea’s Help Sheet on Preparing Solutions
There are several types of stock solutions made in the research lab: Percent (%) solutions, Molar (M)
solutions, X solutions, and mg/ml solutions. First are instructions on how to make % solutions.
First, know the definition of a % solution: 1% = 1g/100ml

That’s the basic formula, and it is logical because “per cent” means “per hundred”.
So, let’s say you are assigned the task of making 1 liter of 20% SDS stock solution, a lab basic.
(On quizzes, you may be told the MW of the chemical; it is not used in % solutions, so ignore it.)
The three steps are:
1: write out the formula: 1% = 1g/100ml
2: scale it up on the percentage: 20% = 20g/100ml
3: scale to the final volume by multiplying by 10: 20% = 200g/1000ml
Write out the description in the following manner:

Place 200g of SDS powder a 1L beaker and add H2O up to 900ml. When dissolved, transfer
solution to a 1L graduated cylinder and add H2O to 1000ml.
This works whether for a % w/v solution (as exampled above) or a % v/v solution. Suppose you
need 100ml of 20% EtOH: 1% = 1ml/100ml
20% = 20ml/100ml
Mix 20ml EtOH with 80ml H2O for a final volume of 100ml.
Next, let’s look at Molar (M) solutions.

First, know the definition of a Molar solution: 1M = 1MW(in grams)/Liter
(MW = molecular wt.) That’s the basic formula, and is logical because Molar is defined as a solution
concentration having one mole of solute per liter of solution.
So, lets say you are assigned the task of making 100ml of 5M NaCl. Here, you will need the
molecular wt for NaCl; it is 58.44.
The three steps are:
1: write out the formula: 1M = 1MW(g)/1000ml
2: plug in the numbers & scale it up on the Molarity: 5 M = 5 (58.44)g/1000ml
& work out the calculations: 5M = 292.2g/1000ml
3: scale to the final volume by dividing by 10: 5M = 29.22g/100ml
Write out the description in the following manner:
Dissolve 29.22g of NaCl in 90ml H2O in a 100ml beaker. Transfer solution to a 100ml graduated
cylinder and add H2O to 100ml.
If you are assigned a specific pH, make sure to titrate the solution in your description:
Making 500ml of 1M Tris, pH 8.2 would go like this (Tris MW = 121.14):
1M = 121.14g/1000ml
1M = 60.57g/500ml
Dissolve 60.57g of Tris in 450ml H2O in a 500ml beaker. Titrate the solution to 8.2 using
either HCl (acid) or NaOH (base) as needed. Transfer the solution to a 500ml graduated
cylinder and add H2O to 500ml.
Next, let’s look at how to make other solutions from our stock solutions:
You have been taught the formula C1V1 = C2V2.
Here’s the formula that I have used for years in the lab, and it is really just the same equation in a
logical order, with the order to punch it in the calculator:
“What you have (stock conc) ÷ what you want (final conc); invert that under 1 with the 1/x key, then
multiply by the volume you want to make.”
That also looks like this: final volume

(stock conc/final conc) dilution factor
That’s the same as the C1V1 = C2V2 formula, solving for V2. The (stock conc. / final conc.) is also
known as your “dilution factor.”
So, let’s say you already have stock solutions of 5M NaCl; 1M Tris, pH 8; and 1M Imidizole, and you
need to make 500ml of 100mM NaCl, 10mM Tris, pH 8, and 20mM Imidizole. Here’ s how you do the
calculations:
Step: NaCl Tris, pH 8 Imidizole
Have Need 5M 100mM 1M 10mM 1M 20mM
(get same units) 5000mM 100mM 1000mM 10mM 1000mM 20mM
Have/need = Dil Fctr 1:50 dilution 1:100 dilution 1:50 dilution
scale up: 10:500 dilution 5:500 dilution 10:500 dilution
add units: 10ml stock:500ml total 5ml stock:500ml total 10ml stock:500ml total
Plugging in the calculator for NaCl would be:
5000 ÷ 100 = 1/x x 500 = , and the result will be 10, or 10ml. Try this method for the other 2
solutions above.
The description of how to make the solution is this:

To a graduated cylinder, measure by pipette 10ml 5M NaCl, 5ml 1M Tris, pH 8.2, 10ml 1M
Imidizole, and water to a total volume of 500ml.
Since all the stock solutions are already liquid, there is none of that tedious “Stir to dissolve” or
transferring to a graduated cylinder later; the solution can be made directly in the grad cylinder in the
first place, and brought to final volume in one easy step. It will mix as you pour it into your bottle for
storage (which you will clearly label…but that’s another lesson!)
You can do the same for %-solutions: suppose you need to make 100ml of a solution with a final
concentration of 1% SDS, and you already have a bottle of 20% stock on your shelf.
20% 1% SDS = 1:20 dilution, and that’s the same as 5:100, so you would use 5ml of the 20%
stock in the total volume of 100ml.
Plugging in the calculator would be:
20 ÷ 1 = 1/x x 100 = , and the result will be 5, or 5ml in a total of 100ml.
The next type of solution we use in the lab is an X-solution. This is a solution that someone has
worked out in the past which may have several fairly dilute components, and we have found it is
generally easier to measure out 10-times the amount of each and prepare the stock solution in a
more concentrated strength. For use, the 10X solution is diluted out to 1X or 0.5X, and is labeled as
such.
For example, a common X-solution is 10X PBS (PBS stands for Phosphate-Buffered Saline). The 1X
concentrations are listed as:
137mM NaCl,
2.7mM KCl,
10mM Na2HPO4,
2mM KH2PO4.
The recipe for this would be 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 per 1000ml total
volume, pH 7.4. (You should practice those calculations yourself, using MWs of 58.44 for NaCl, 74.55
for KCl, 141.96 for Na2HPO4 and 136.09 for KH2PO4.) PBS is generally used in large volumes (500ml-
1000ml at a time), so it would be a waste of time to make 1 liter of 1X PBS over and over again.
However, making a concentrated (10X) solution is more time efficient; the larger masses are easier
to weigh out; the pH titration is done only once per 10 liters total volume if the solution is made at
the 10X concentration. Additionally, it is a simple process to dilute the 10X solution to 1X as it is
needed.
So, to prepare 1L of 10X PBS (notice how the weights are all 10x as listed above):
Dissolve 80g NaCl, 2g KCl, 14.4g Na2HPO4, and 2.4g KH2PO4 in 950ml H2O, and titrate to pH 7.4
with acid. Pour the solution into a 1000ml graduated cylinder, and add H2O to a total volume of
1000ml.
{This particular solution needs to be sterilized by autoclaving or filter sterilization before use and
storage, so you would either pour it into a sterile bottle through a 0.22um sterile filter, or put it into
glass bottles and autoclave it for 20 minutes at 15psi on the liquid cycle.}
There is one other type of solution to make, and is mostly used for things like antibiotics: it is a
simple mg/ml or μg/ml solution (the same as a w/v solution). For instance, Ampicillin is
generally made at a stock concentration of 50mg/ml. Suppose you need to make some of this stock
for use over the next few months. I’d make at least 50ml, so the calculation is like this:
50mg/ml = 500mg/10ml = 2500mg/50ml, or 2.5g/50ml.
You would weigh out 2500mg of Ampicillin, put it into a 50ml graduated cylinder, and add water to
50ml. Capping and sealing the cylinder with Parafilm (a handy lab supply) allows you to rock or
invert the cylinder in your hand until the powder is dissolved. Even though the antibiotic does kill
non-resistant bacteria, it needs to be sterile-filtered (autoclaving it would inactivate its activity) to be
used in sterile plates and media.
Once you have your sterile stock of 50mg/ml, you find that you need to make some LB media with
Ampicillin at 50μg/ml; diluting mg to μg is a 1:1000 dilution. So if you want to make 25ml of LB with
Ampicillin at 50μg/ml, you would add 1μl Amp per ml of media (that’s a 1:1000 dilution), or 25μl
Amp/25ml LB. The volume of Ampicillin being added (25μl) is inconsequential to the total volume
(25ml), so don’t worry about off-setting that in the preparation.
King Saud University
College of Science
Department of Biochemistry
Biochemical Calculation
BCH 231
Sample Exam and Answer Key
Q# 1 2 3 4 5 6 7 8 9 10 Total
Q (pts) 100
Y (pts)
Serial #:.......................Name: Answer Key I. D. #.......................................
1. (10 pts). Define the following:
(1) Aqueous solutions
Aqueous solutions are solutions in which water is the solvent

Aqueous solutions are the most common type of solution
(2) Normality
Normality (N) is the number of equivalents of solute per liter of solution

or
#

2. (10 pts). How would we make 1500 mL of a 1.5% w/v solution of NaCl in water, MW
of NaOH = 40?
Solution
We find out how many grams (g) of NaCl there are in this solution by
taking the percentage:
1.5 g NaCl →100 mL water
x g NaCl → 1500 mL
#.% & #%''
x g NaCl

22.5 *
#''
We then put 22.5 g of NaCl into a beaker, add some distilled water, and
stir until the NaCl is all dissolved. We then transfer the solution to a 1500-
mL volumetric flask and complete the final volume with distilled water
into 1500 mL. Please note that we don’t know exactly how much water
we have added, however, we do know that the total volume is 1500
mL.
1
3. (10 pts). How do we prepare 2.0 L of a 0.15 M solution of NaOH, MW = 40?
Solution
First we find out how many moles of NaOH there will be in this solution:
#
+

# , -.
/ 01-
# , -.
0.15 / 2.0
0.30 - , 56
We need 0.30 mole of NaOH to make up this solution. How many grams
is this? To convert moles to grams, we multiply by the appropriate
conversion factor:
7 8
# , -
9:
.. , .1-
- / +;
0.30 / 40
12.0 * 56
We, therefore, weight out 12.0 g of NaOH, put it into a beaker, add some
distilled water, and stir until the solid NaOH is all dissolved. We then
transfer the solution to a 2000-mL volumetric flask and complete the final
volume with distilled water into 2000 mL. Please note that we don’t
know exactly how much water we have added, however, we do know
that the total volume is 2000 mL.
4. (10 pts). The concentration of NaCl in serum is approximately 0.15 M. What volume
of serum contains 4.0 g of NaCl, MW of NaCl = 58.5?
Solution
Since we know the concentration in M (moles/L), we must first find out
how many moles there are in 4.0 g of NaCl by multiplying by the
appropriate conversion factor:
7 =7> 8
# , -
9: =7>
?.'
# , -
%@.%
0.068 - C
We next find the volume of serum:
#

D
'.'E@
0.15
D
'.'E@
01- , .-1
= 0.453 L = 453 mL of serum
'.#%
5. (10 pts). If the molality of concentrated H2SO4 is 16.2 m. Calculate its molarity,
density of H2SO4 = 1.84 g/mL, and MW = 98?
Solution
First we find out how many moles of H2SO4 there will be in this solution
by taking the percentage:
# FG HIJ
+
7 7 K8
# , -. , 6L M5?
/ .. , .1N
# , -. , 6L M5?
16.2 / 1 O* , .1N
16.2 -.
2
7 FG HIJ
# , -. , 6L M5?
9: FG HIJ
.. , 6L M5?
# , -. , 6L M5? / +;
.. , 6L M5?
16.2 / 98
1587.6 *
Total mass solution = 1587.6 g + 1000 g = 2587.6 g
1.84 g → 1 mL
x g R 1000 S
x g = 1000 x 1.84 = 1840 g/L
1840 g R 1 S
2587.6 g → x L
L%@U.E 8
T 01- , .1N

1.41 S
#@?' 8/
# FG HIJ #E.L
+ , 6L M5? .1N

#.?#

11.41 +
6. (10 pts). Describe the preparation of 3.0 L of 0.5 M HCl starting with a concentrated
HCl solution, 28%w/w. SG = 1.15, MW = 36.5?
Solution
We find out how many moles of HCl there are needed to prepare 3 L of
0.5 M HCl solution by taking the percentage:
#
+

# of moles HCl needed = volume (L) x molarity
# of moles HCl needed = 3 x 0.5 = 1.5 moles
7 F>
# , -. 6C N--W-W
9: F>
.. , 6C N--W-W
# , -. / +;
1.5 / 36.5
54.75 *
The stock solution is not pure HCl, but only 28% HCl by weight
Therefore,
28 g HCl → 100 g stock solution
54.75 g HCl needed → x g stock solution

#'' & %?.U%
x g stock solution = L@
= 195.54 g
Instead of weighting out 195.54 g of stock solution, we can calculate the
volume required
1.15 g → 1 mL of stock solution
195.54 g → x mL of stock solution
#X%.%?
x mL of stock solution needed = #.#% = 170 mL
We, therefore, measure out 170 mL of stock solution and transfer it into to
a 2000-mL volumetric flask and complete the final volume with distilled
water into 2000 mL.
3
7. (10 pts). 0.89% w/v NaCl solution is referred to as a physiological saline solution
because it has the same concentration of salts as normal human blood. Although blood
contains several salts and saline salutation has only by NaCl. What is the osmolarity of
this solution, MW of NaCl = 58.5?
Solution
We find out how many g of NaCl there are in 1 L of blood by taking the
percentage:
0.89 g NaCl →100 mL of blood
x g NaCl → 1000 mL of blood
'.@X & #'''
x g NaCl
#''

8.9 *
Second we find out how many moles of NaCl there will be in 8.9 g:
7 8 @.X 8
# , -. , C

0.15 -.
9: =7> %@.%
TY- , Y- Y1N ZW . 0.15 +
# , [\-. N C . 2 N.
TY- . , Y1N ZW
2 / 0.15
0.30 .
8. (10 pts). How do we prepare 400 mL of a 4.5 M solution of acetate buffer if we have a
bottle of concentrated acetate buffer solution, 10.0 M?
Solution
We use the following equation:
M1 x V1 = M2 x V2
10.0 x V1 = 4.5 x 0.4
?.% & '.?
]#
#'
0.18 S
We therefore, measure out 0.18 L or 180 mL the concentrated acetate
buffer solution and transfer it into to a 400-mL volumetric flask and
complete the final volume with distilled water to the mark 400 mL.
9. (10 pts). How do we prepare 500 mL of a 0.5% w/v solution of NaOH if we have a
stock solution of 50% w/v NaOH on hand?
Solution
We use the following equation:
% x V1 = % x V2
50% x V1 = 0.5% x 500 mL
'.%% & %''
]# S
%'%

5.0 S
We therefore, add 5.0 mL the concentrated solution to a 500-mL
volumetric flask, and some distilled water and mix well and then fill to the
mark 500-mL with distilled water.
4
10. (10 pts). Complete the following table for water solution of sodium sulfate (Na2SO4,
MW = 142):
Mass Solute Mole Solute V Solution Molarity

20.8 g ---------- 540 mL -------
--------- 0.039 --------- 0.15
--------- ---------- 492 mL 0.25
Solution:
(ROW # 1)
Since we know the mass of solute, we must first find out how many moles
there are in 20.8 g of solute by multiplying by the appropriate conversion
factor:
7 8
# , -
9:
L'.@
# , -

_. `ab cdef
#?L
We next find the molarity of the solution:
#

'.#?E

_. gh_ i
'.%%L
(ROW # 2)
Since the molarity of the solution and the moles of solute are given, we
must first find out how many grams there are in 0.039 mole of solute by
multiplying by the appropriate conversion factor:
7 8
# , -
9:
.. ,.1- *
-. / +;
0.039 / 142
j. ja k
We next find the volume of the solution:
#

# '.'mX
01- S
7Dl

'.#%

0.26 S
gb_ cn
(ROW # 3)
Since we know the molarity of the solution and the volume of solution, we
must first find out how many moles there are in 0.25 M of final volume is
492 mL by multiplying by the appropriate conversion factor:
#

# , - ,.1-
/ 01- S
# , - , .1-
0.25 / 0.492
_. `go cdef
5
We next find the mass of the solute:
7
# -. , .1-
9:
.. , .1-
# , -. / +;
0.123 / 142
`h. ah k
Mass Solute Mole Solute V Solution Molarity

20.8 g 0.146 541 mL 0.270
5.54 g 0.039 260 mL 0.15
17.47 0.123 492 mL 0.25
Good luck!
6
Biological
Buffers
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Biological Buffers • AppliChem © 2008

c o n t e n t s
Introduction 2
• The buffer concept • Buffer capacity • The pH value • The pKa value • Biological buffers
Requirements of biological buffers 3
• Solubility • Permeability • Ionic strength 3
• Dependence of pKa value • Complex formation • Inert substances • UV absorption
• Purity – simple method of manufacture • Costs 4
• Overview of most important properties of buffers 4
Recommendations for the setting of the pH value of a buffer 5
• Temperature • Titration • Ionic strength • Buffer additives • pH meter control
Criteria for the selection of a buffer 6
• Selection of a buffer for the correct pH range • Determination of the pH optimum of an enzyme
• Determination of the optimum buffer concentration • Application-dependent choice of buffer substance 6
• Tris buffer: not always the best choice! 7
• Volatile buffers • Buffer mixtures • Buffers for gel electrophoresis 8
• Electrophoresis buffers 9

Technical tips 10
• How can microbial contamination of buffer solutions be prevented?
• How can precipitation in concentrated TBE buffers be prevented?
• What is the best way of obtaining solutions of the free acids of PIPES, POPSO and ADA?
• What is the importance of water as a solvent?
• (Disturbing) Effects of biological buffers in different assays11
• Concentration limits for buffers in protein assays12
• Saturated concentrations of buffers in solution at 0°C
• “Old” buffers replaced by buffers with better properties
Alphabetical list of biological buffers 13
Temperature dependence of the pKa value of biological buffers (100 mM) 14
pKa values of biological buffers (25°C, 100 mM), alphabetical list 16
References 16
Alphabetical product list of biological buffers supplied by AppliChem 17
© 2008 AppliChem • Biological Buffers

i n t r o d u c
Introduction
The buffer concept

Many biochemical processes are markedly impaired by even small changes in the concentrations of free H+ ions. It is
therefore usually necessary to stabilise the H+ concentration in vitro by adding a suitable buffer to the medium, without,
however, affecting the functioning of the system under investigation. A buffer keeps the pH of a solution constant by taking
up protons that are released during reactions, or by releasing protons when they are consumed by reactions. The observation
that partially neutralised solutions of weak acids or bases are resistant to changes in pH when small amounts of strong acids
or bases are added led to the concept of the ‘buffer’.
Buffer capacity
Buffers consist of an acid and its conjugated base. The quality of a buffer is determined by its buffer capacity, i.e. its resistance
to changes in pH when strong acids or bases are added. In other words: the buffer capacity corresponds to the amount
of H+ or OH– ions that can be neutralised by the buffer. The buffer capacity is related to the buffer concentration. The
graph described by the relation of the pH to the addition of H+/OH– ions is called the titration curve. The point of inflection
of the curve corresponds to the pKa value. At this point, the buffer capacity is at its maximum at the pKa value. This point
therefore corresponds to the mid-point of the pH range covered by the buffer and is where the concentration of acid and
base is the same. In the area of this pH range, therefore, relatively large amounts of H+/OH– ions result in only small changes
in pH.
A basic principle is that a buffer that has a pH value of one pH unit above or below the pKa value loses so much buffer capacity
that it no longer has any real buffer function. Based on the Henderson-Hasselbalch equation
pH = pKa + log [A–]/[HA]
for the calculation of the pH of a weak acid or alkaline solution, the ions in the water must also be taken into account when
working in pH ranges below 3.0 and above 11.0. Most biochemical reactions, however, take place in the pH range between
6.0 and 10.0.
The pH value
Conductivity can also be detected in highly purified water due to the OH– and H3O+ ions resulting from the autoprotolysis of
water. This intrinsic dissociation of the water is an equilibrium reaction and the product of the concentrations of the two
ions represents a constant:
K = [H3O+] x [OH–].
This value is depends on the temperature only and is 10–14 for purified water at 22°C. Depending on which of the two ions
is present in a higher concentration in a solution, the solution is called to be acidic or alkaline. To express this fact in terms
of a simple number, the negative exponent of the easily measurable hydronium ion concentration [H3O+] was chosen. This
dimensionless number is called the pH value. The pH can also be described as the negative, decadic logarithm of the hydro-
nium ion concentration of a solution:
pH = – log [H3O+]
The hydronium ion concentration of pure water is 10–7 mol/L, as can be derived from the above equation for the ion product.
Therefore, the pH value is 7.
In an acidic solution, the concentration of H3O+ ions is increased (e.g. from 10–7 mol/L to 10–2 mol/L) and the pH is there
fore < 7. In an alkaline solution, the hydronium ion concentration is decreased and this results in a pH > 7.
The pKa value

To change the pH value of a solution, substances are dissolved in the solution which release H+ ions into the water (acids),
thus raising the H3O+ ion concentration and lowering the pH value, or which decrease the H3O+ ion concentration and thus
increase the pH value by taking up H+ ions (bases). As always in chemistry, this reaction is an equilibrium reaction, and the
capacity of a substance to shift this equilibrium in one direction or another is determined by the potency of the acid. It is
calculated from the equilibrium constants using the following equation
K = [A–] x [H3O+] / [HA],

t i o n
as the negative decadic logarithm of the constants and, in analogy to the pH value, is termed the pKa value. The pKa value is
therefore a simple number that describes the acid potency of a substance.
This calculation shows that hydrochloric acid, as one of the most potent acids, has a pKa value of -6, and all HCl molecules
form hydronium ions with water. For a weak acid, such as acetic acid, a pKa value of only 4.75 is calculated (i.e. only very
few molecules form an H3O+ and a CH3COO– ion), and the alkaline HPO42– ion has a pKa value of 12.32.
Biological buffers
Different inorganic substances were originally used as buffers (e.g. phosphate, cacodylate, borate, bicarbonate), and later
weak organic acids were also used. Many of these buffer substances, however, have the disadvantage that they are not inert
and have lasting effects on the system under investigation (e.g. inhibition of enzymes, interactions with enzyme substrates
etc.). Most biological buffers in use today were developed by NE Good and his research team (Good et al. 1966, Good &
Izawa 1972, Ferguson et al. 1980; “Good buffers”) and are N-substituted taurine or glycine buffers. These zwitterionic
buffers meet most of the requirements that biological buffers have to fulfil.
Buffer systems described in the literature are usually used for experiments to enable direct comparison of results. Again and
again, it is shown that the conditions in experiments – even in standard systems – could be optimised (Spectrophotometric
Assessment of Nucleic Acid Purity: Wilfinger et al. 1997, pK-Matched Running Buffers for Gel Electrophoresis: Liu et al.
1999, Buffer Effects on EcoRV Kinetics: Wenner & Bloomfield 1999).
We have put together the information from the literature that we believe will be of assistance to you in solving your everyday
problems and in the development and optimisation of your test systems.
Requirements of biological buffers

Solubility
The buffer should be freely soluble in water and poorly soluble in other solvents. The higher the water solubility, the simpler
it is to prepare concentrated stock solutions (frequently 10X, 50X or 100X stock solutions). The pH of concentrated stock
requirements
solutions may change on dilution. For example, the pH value of an 100 mM sodium phosphate buffer increases from 6.7 to
6.9 with 10fold dilution and to 7.0 with 100fold dilution (Tipton & Dixon 1979). The pH value of a Tris solution decreases
by 0.1 pH units per 10fold dilution.
Permeability
The buffer should not be able to permeate biological membranes to prevent concentration in the cell or organelles. Tris has
a relatively high degree of fat solubility and may therefore permeate membranes. This also explains its toxicity for many
mammalian cells in culture.
Ionic strength
The buffer should not alter the ionic strength of the system as far as possible. The physiological ionic strength is between
100 – 200 mM KCl or NaCl. This can be very important, especially when investigating enzymatic reactions, because the ionic
strength of the solution is a measure of the ionic milieu, which may also affect the catalytic activity of an enzyme. The
protonisation and deprotonisation depending on the ionic composition of the surrounding medium in the reaction set-up
affects the binding and conversion of an enzyme substrate by the enzyme. Under non-physiological conditions in altered
protonised and deprotonised forms, both the amino acid residues in proteins that interact with the substrate and the sub-
strate itself will not be able to interact in the same way as under physiological conditions (Ellis & Morrison 1982). At a pH
of 7.5, for example, phosphate buffers add about 7x more ions to the medium than zwitterionic Tricine buffers at the same
pH (Good & Izawa 1972). The Tris buffers for the preparation of the separation and stacking gels for SDS-PAGE are made
from Tris base and HCl because of the ionic strength. If Tris · HCl is used and the pH value is adjusted using NaOH, NaCl
forms, resulting in an increased salt concentration that causes abnormal migration of protein and diffuse bands (Ausubel et
al. 1995).

requirements
Dependence of the pKa value
The pKa value of a buffer should be influenced as little as possible by the buffer concentration, the temperature and the ion
composition of the medium. Amongst the buffers with temperature dependent pKa values, for example, are the amine buffers,
whilst carboxylic acid buffers generally react less sensitively to changes in temperature. The pH value of a Tris solution set
at a pH of 7.8 at room temperature is 8.4 at 0°C and 7.4 at 37°C.
Complex formation
When a buffer forms complexes with metal ions, protons are released, which causes the pH value to decrease. The formation
of insoluble precipitates usually represents a greater problem, however. If enzymes need the metal ions for their activity,
these would be inhibited. Complexes should therefore be soluble and their binding constant should be known. Phosphates,
for example, form insoluble salts with bivalent metals and precipitate. Phosphate buffered salt solution (PBS) is never
autoclaved with Ca2+ or Mg2+ for this reason. Good buffers, such as PIPES, TES, HEPES and CAPS have very low metal-binding
constants and are therefore particularly suited to investigate metal-dependent enzymes (Good & Izawa 1972, Blanchard
1984).
Inert substances
The buffer should not be subject to either enzymatic or non-enzymatic changes, i.e. it should not be an enzyme substrate or
enzyme inhibitor and should not react with metabolites or other components. The buffer should therefore be inert.
Phosphate and pyrophosphate are both substrates and inhibitors of different enzymatic reactions (inhibition of carboxypep-
tidase, urease, various kinases, various dehydrogenases). Borate forms covalent complexes with mono- and oligo
saccharides, ribose subunits of nucleic acids, glycerol and pyridine nucleotides. Bicarbonate is in equilibrium with CO2 and
therefore needs a closed system. Tris and other primary amines can form Schiff’s bases with aldehydes and ketones. They
also interfere with the Bradford protein assay (e.g. Tris and glycine). Tricine is photo-oxidised by flavins, and daylight is
therefore sufficient to reduce the activity of flavone enzymes. HEPES, HEPPS and Bicine interfere with Lowry (Folin) protein
assays. Buffers that are chemically based on the piperazine ring may form radicals under certain circumstances (see
below).
UV absorption
Buffers should not absorb any light at wave-lengths longer than 230 nm, since many spectrophotometric investigations are
performed in this range (determination of the concentrations of DNA, RNA and proteins). ADA, for example, has an absorp-
tion of 0.1 at 260 nm. If buffers interfere with photometric analyses, they should be neutralised or set at the pH optimum
for the test system used (Lowry pH 10; BCA pH 11; Bradford pH 1; colloidal gold pH 3). If this is not possible, proteins can
be precipitated with trichloroacetic acid, perchloric acid or acetone, for example, and can then be redissolved in a solvent
that does not interfere.
Purity – simple method of manufacture

Buffers should be as easy to manufacture and purify as possible. Purity is extremely important, since contaminations (e.g.
heavy metals) can easily interfere with sensitive biological systems.
Costs
When purifying proteins, large amounts of buffer are often need for centrifugation, chromatography steps or dialysis. The
costs for materials therefore affect the planning of an experiment.
Overview of the most important properties of buffers

(Good & Izawa 1972, Scopes 1994):
1.) Solubility
2.) Permeability through biological membranes
3.) pKa value at the mid-point of the range of the test system
4.) Change in pKa value dependent on temperature
5.) Change in pKa value dependent on dilution
6.) Interaction with other components (e.g. metal ions, enzymes)
7.) UV absorption
8.) Non-toxic
9.) Costs

Recommendations for the setting of the pH value of a buffer
Temperature
Depending on the buffer substance, its pH may vary with temperature. It is therefore advisable, as far as possible, to set the
pH at the working temperature to be used for the investigation. The physiological pH value for most animal cells at 37°C is
between 7.0 and 7.5. One buffer particularly susceptible to changes in temperature is Tris (see above). If set at 7.5 at 37°C,
it increases to about 8.5 if a test system with a temperature of 0°C is used. In vitro tests on cell extracts are often performed
at 0°C (Scopes, 1994). Good buffers generally have a low degree of temperature sensitivity, and carboxylic acid buffers (citrate,
formate, succinate) are even less sensitive. For practical work, this means that the buffer should be brought to the working
temperature (Scopes 1994, Chapter 12.3) and that the pH electrode should also be calibrated at the working temperature.
Nowadays, many pH meters have an integrated function that enables setting of the pH value at room temperature and allows
for different working temperatures (e.g. +4°C or +37°C). A limitation on this function, however, is that the dpKa/dT value,
i.e. the value for the change in the pKa value (dpKa) dependent on the temperature change (dT), is not the same for all
settings
buffers. For example, the change in the pKa value for Tris with an increase of 1°C amounts to 0.028 units, whilst the value
for HEPES changes by only 0.014. Imprecision is unavoidable with this approach, since such changes should actually be
accounted for with the pH meter.
Titration
Generally, the pH value is set using NaOH/KOH or HCl. Slow addition of the acid or base whilst stirring vigorously avoids
local high concentrations of H+ or OH– ions. If this is not done, the buffer substances may undergo chemical changes that
inactivate them or modify them so that they have an inhibitory action (Ellis & Morrison 1982). If a buffer is available in the
protonised form (acid) and the non-protonised form (base), the pH value can also be set by mixing the two substances.
If monovalent cations interfere with the reaction or are to be investigated, the pH value can be set with tetramethyl or tetra
ethylammonium hydroxide. Acetate, sulphate or glutamate can be used instead of HCl, although here the risk of interference
with an enzyme is particularly high.
Ionic strength
The setting of the ionic strength of a buffer solution (if necessary) should be done in the same way as the setting of the
pH value when selecting the electrolyte, since this increases depending on the electrolyte used. The salts of tetramethyl
ammonium or tetraethylammonium are suitable for the setting of the ionic strength, since the larger cations do not interact
so well with the negative charges of the enzymes. Acetate, as a large anion, has a poor interaction with the alkali metals (Ellis
& Morrison 1982). The following example with the buffer triethanolamine (20 mM, pH 7.5) illustrates how the different
setting of a buffer can affect the ionic strength (I). If 20 mM triethanolamine are set at a pH of 7.5 with HCl, the resulting
ionic strength is I = 0.012, with the ions H-triethanolamine+ and Cl–. However, if 20 mM triethanolamine hydrochloride are
set at a pH of 7.5 with NaOH, the resulting ionic strength is I = 0.020, since the buffer solution also contains 8 mM NaCl
(Scopes 1994). A further example is the electrophoresis buffer for SDS-PAGE, which is prepared using Tris base with HCl
and not Tris hydrochloride and NaOH (Ausubel et al. 1995).
Buffer additives
If other components are added to the buffer (e.g. EDTA, DTT, Mg2+), changes in the pH should also be expected and it should
be retested. In living cells, particularly the oxidisation of proteins by different substances is inhibited by glutathione. Usually,
therefore, if cells are being disrupted, a reducing agent such as β-mercaptoethanol (5 – 20 mM) or DTT (1 – 5 mM) has
to be added. β-mercaptoethanol is oxidised within 24 hours after addition to the buffer (Bollag & Edelstein 1992, Scopes
1994. It is therefore advisable to add this substance only to the buffer while the proteins are being processed and to use DTT
for longer storage periods of proteins.
To prevent the growth of bacteria or fungi, particularly in buffers in the pH range of 6.0 – 8.0, sterile filtration (0.22 µm)
and/or the addition of 0.02 % (3 mM) sodium azide is recommended. If added to concentrated stock solutions, the latter
is diluted to such an extent in the working solution that it usually does not interfere with the reaction.
pH meter control
Nowadays, accurate pH meters with a digital display are usually available for the setting of the pH value of a buffer. The
pH meter is calibrated using two pH standards which cover the range of the buffer to be set. If there are any doubts about
the precision of the device, this can simply be resolved by standardising the pH meter using 50 mM phosphate buffer, which
is then diluted 10fold. The pH value should then be 0.2 pH units higher (Scopes 1994).

s e l e c t i o n Criteria for the selection of a buffer
As already described, buffers can also have a decisive influence on the activity of an enzyme. Together with other factors,
such as the ionic strength and the salt concentration, the activity of the restriction enzyme EcoRV can also be improved
(Wenner & Bloomfield, 1999). We shall therefore take a closer look at a range of factors at this point.
Selection of the buffer for the correct pH range

The pKa value of the buffer should be in the range of the pH optimum for the test system, as far as possible. If the pH is likely
to increase during the experiment, then a buffer should be chosen with a pKa value that is slightly higher than the optimum
at the beginning of the experiment. Conversely, if the pH value is expected to decrease during the experiment, a buffer with
a slightly lower pKa value should be chosen.
Determination of the pH optimum of an enzyme

If an enzyme is to be investigated, the first step is usually to determine the conditions under which the enzyme will show the
highest possible degree of stability and activity. The determination of the pH optimum is important as the initial step in this.
It is advisable to test chemically similar buffers first of all, which cover overall a wide pH spectrum, e.g. MES, PIPES, HEPES,
TAPS, CHES and CAPS for the pH range ~5.5 – 11.0 (Viola & Cleland 1978, Cook et al. 1981, Blanchard 1984). Once the pH
optimum has been found, different buffers (e.g. for the pH value 7.5: TES, TEA or phosphate; Blanchard 1984) can be tested,
in order to be able to rule out or minimise non-specific buffer effects for later investigations. The pKa value of a buffer, i.e.
the mid-point of its pH range, should be as near as possible to the desired pH value for the buffer being used, in other words,
it should correspond to the pH optimum of the enzyme under testing. The protonised (ionised) forms of amine buffers have
less inhibitory effects than the non-protonised forms. For Tris and zwitterionic buffers, therefore, a working range slightly
lower than the pKa value is usually more suitable, whilst in contrast to this, carboxylic acid buffers with a working range
slightly above their pKa values are better suited, since these buffers consist mainly of the ionised form (Good & Izawa 1972).
Determination of the optimum buffer concentration

An adequate buffer capacity is often only reached at concentrations higher than 25 mM. However, higher buffer concen
trations and related high ionic strengths can inhibit enzyme activity. Suitable initial concentrations are therefore between
10 and 25 mM. If, after addition of the protein or enzyme, the pH value changes by more than 0.05 units, the concentration
of the buffer can first be increased to 50 mM. Up to this concentration, no interference was observed with the Good buffers
in cell culture experiments, for example (Ferguson et al. 1980). In order to form complexes with heavy metals, EDTA can
be added in small amounts to buffers, if desirable (10 – 100 µM; Stoll & Blanchard 1990). Between 0.1 and 5.0 mM
chelating agents can be added to achieve the complete removal of multivalent cations.
Application-dependent choice of buffer substances

The decision for or against a buffer is also dependent on the method for which it is used. In addition to the measurement
of activity, the concentration is also usually determined when proteins or enzymes are undergoing purification. In assays
using reagents for protein determination, many of the buffer substances based on amino acids can lead to false-positive
results due to interactions with the reagents or absorption of the buffer substance itself in the range above 230 nm. For
example, various buffers interfere with the Lowry protein assay (see below). Such interference can, however, usually be
relatively easily abolished by inclusion of the buffer in the blank control (Peterson 1979).
Many buffers are basically suitable for gel filtration. Cationic buffers such as Tris are preferred for anion exchange
chromatography. Anionic buffers (such as phosphate or MES) should be preferred for cation exchange chromatography or
hydroxylapatite chromatography, i.e. the buffer should have the same charge as the ion exchange material, to prevent it
binding itself to the ion exchanger (Blanchard 1984; Scopes 1994). The buffer requirements for ion exchange chromato-
graphy are discussed in detail by Scopes (1984).
For example, borate is not suitable for the isolation of glycoproteins or systems that include nucleotides, since it interacts
with the cis-hydroxyl group of sugars. If electrophoresis is performed subsequent to dissolution of the protein in protein
purification systems, a buffer with a low ionic strength should be used, since a high ionic strength would heat up the gel
(Hjelmeland & Chrambach, 1984).
The Good buffers based on the piperazine ring – HEPES, HEPPS, HEPPSO and PIPES – are not suitable for the investigation
of redox processes, since, in the presence of H2O2, oxygen radicals, autooxidising iron or, under certain electrolytic condi-
tions, they easily form radicals. In contrast to this, the Good buffer based on a morpholine ring, MES, does not form any
radicals (Grady et al. 1988).

Tris buffer: not always the best choice! (Sambrook & Russell 2001) pH value of a
Tris (tris-(hydroxymethyl)-aminomethane) is probably the most frequently used buffer substance in 50 mM Tris solution
biological experiments. The reasons for this are that Tris is comparatively inexpensive, very freely soluble 5°C 25°C 37°C
in water, is inert in many enzymatic systems (no interactions with other components) and has a high 9.5 8.9 8.6
buffer capacity. Since, however, Tris may have a series of negative characteristics, these are presented here
in detail. Temperature dependency
1.) The pKa value of Tris is 8.06 at 25°C. This means that it is already at the upper end of the pH range of the pH value of a
of many biological systems (pH 6.0 – 8.0) and that it has a relatively low buffer capacity in the 50 mM Tris solution
actual physiological pH range (7.0 – 7.5). (pH value set at 25°C)
2.) Tris buffers have a significantly high degree of temperature sensitivity. The effects are therefore very The pH values to be expected
different when Tris is used in the cold room, at room temperature, or at 37°C. This means that the at +4°C and +37°C are given.
pH value has to be set for the ambient temperature at which it is used. 4°C 25°C 37°C
3.) Tris reacts with many pH electrodes that have a linen-fibre junction. This results in high liquid-
7.79 7.20 6.86
junction potentials, electromotive force drifts (emf) and long equilibration times. This means that
only electrodes with ceramic or glass junctions can be used which are declared as suitable by the 7.89 7.30 6,96
manufacturer. 7.99 7.40 7,06
4.) The pH value of a Tris solution is concentration-dependent. On dilution, the pH value decreases by 8.09 7.50 7.16
0.1 pH unit, when diluted from 100 mM to 10 mM. 8.19 7.60 7.26
5.) Tris is toxic for many mammalian cells, since it penetrates cells due to its relatively good fat solubility.
8.29 7.70 7.36
6.) Tris is a primary amine. It cannot be used with fixation reagents such as glutaraldehyde or form
aldehyde. It also reacts with glyoxal and DEPC. In such cases, phosphate, HEPES or MOPS buffers are 8.39 7.80 7.46
used instead. 8.49 7.90 7.56
8.59 8.00 7.66
Preparation of Tris solutions in a concentration of 50 mM 8.69 8.10 7.76
for 100 ml 50 mM Tris solution* for 1 liter 50 mM Tris solution 8.79 8.20 7.86
50 ml 100 mM Tris (12.11 g Tris base per liter dH2O) 6.057 g Tris base/900 ml liter dH2O 8.89 8.30 7.96
x ml 0,1 N HCl z ml 1.0 N HCl 8.99 8.40 8.06
y ml dH2O ad 100 ml dH2O ad 1 liter
9.09 8.50 8.16
9.19 8.60 8.26
pH 100 mM x ml y ml pH z ml
Tris 0.1 N HCl dH2O 1,0 N HCl 9.29 8.70 8.36
7.10 50 ml 45.7 4.3 7.10 45.7
7.20 50 ml 44.7 5.3 7.20 44.7
Preparation of
7.30 50 ml 43.4 6.6 7.30 43.4
1 M Tris solutions (1 liter)
7.40 50 ml 42.0 8.0 7.40 42.0 121.14 g Tris base in 800 ml
7.50 50 ml 40.3 9.7 7.50 40.3 dH2O set pH with concentrated
7.60 50 ml 38.5 11.5 7.60 38.5 hydrochloric acid dH2O ad
7.70 50 ml 36.6 13.4 7.70 36.6 1 liter
for 1 liter 1 M Tris
7.80 50 ml 34.5 15.5 7.80 34.5
pH x ml conc. HCl
7.90 50 ml 32.0 18.0 7.90 32.0
7.2 76.10
8.00 50 ml 29.2 20.8 8.00 29.2
7.5 69.10
8.10 50 ml 26.2 23.8 8.10 26.2
8.0 48.30
8.20 50 ml 22.9 27.1 8.20 22.9
8.5 23.90
8.30 50 ml 19.9 30.1 8.30 19.9
9.0 8.25
8.40 50 ml 17.2 32.8 8.40 17.2
8.50 50 ml 14.7 35.3 8.50 14.7
8.60 50 ml 12.4 37.6 8.60 12.4
8.70 50 ml 10.3 39.7 8.70 10.3
8.80 50 ml 8.5 41.5 8.80 8.5
8.90 50 ml 7.0 47.0 8.90 7.0
* Dawson, R.M.C. et al. (1986) Data for Biochemical Research. p. 436. Clarendon Press, Oxford.

TE buffer
10 mM Tris · HCl (pH 7.4, 7.5 or 8.0)
1 mM EDTA (pH 8.0)
This buffer has become the standard buffer for the storage of nucleic acids. It is used at different pH values. It is generally
prepared by mixing Tris buffer stock solutions (1 M) with an EDTA stock solution (0.5 M; pH 8.0). The prepared buffer can
also be stored at room temperature following autoclaving. TE stock solutions are prepared in concentrations of 100X to 1X.
Volatile buffer systems Volatile buffers

effective Description Counter ion pK-value A number of buffers are available that can be easily and
pH range completely removed. These buffers are used particularly
when subsequent reactions must not contain any
3.3 – 4.3 Formic acid H+ 3.75
disturbing components. They are useful for electro
3.3 – 4.3 Pyridine / formic acid HCOO– 3.75 phoresis, ion exchange chromatography, or for digestion
3.3 – 4.3 Trimethylamine / formic acid HCOO– 4.75 of proteins with subsequent removal of peptides and
3.3 – 4.3 Ammonia / formic acid HCOO– 3.75 amino acids. These buffer substances include: formic
4.3 – 5.3 Trimethylamine / acetic acid CH3CO– 4.75 acid, ammonia, ammonium carbonate, acetic acid, pyri-
dine and triethanolamine. A pH range of 1.9 – 8.9 can be
4.3 – 5.3 Ammonia / acetic acid CH3COO– 4.75
covered with appropriate mixtures of these substances.
4.3 – 5.3 N-ethylmorpholine / acetate HCOO– 4.75
4.3 – 5.8 Pyridine / acetic acid CH3COO– 4.75; 5.25
4.8 – 5.8 Pyridine / formic acid HCOO– 5.25 Buffer mixtures
5.9 – 6.9 Trimethylamine / carbonate CO32– 6.35 Since the maximum buffer range of a weak acid or base
is relatively narrow, namely one pH unit above and below
5.9 – 6.9 Ammonium bicarbonate HCO3– 6.35
the pKa value, it is necessary under certain circumstances
5.9 – 6.9 Ammonium carbonate / ammonia CO32– 6.35 to prepare mixtures of buffers that cover a wider pH
5.9 – 6.9 Ammonium carbonate CO32– 6.35 range and therefore have a constant buffer capacity in
6.8 – 8.8 Trimethylamine / hydrochloric acid Cl– 9.25 this range. For such mixtures, it is advisable to use buf-
7.0 – 8.2 N-ethylmorpholine / acetate HCOO– 7.72 fers with a similar structure that have overlapping
optimal buffer ranges (pH ranges) (e.g. MES/acetate/
8.8 – 9.8 Ammonia / formic acid HCOO– 9.25
Tris, pH 4.0 – 9.0). The pKa values should not be separa-
8.8 – 9.8 Ammonia / acetic acid CH3COO– 9.25 ted more than 1 – 2 pH units (Williams & Morrison
8.8 – 9.8 Ammonium bicarbonate HCO3– 9.25 1981, Blanchard 1984, Stoll & Blanchard 1990). The
8.8 – 9.8 Ammonium carbonate / ammonia CO32– 9.25 buffer capacities are additive where the ranges overlap.
8.8 – 9.8 Ammonium carbonate CO32– 9.25 These systems do, however, have disadvantages in some
situations. Since each of the components of the buffer
9.3 – 10.3 Trimethylamine / formic acid HCOO– 9.81
only buffers in a very narrow pH range, it is also present
9.3 – 10.3 Trimethylamine / acetic acid CH3COO– 9.81 outside the buffer range in its ionised form, and this
9.3 – 10.3 Trimethylamine / carbonate CO32– 9.81 ionised form may have inhibitory effects. In addition, the
from Dawson et al. 1986 and Stoll & Blanchard 1990 presence of different additional buffer substances
increases the ionic strength.
Buffer series or multicomponent buffers are used for the
determination of the pH-dependency of enzyme activity, for example. Examples for the use of buffer series are assays on
hexokinase from yeast (Viola & Cleland 1978), muscle creatine kinase from rabbits (Cook et al. 1981), dihydrofolate
reductase from S. faecium (Williams & Morrison 1981), chymase from humans (McEuen et al. 1995) and trehalase from
silkworm moths (Ando et al. 1995).
If the ionic strength is important, it can be reduced by choosing the appropriate buffer substances. The amount of acid or
alkali (electrolyte) that has to be added to set the pH value can be reduced by combining a weak acid with a weak base (Ellis
& Morrison 1982). And the ionic strength can also be maintained constant over wide pH ranges by choosing the right
buffers. Ellis & Morrison (1982) describe examples of three-component buffers of this type that can cover up to 4 pH
units.
Buffer mixtures are also used for high performance chromatofocusing. This chromatographic method enables the
separation, also of protein isoforms, for example, according to the surface charge of the protein in pH gradients which are
created by applying an electrical field. The focussing buffers used can have a very complex composition (31 components;
Hutchens et al. 1986).

Buffers for gel electrophoresis
Gel electrophoresis has become one of the most important methods in the analysis of nucleic acids and proteins. Three
principal buffers have established themselves as the standards for the techniques of polyacrylamide gel electrophoresis and
agarose gel electrophoresis: TAE buffer (Tris-acetate-EDTA), TBE buffer (Tris-borate-EDTA) and Tris-glycine buffer.
Depending on the application, other substances may be added to these, such as urea and SDS. Since there are many derived
methods based on these electrophoresis techniques, there is a correspondingly high number of modified buffers.
Electrophoresis buffers
TAE buffer (Tris-acetate-EDTA) Order No. A1691 Tris-glycine buffer (TG) Order No. A1418
50X stock solution 10X stock solution
(usual working concentration 0.5X–1X) 15.1 g Tris
242 g Tris 72.0 g Glycine
57.1 ml Glacial acetic acid add 5 liter dH2O
37.2 g EDTA – disodium salt - dihydrate Storage for up to 1 month at +4°C
set pH to 8.5
add 1 liter dH2O
SDS-Tris-glycine buffer Order No. A1415
TBE buffer (Tris-borate-EDTA) Order No. A0972 (Laemmli buffer)
10X stock solution 10X stock solution
(usual working concentration 1X) 30.29 g Tris (0.25 M)
108 g Tris (890 mM) 144.13 g Glycine (1.92 M)
55 g Boric acid (890 mM) 10.00 g SDS (1 %)
40 ml 0.5 M EDTA – disodium salt – dihydrate pH should be 8.3!
(pH 8.0) add 1 liter dH2O
set pH to 8.3
add 1 liter dH2O
The following buffers are also used for this electrophoresis system (Laemmli):
4X Tris/SDS pH 6.8 stacking gel buffer 6X SDS sample buffer
1. dissolve 6.05 g Tris-base in 40 ml H2O • 7 ml 4X Tris/SDS pH 6.8
2. adjust pH to 6.8 with 1 N HCl stacking gel buffer
3. add H20 to 100 ml • 3.0 ml glycerol
4. add 0.4 g SDS • 1 g SDS
store at room temperature • 0.93 g DTT (dithiothreitol)
• 1.2 mg bromophenol blue
4X Tris/SDS pH 8.8 resolving gel buffer • add dH2O to 10 ml
1. dissolve 91 g Tris-base in 300 ml H2O store in 1 ml aliquots at -20 °C
2. adjust to pH 8.8 with 1 N HCl
3. add H2O to 500 ml Tris-Tricine buffer
4. add 2 g SDS Working concentration (do not adjust pH)
store at room temperature 12.11 g Tris (0.1 M)
17.92 g Tricine (0.1 M)
1 g SDS ultrapure (0,1 %)
add 1 liter dH2O
Storage for up to 1 month at +4°C

t i p s
Technical tips
How can microbial contamination of buffer solutions be prevented?
1.) Sterilisation by filtration or autoclaving
2.) addition of 0.02 % (3 mM) sodium azide
3.) Storage at +4°C
4.) High-concentration stock solutions
Special note for buffers containing sodium hydrogen carbonate (sodium bicarbonate): this buffer substance requires a
closed system. In aqueous solutions, sodium hydrogen carbonate degrades into CO2 and sodium carbonate above 20°C.
Complete degradation occurs at 100°C. Solutions containing sodium hydrogen carbonate cannot therefore be autoclaved,
but have to be sterile filtered. When preparing, they should not be stirred too vigorously and too long. The pH of a freshly
prepared 100 mM solution is 8.3 at 25°C.
How can precipitation in concentrated TBE buffers be prevented?

Precipitation tends to occur in concentrated TBE buffer solutions (usually 10X) very soon after they are prepared. This can
be prevented by filtering the solution using a cellulose acetate or cellulose nitrate filter (0.2 – 0.45 µm). The vessels into
which the buffer is filtered must be dust-free. Salt crystals appear to be responsible for the precipitation, which form as
crystallisation buds on dust particles or other microscopically small particles. Concentrated TBE buffer solutions that have
become turbid can also be autoclaved (Mayeda & Krainer 1991).
What is the best way of obtaining solutions of the free acids of PIPES, POPSO or ADA?
The free acid of PIPES is very poorly soluble in water (only 1 g/L; see Good et al. 1966 [page 469]). By conversion to the
sodium salt with NaOH, the pH of the solution increases to higher than 6 and the salt is easily soluble. The same applies to
POPSO and ADA, which are very poorly soluble and are not soluble until converted to the sodium salt.
What is the importance of water as a solvent?

The buffer substances that are commercially available today are usually of the highest quality. For example, they are tested
for low heavy metal content, absence of endotoxins and enzyme contamination (DNases, RNases, proteases, phosphatases).
The water in which the buffer substances are dissolved is usually from the user’s laboratory where the buffer solutions are
prepared. Here, too, attention must be paid to using only the highest quality. Water that stands too long in pipes increases
the risk of contamination of the buffer solution. Gases may be dissolved into the water and contaminating agents may adhere
to the taps. The water should therefore be run for a short time before using it to prepare the buffer solution.
a
BCA Kaushal, V. & Barnes, L.D. (1986) Anal. Biochem. 157, 291-294 – Bicinchoninic Acid – protein detection:
the buffers were used in a concentration of 50 mM.
b
Lowry Peterson, G.L. (1979) Anal. Biochem. 100, 201-220 – with recommendations on how to minimise and rule out
disturbing factors and information on tolerable final concentrations. In some cases it is sufficient to include the substance
concerned as a control.
c
Radical formation Grady, J.K. et al. (1988) Anal. Biochem. 173, 111-115. Under certain conditions, the piperazine ring
system forms radicals. These buffers are therefore not suitable for the investigation of redox processes in biochemistry.
d
absence of any comments does not indicate that there is no influence on results.
10 Biological Buffers • AppliChem © 2008

(Disturbing) effects of biological buffers in different assays*
Buffer substance BCA a, d Lowry b, d Comments
(folin)
ACES + significant absorption of UV light at 230 nm, binds Cu2+
ADA + + marked absorption in UV range below 260 nm; binds metal ions
AMP
BES – + binds Cu2+
Bicarbonate limited solubility; needs closed system, since in equilibrium with CO2
Bicine + + slowly oxidised by ferricyanide; strongly binds Cu2+
Bis-Tris + substitute for cacodylate
Bis-Tris-Propane
s
Borate forms covalent complexes with mono- and oligosaccharides,
ribose subunits of nucleic acids, pyridine nucleotides, glycerol
Cacodylate very toxic; nowadays usually replaced by MES
CAPS – +
CAPSO
CHES +
Citrate binds to some proteins, forms complexes with metals; replaced by MES
DIPSO +
Glycine + interferes with Bradford protein assay
p
Glycylglycine + binds Cu2+
HEPES – + can form radicals, not suitable for redox studies
HEPPS, EPPS – + can form radicals, not suitable for redox studies
HEPPSO – + can form radicals, not suitable for redox studies
Imidazole forms complexes with Me2+, relatively instable
Maleic acid absorbs in the UV range; replaced by MES or Bis-Tris
MES – + substitute for cacodylate
MOPS – + partly degraded on autoclaving in the presence of glucose;
negligible metal ion binding
MOPSO
Phosphate
PIPES –
+
+
substrate/inhibitor of various enzymes
(inhibits many kinases and dehydrogenases, enzymes with phosphate
esters as substrate; inhibits carboxypeptidase, fumarase, urease);
precipitates/binds bivalent cations; pK increases on dilution;
can form radicals, not suitable for redox studies
i
POPSO +
TAPS +
t
TAPSO +
TEA
TES – + binds Cu2+
Tricine + + strongly binds Cu2+; addition of Cu2+ in the Lowry assay enables it to be
used; is photooxidised by flavines; substitute for barbital (Veronal)
Tris + + high degree of temperature-sensitivity; pH decreases by 0.1 unit with
each 10fold dilution; inactivates DEPC, can form Schiff’s bases with
aldehydes/ketones, as it is a primary amine; is involved in some
enzymatic reactions (e.g. alkaline phosphatase)
* partly taken from Bollag, D.M. & Edelstein, S.J. (1992) Protein Methods, Chapter 1, II (pp. 3-9). Wiley-Liss, New York.
© 2008 AppliChem • Biological Buffers11

Concentration limits for buffers in protein assays *
Buffer substance Lowry (Folin) BCA Bradford Colloidal Gold UV UV
280 nm 205 nm
Acetate 0.2 M 0.6 M 0.1 M 10 mM
Borate 10 mM >100 mM
Citrate 2.5 mM < 1 mM 50 mM 5 % <10 mM
Glycine 2.5 mM 1 M 0.1 M 100 mM 1 M 5 mM
HEPES 2.5 µM 100 µM 100 mM 20 mM <20 mM
Phosphate 250 mM 250 µM 2 M 100 mM 1 M 50 mM
Tris 250 mM 0.1 M 2 M 0.5 M 40 mM
* according to Stoscheck, C.M. (1990) Methods Enzymol. 182, 50-68 – The values given correspond to the final
concentration. In case of the UV absorption, the final concentration of the chemical corresponds to an absorption value
smaller than 0.5 above water. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254
Saturated concentrations of buffers in solution at 0°C

(according to Good et al. 1966, Good & Izawa 1972, Ferguson et al. 1980)
Buffer substance Concentration (M) Buffer substance Concentration (M)
ACES 0.22 HEPPSO 2.20
ADA 0.09 Potassium phosphate 2.50
BES 3.20 MES 0.65
Bicine 1.10 MOPS 3.00
CAPS 0.47 MOPSO 0.75
CHES 1.14 PIPES 2.30
DIPSO 0.24 TES 2.60
Glycylglycine 1.10 TAPSO 1.00
HEPES 2.25 Tricine 0.80
HEPPS 4.58 Tris 2.40
“Old” buffers replaced by buffers with better properties

(according to Scopes 1994)
“Old” Buffer Unwanted properties Recommended substitute

Veronal (5,5-Diethylbarbituric acid; Barbital) toxic Tricine, Tris
Cacodylic acid, Cacodylate toxic MES, Bis-Tris
Citric acid, Citrate complexes metal ions MES, Bis-Tris
Maleic acid UV-Absorption MES, Bis-Tris
t i p s
l i s t
Alphabetical list of biological buffers
Trivial name Name
ACES N-(2-Acetamido)-aminoethanesulfonic acid
Acetate Salt of acetic acid
ADA N-(2-Acetamido)-iminodiacetic acid
AES 2-Aminoethanesulfonic acid, Taurine
Ammonia –
AMP 2-Amino-2-methyl-1-propanol
AMPD 2-Amino-2-methyl-1,3-propanediol, Ammediol
AMPSO N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid
BES N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid
Bicarbonate Sodium hydrogen carbonate
Bicine N,N’-Bis(2-hydroxyethyl)-glycine
BIS-Tris [Bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane)
BIS-Tris-Propane 1,3-Bis[tris(hydroxymethyl)-methylamino]propane
Boric acid –
Cacodylate Dimethylarsinic acid
CAPS 3-(Cyclohexylamino)-propanesulfonic acid
CAPSO 3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid
Carbonate Sodium carbonate
CHES Cyclohexylaminoethanesulfonic acid
b u f f e r
Citrate Salt of citric acid
DIPSO 3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid
Formate Salt of formic acid
Glycine –
Glycylglycine –
HEPES N-(2-Hydroxyethyl)-piperazine-N’-ethanesulfonic acid
HEPPS, EPPS N-(2-Hydroxyethyl)-piperazine-N’-3-propanesulfonic acid
HEPPSO N-(2-Hydroxyethyl)-piperazine-N’-2-hydroxypropanesulfonic acid
Imidazole –
Malate Salt of malic acid
Maleate Salt of maleic acid
MES 2-(N-Morpholino)-ethanesulfonic acid
MOPS 3-(N-Morpholino)-propanesulfonic acid
MOPSO 3-(N-Morpholino)-2-hydroxypropanesulfonic acid
Phosphate Salt of phosphoric acid
PIPES Piperazine-N,N’-bis(2-ethanesulfonic acid)
POPSO Piperazine-N,N’-bis(2-hydroxypropanesulfonic acid)
Pyridine –
Succinate Salt of succinic acid
TAPS 3-{[Tris(hydroxymethyl)-methyl]-amino}-propanesulfonic acid
TAPSO 3-[N-Tris(hydroxymethyl)-methylamino]-2-hydroxypropanesulfonic acid
Taurine 2-Aminoethanesulfonic acid, AES
TEA Triethanolamine
TES 2-[Tris(hydroxymethyl)-methylamino]-ethanesulfonic acid
Tricine N-[Tris(hydroxymethyl)-methyl]-glycine
Tris Tris(hydroxymethyl)-aminomethane

Temperature dependence of the pKa value
temperature dependence
of biological buffers (100 mM)
effective Description d(pKa)/dT pKa (0°C) pKa (4°C) pKa (20°C) pKa (25°C) pKa (37°C)
pH range
1.2 – 2.6 Maleate (pK1) 1.97
1.7 – 2.9 Phosphate (pK1) 0.0044 2.15
2.2 – 3.6 Glycine (pK1) 2.35
2.2 – 6.5 Citrate (pK1) 3.13
2.5 – 3.8 Glycylglycine 3.14
2.7 – 4.2 Malate (pK1) 3.40
3.0 – 4.5 Formate 0.0 3.75
3.0 – 6.2 Citrate (pK2) -0.0016 4.79 4.77 4.76 4.74
3.2 – 5.2 Succinate (pK1) -0.0018 4.21
3.6 – 5.6 Acetate 0.0002 4.76
4.0 – 6.0 Malate (pK2) 5.13
4.9 – 5.9 Pyridine -0.014 5.23
5.0 – 7.4 Cacodylate 6.27
5.5 – 6.5 Succinate (pK2) 0.0 5.64
5.5 – 6.7 MES -0.011 6.38 6.33 6.15 6.10 5.98
5.5 – 7.2 Maleate (pK2) 6.15 6.24
5.5 – 7.2 Citrate (pK3) 0.0 6.40
5.8 – 7.2 BIS-Tris -0.017 6.82 6.54 6.46 6.25
5.8 – 8.0 Phosphate (pK2) -0.0028 7.26 7.21 7.20 7.16
6.0 – 7.2 ADA -0.011 6.85 6.80 6.60 6.59 6.45
6.0 – 8.0 Carbonate (pK1) -0.0055 6.30 6.35
6.1 – 7.5 PIPES -0.0085 7.02 6.94 6.80 6.76 6.66
6.1 – 7.5 ACES -0.020 7.32 7.20 6.90 6.78 6.56
6.2 – 7.6 MOPSO -0.015 6.95 6.87
6.2 – 7.8 Imidazole -0.020 7.37 7.05 6.95 6.71
6.3 – 9.5 BIS-Tris-Propane 6.80
6.4 – 7.8 BES -0.016 7.50 7.41 7.15 7.09 6.90
6.5 – 7.9 MOPS -0.011 7.41 7.20 7.14 6.98
6.8 – 8.2 TES -0.020 7.92 7.82 7.50 7.40 7.14
6.8 – 8.2 HEPES -0.014 7.85 7.77 7.55 7.48 7.31
7.0 – 8.2 DIPSO -0.015 7.60 7.52
7.0 – 8.2 TAPSO -0.018 7.70 7.61
7.0 – 8.3 TEA -0.020 7.76
7.1 – 8.5 HEPPSO -0.010 7.90 7.85
7.2 – 8.5 POPSO -0.013 7.85 7.78
7.4 – 8.8 Tricine -0.021 8.60 8.49 8.15 8.05 7.80
7.5 – 8.9 Glycylglycine -0.025 9.00 8.85 8.40 8.25 7.90
7.5 – 9.0 Tris -0.028 8.90 8.80 8.30 8.06 7.70
7.6 – 8.6 HEPPS. EPPS -0.015 8.18 8.10 8.00 7.81
7.6 – 9.0 Bicine -0.018 8.70 8.64 8.35 8.26 8.04

continued
effective Description d(pKa)/dT pKa (0°C) pKa (4°C) pKa (20°C) pKa (25°C) pKa (37°C)
pH range
7.7 – 9.1 TAPS +0.018 8.02 8.31 8.40 8.62
7.8 – 9.7 AMPD -0.029 8.80
8.3 – 9.7 AMPSO 9.10 9.00
8.4 – 9.6 Taurine (AES) -0.022 9.06
8.5 – 10.2 Boric acid (pK1) -0.008 9.23
8.8 – 9.9 Ammonia -0.031 9.25
8.6 – 10.0 CHES -0.011 9.73 9.55 9.50 9.36
8.7 – 10.4 AMP -0.032 9.69
8.8 – 10.6 Glycine (pK2) -0.025 10.30 9.90 9.78 9.48
8.9 – 10.3 CAPSO 9.60
9.5 – 11.1 Carbonate (pK2) -0.009 10.33
9.7 – 11.1 CAPS -0.009 10.40
Phosphate (pK3) -0.026 12.33
Boric acid (pK2) 12.74
Boric acid (pK3) 13.80
d(pKa)/dT from Ellis & Morrison 1982 and Good & Izawa 1972 and Dawson et al. 1986
pKa 25°C from Stoll & Blanchard 1990 and Dawson et al. 1986
pKa 20°C from Good et al. 1966 and Good & Izawa 1972 and Ferguson et al. 1980
pKa 0°C and 37°C from Good et al. 1966
Depending on the author small differences may occur!
temperature
dependence

pK

pKa
(25°C)
a
pKa values of biological buffers (25°C, 100 mM), alphabetical list
Description effective pH
range
values
Description pKa
(25°C)
effective pH
range
Description

pKa
(25°C)
effective pH
range
ACES 6.78 6.1 – 7.5 CHES 9.50 8.6 – 10.0 MOPS 7.14 6.5 – 7.9
Acetate 4.76 3.6 – 5.6 Citrate (pK1) 3.13 2.2 – 6.5 MOPSO 6.87 6.2 – 7.6
ADA 6.59 6.0 – 7.2 Citrate (pK2) 4.76 3.0 – 6.2 Phosphate (pK1) 2.15 1.7 – 2.9
Ammonia 9.25 8.8 – 9.9 Citrate (pK3) 6.40 5.5 – 7.2 Phosphate (pK2) 7.20 5.8 – 8.0
AMP 9.69 8.7 – 10.4 DIPSO 7.52 7.0 – 8.2 Phosphate (pK3) 12.33
AMPD 8.80 7.8 – 9.7 Formate 3.75 3.0 – 4.5 PIPES 6.76 6.1 – 7.5
AMPSO 9.00 8.3 – 9.7 Glycine (pK1) 2.35 2.2 – 3.6 POPSO 7.78 7.2 – 8.5
BES 7.09 6.4 – 7.8 Glycine (pK2) 9.78 8.8 – 10.6 Pyridine 5.23 4.9 – 5.9
Bicine 8.26 7.6 – 9.0 Glycylglycine 3.14 2.5 – 3.8 Succinate (pK1) 4.21 3.2 – 5.2
BIS-Tris 6.46 5.8 – 7.2 Glycylglycine 8.25 7.5 – 8.9 Succinate (pK2) 5.64 5.5 – 6.5
BIS-Tris-Propane 6.80 6.3 – 9.5 HEPES 7.48 6.8 – 8.2 TAPS 8.40 7.7 – 9.1
Boric acid (pK1) 9.23 8.5 – 10.2 HEPPS, EPPS 8.00 7.6 – 8.6 TAPSO 7.61 7.0 – 8.2
Boric acid (pK2) 12.74 HEPPSO 7.85 7.1 – 8.5 Taurine (AES) 9.06 8.4 – 9.6
Boric acid (pK3) 13.80 Imidazole 6.95 6.2 – 7.8 TEA 7.76 7.0 – 8.3
Cacodylate 6.27 5.0 – 7.4 Malate (pK1) 3.40 2.7 – 4.2 TES 7.40 6.8 – 8.2
CAPS 10.40 9.7 – 11.1 Malate (pK2) 5.13 4.0 – 6.0 Tricine 8.05 7.4 – 8.8
CAPSO 9.60 8.9 – 10.3 Maleate (pK1) 1.97 1.2 – 2.6 Tris 8.06 7.5 – 9.0
Carbonate (pK1) 6.35 6.0 – 8.0 Maleate (pK2) 6.24 5.5 – 7.2
Carbonate (pK2) 10.33 9.5 – 11.1 MES 6.10 5.5 – 6.7
References
literature
(1) Ando, O. et al. (1995) Biochim. Biophys. Acta 1244, 295-302
(2) Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.
& Struhl, K. (eds.) (1995) Current Protocols in Molecular Biology. Greene
(14) Hutchens, T.W. et al. (1986) J. Chromatogr. 359, 157-168
(15) Kaushal, V. & Barnes, L.D. (1986) Anal. Biochem. 157, 291-294
(16) Liu, Q. et al. (1999) Anal. Biochem. 270, 112-122
Publishing & Wiley-Interscience, New York (17) Mayeda, A. & Krainer A.R. (1991) Biotechniques 10, 182
(3) Blanchard, J.S. (1984) Methods Enzymol. 104, 404-414 (18) McEuen, A.R. et al. (1995) Biochim. Biophys. Acta 1267, 115-121
(4) Bollag, D.M. & Edelstein, S.J. (1992) Protein Methods. Chapter 1, II. Wiley-Liss. (19) Peterson, G.L. (1979) Anal. Biochem. 100, 201-220
New York. (20) Sambrook, J. & Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual.
(5) Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 3rd Edition, Page A1.3. CSHL Press Cold Spring Harbor. New York
(6) Cook, P.F. et al. (1981) Biochemistry 20, 1204-1210 (21) Scopes, R.K. (1994) Protein Purification, Principles and Practice 3rd ed.,
(7) Dawson, R.M.C. et al. (1986) Data for Biochemical Research. Clarendon Press, Springer-Verlag New York Berlin Heidelberg
Oxford. (22) Stoll, V.S. & Blanchard, J.S. (1990) Methods Enzymol. 182, 24-38
(8) Ellis, K.J. & Morrison, J.F. (1982) Methods Enzymol. 87, 405-426 (23) Stoscheck, C.M. (1990) Methods Enzymol. 182, 50-68
(9) Ferguson, W.J. et al. (1980) Anal. Biochem. 104, 300-310 (24) Tipton, K.F. & Dixon, H.B.F. (1979) Methods Enzymol. 63, 183-234
(10) Good, N.E. et al. (1966) Biochemistry 5, 467-477 (25) Viola, R.E. & Cleland, W.W. (1978) Biochemistry 17, 4111-4117
(11) Good, N.E. & Izawa, S. (1972) Methods Enzymol. 24, 53-68 (26) Wenner, J.R. & Bloomfield, V.A. (1999) Anal. Biochem. 268, 201-212
(12) Grady, J.K. et al. (1988) Anal. Biochem. 173, 111-115 (27) Wilfinger, W.W. et al. (1997) Biotechniques 22, 474-481
(13) Hjelmeland, L.M. & Chrambach, A. (1984) Methods Enzymol. 104, 305-318 (28) Williams J.W. & Morrison, J.F. (1981) Biochemistry 20, 6024-6029

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taining
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eMail service@applichem.com internet www.applichem.com
STRATAGENE
Product Update
High-level expression of heterologous proteins in E. coli
BL21-CodonPlus Cells Correct Expression ™
Problems Caused by Codon Bias

Carsten-Peter Carstens • Julie Bonnardel • Ronda Allen • Anna Waesche
Stratagene
To resolve the codon bias problem that can hinder the expression of of tRNA genes that are rare in E. coli but used more frequently in
heterologous proteins in E. coli hosts, Stratagene has developed other organisms, dramatically improving heterologous protein
BL21-CodonPlus competent cells.* These specially engineered
™
Figure 1
derivatives of BL21-Gold cells improve protein expression by supplying Expression of AT-Rich Genes
additional copies of specific tRNA genes that are rare in E. coli.
Stratagene offers several varieties of BL21-CodonPlus cells, so that
high-level expression can be customized for different genomes, expression
vectors, and applications. BL21-CodonPlus-RIL and
BL21-CodonPlus-RP competent cells rescue expression of genes derived
from AT- and GC-rich genomes, respectively.
BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP cells
feature IPTG- inducible expression of T7 promoter controlled genes. ‡‡
BL21-CodonPlus(DE3)-RIL-X and BL21-CodonPlus(DE3)-RP-X cells are

methionine auxotrophs for abundant production of methionine-labeled proteins
for structural analysis by crystallography. Here, we compare expression
levels of recombinant proteins produced in conventional BL21 versus
BL21-CodonPlus competent cells.
Cultures of BL21-(DE3) or BL21-CodonPlus (DE3)-RIL cells that
Expression of recombinant proteins in E. coli offers the contained expression vectors with one of three different proteins (A, B,
and C) were induced at log phase growth with IPTG. Cell lysates were
advantages of speed, simplicity, and high-level expression. separated by SDS-PAGE and visualized by staining with Coomassie®
However, expression of heterologous genes in E. coli is hardly Blue dye.
infallible. The genomes of certain organisms favor sequences
with codons that occur infrequently in E. coli hosts. Forced 2,3 production within these E. coli expression systems.
high-level expression of rare codon-containing genes in E. coli BL21-CodonPlus-RIL cells contain extra copies of the argU,
depletes the endogenous pool of corresponding tRNAs. The ileY, and leuW tRNA genes, which recognize the AGA/AGG,
deficit of tRNA molecules disrupts translation, leading to AUA, and CUA codons, respectively. These codons are a
truncated protein expression or no protein expression. Other problem predominantly in organisms with AT-rich genomes.
symptoms of this problem include frameshifts, codon skipping, BL21-CodonPlus-RP competent cells contain extra copies of
and misincorporations. Ultimately, protein expression is slowed the tRNA gene argU, which recognizes the AGA and AGG
or aborted and mRNA is degraded. 4 arginine codons, and the tRNA gene proL, which recognizes the
Resolving codon bias is challenging. Typical remedies include proline codon, CCC. These codons appear frequently in GC-rich
altering the codon specifications of the heterologous gene by genomes, such as mammals.
site-directed mutagenesis or recloning the gene into eukaryotic BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP
expression systems. These solutions are not only inconvenient, cells offer IPTG-inducible protein expression, as they contain
they are also expensive and require a significant output of labor, T7 RNA polymerase under the control of the lacUV5 promoter.
time, and reagents. When used in conjunction with T7 promoter-driven expression
vectors, such as Stratagene’s Affinity pCAL and pET-type
®
Modified Strains Contain Supplemental tRNA Genes vectors, the expressed T7 RNA polymerase provides significant
Stratagene has developed a better solution to the codon bias levels of transcription of heterologous genes. The newest
problem by engineering special BL21 strains that supply extra
1
m e m b e r s of the BL21-CodonPlus family are
copies of tRNA genes that are rare to E. coli. BL21-CodonPlus BL21-CodonPlus(DE3)-RP-X and BL21-CodonPlus(DE3)-RIL-X
competent cells maintain important features of their parental competent cells, which are methionine auxotrophs. These cells are
BL21-Gold cells, such as Lon and OmpT protease deficiencies designed to metabolically label proteins with methionine for analysis of
for preserving protein integrity, the endA 1 deficiency for protein structure by crystallography (data not shown).
ensuring nondegraded miniprep DNA, and the Hte phenotype
for increasing the transformation efficiency. However, the Rescuing Expression of Genes with Rare Codons
BL21-CodonPlus series of competent cells contains extra copies
50 STRATEGIES • Volume 14
STRATAGENE
Figure 2 problem, such as site-directed mutagenesis or recloning into a

Expression of GC-Rich Genes eukaryotic expression system. We compared the expression of
heterologous proteins in BL21 parental strains versus
BL21-CodonPlus cells. Our results confirm that
BL21-CodonPlus-RIL cells rescue expression of AT-rich genes
and that BL21-CodonPlus-RP cells rescue gene expression from
GC-rich organisms. All members of the BL21-CodonPlus family
correct codon bias problems and preserve high-level protein
expression. Therefore, these competent cells are valuable
expression hosts, even in the absence of a detected codon bias
problem.
REFERENCES
1. Jerpseth, M., Jerpseth, B., Breister, L., and Greener, A. (1998)
Strategies 11: 3-4.
2. Kane, J. (1995) Curr. Opin. Biotechnol. 6: 494-500.
3. Bonekamp, F., Andersen, H.D., Christensen, T., and Jensen, K.F.
(1985) Nucleic Acids Res. 13: 4113-4123.
4. Deana, A., Ehrlich, R., and Reiss, C. (1998) Nucleic Acids Res.
26: 4778-4782.
5. Carstens, C.-P. and Waesche, A. (1999) Strategies 12: 49-51.
6. Hu, X., Shi, Q., Yang, T., and Jackowski, G. (1996) Protein Expr. Purif.
7: 289-293.
* Patents Pending
‡‡ See inside front cover.
The hcTnT-wt (argU dependent) or CBP-3xP-Cre (proL dependent) test
genes were expressed in BL21-Gold(DE3) and BL21-CodonPlus(DE3)-RP
cells. Lanes 2 and 3: BL21-Gold(DE3) cells. Lanes 4 and 5:
BL21-CodonPlus(DE3)-RP cells.
Lane M: molecular weight markers; Lane 2: hcTnT-wt (human cardiac
troponin T) in BL21-Gold(DE3); Lane 3: CBP-3xP-Cre in
BL21-Gold(DE3); Lane 4: hcTnT-wt (human cardiac troponin T)
i n B L 2 1 - C o d o n P l u s - R P ( D E 3 ) ; L a n e 5 : C B P - 3 x P - C r e in
BL21-CodonPlus-RP(DE3). The band around 30 kb is the
chloramphenicol gene.
We transformed BL21-CodonPlus(DE3)-RIL cells and

BL21(DE3) cells with plasmids that encode three different genes
for which protein expression in E. coli would be affected by
codon bias. For each plasmid, a T7 RNA polymerase responsive
promoter regulated gene expression (Figure 1). Expression of
each recombinant gene was greatly increased in the
BL21-CodonPlus(DE3)-RIL cells when compared to expression
in BL21(DE3) parental cells. Moreover, because the presence of
extra copies of tRNA genes had no deleterious effects to the host
cells, we conclude that BL21-CodonPlus cells can also be used
for expressing genes with codons that are compatible with
conventional E. coli strains.
In a similar experiment, we transformed BL21-
CodonPlus(DE3)-RP and BL21-Gold (DE3) cells with plasmids
encoding the hcTnT-wt (argU dependent) gene or a proL dependent See next page for a list of
derivative of the Cre Recombinase gene, CBP-3xP-Cre (Figure 2). The
BL21-CodonPlus(DE3)-RP cells, but not the parental BL21-CodonPlus™ competent cells!
BL21-Gold(DE3) cells, demonstrated expression of both genes. These v
data demonstrate that BL21-CodonPlus(DE3)-RP cells are ideal
for high-level expression of heterologous genes restricted by the
presence of AGG/AGA and CCC codons in conventional BL21
hosts.
Conclusions
Stratagene’s family of BL21-CodonPlus competent cells allow
high-level expression of proteins that are difficult to express in
conventional E. coli hosts due to the codon bias of the
heterologous gene. All BL21-CodonPlus cells contain extra
copies of tRNA genes that rescue protein expression that would
otherwise be restricted. These cells offer a time- and
money-saving alternative to previous solutions to the codon-bias
STRATEGIES • Volume 14 51
STRATAGENE
BL21-CodonPlus™ Competent Cells

• Use for non-T7 polymerase
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10 x 0.1-ml aliquots extremely toxic proteins
• 1 x 10 cfu/µg of supercoiled
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7
in E. coli!
BL21-CodonPlus™ (DE3)-RP • Use with pET or pCAL
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#230255
10 x 0.1-ml aliquots • 1 x 107 cfu/µg of supercoiled When you need to produce recombinant protein, an
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• Use for labeling cells #230265
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• endA- for direct cloning
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bias is not an issue — this strain is endA minus to
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• Use with non-T7 Offer valid for Stratagene direct customers; all others, please contact
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5 x 0.2-ml aliquots #200133 your local distributor for more information.
• Use with lambda CE6 for for
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52 STRATEGIES • Volume 14
Buffer Preparation (Gozani Lab)
1. 1 M Tris-HCl Buffers
pH Volume (L) TrisBase (g) HCl (ml)

pH 7.0 2 242.2 150-155
pH 7.5 2 242.2 120-125
pH 8.0 2 242.2 80-85
Autoclavable.
2. EDTA 0.5 M (pH8.0)
0.5M, 1L: 148 g EDTA

+ ~30-40 g NaOH to adjust pH
(or 186 g EDTA-Na.2H2O + ~20 g NaOH)
Note: pH adjusted by NaOH is essential for solubility. Autoclavable.
3. TAE DNA Electrophoresis Buffer (50 X)

(2 M Tris, 50 mM EDTA)
2L
484 g Tris
114.2 ml glacial acetic acid
200 ml 0.5 M EDTA 8.0
To make 1x TAE 20 L, add 400 ml 50X buffer into 19.6 L ddH2O.
4. SDS-PAGE Gel Solutions
Vol (L) Tris (g) HCl (ml) 10% SDS (ml)

4x Lower gel buffer
1.5 M Tris-Cl, pH 8.8, 2 363.3 50-60 80 ml
0.4% SDS
4x Upper gel buffer

0.5 M Tris-Cl, pH 6.8, 2 121.1 70-80 80 ml
0.4% SDS
4.1 10% SDS

1L:
100g SDS into 1 L, heat to 68oC for solubility. pH ~6.6.
5. 5X SDS Loading Sample Buffer
100 ml
Stock solution Add volume
250 mM TrisHCl pH6.8 2M 12.5 ml
10% SDS 10 g
30% Glycerol 30 ml
5% β-mercapitalethanol (or 0.5M DTT) 5 ml
0.02% bromophenol blue 0.04% 52 ml
6. 6X DNA loading sample buffer:

(40% sucrose, 0.01-0.02% BPB)
100 ml
Add 40 g sucrose to 50 ml 0.04% BPB solution, adjust final volume 100 ml.
7. SDS-PAGE Electrophoresis Running Buffer (10x)

(1x: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH8.3)
10 L.
303 g Trisbase (FW 121.1)
1440 g glycine (FW 75.07)
100 g SDS
No need to adjust pH
8. Transfer Buffer without SDS (10x)

(1x: 25 mM Tris, 192 mM glycine, pH8.3)
10 L
303 g Trisbase,
1440 g glycine
No need to adjust pH
8.1 Transfer Buffer (1x)
500 ml
50 ml of 10x Transfer buffer (without SDS) or 10x SDS-PAGE running buffer (w/ SDS)
100 ml of Methanol (final 20% methanol)
350 ml ddH2O
9. TBS (10x)
(1x: 150 mM NaCl, 10 mM Tris pH8.0)
10 L
876.6 g NaCl (FW 58.44),
121.1 g Tris,
~40 ml HCl
to pH8.0
9.1 TBS-T (1x)
1L
100 ml 10x TBS
10 ml 10% Tween20 (final 0.1% v/v)
890 ml ddH2O
9.2 Block buffer

(5% Nonfat milk in TBS-T)
5g milk in 100 ml TBST
10. NaCl 4 M
2 L: 467.5 g NaCl. Autoclavable.
11. NaOH 10 M
0.5 L: 200 g
12. NaAc 3 M
500 ml: add 204 g NaAc.3H2O (FW 136), adjust pH by glacial acetic acid (~60 ml)
to pH5.2. Autoclavable.
13. MgCl2 1M
500 ml: Add 101.65 g MgCl2.6H2O into 500 ml ddH2O. Autoclavable.
14. CaCl2 1M
400 ml: Add 58.8 g CaCl2.2H2O (FW 147), filter for sterilization.
Dilute 10x to make 100 mM CaCl2.
15. MgSO4 1M
500 ml: Add 123.3 g MgSO4.7H2O into 500 ml ddH2O. Autoclavable.

16. ZnCl 0.5M
50 ml: 3.4 g ZnCl to 50 ml.

Stock in -20oC
1. IPTG (1 M)
1 g IPTG (FW 238.3) resolved in 4.2 ml (~4 ml) ddH2O, filter through 0.22 µm
filters, aliquot 1 ml in eppendorf. Store at -20oC.
2. DTT (1 M)
5 g DTT (FW 154.25) resolved in 32.5 ml (~30 ml)10 mM NaAc (pH 5.2), filter
through 0.22 µm filters, aliquot 1 ml in eppendorf. Store at -20oC.
3. X-gal (20mg/ml)
Add 5 ml (~4.8 ml) DMSO into 100 mg X-gal bottom (FW 408.24). Store at -20oC.
4. PMSF (100 mM, =17.4 mg/ml)

Resolve 1.74g PMSF (MW 174) in isoproponal, total 100 ml. Aliquot and store at -
20oC or R.T..
5. Carbencillin or Ampcillin (50 mg/ml) in water. 1000x

2.5 g 50 ml.
6. Kanamycin (10 mg/ml) in water. 200x

0.5 g 50 ml.
7. Chloramphenicol (34 mg/ml) in ethanol. 200x

1.7 g/ 50 m l.
8. lysozyme 50 mg/ml, 1000x.

2.5 g/ 50 ml.
9. TSA (MW 303):
Add 1.32 ml Ethanol into each vial (1 mg?) to make the TSA stock 2.5 mM, 5000x.
Final concentration of TSA in the cell culture is 0.5 μM (~150 ng/ml).
Solutions.
1. Bacteria lysis buffer (GST pull-dwon binding buffer)

(50 mM Tris 7.5, 150 mM NaCl, 0.05% NP-40.)
1L
50 ml 1M Tris HCl 7.5;
37.5 ml 4 M NaCl;
5 ml 10% NP-40.
ddH2O to 1L.
1.2. GST pull-dwon binding buffer (1 M)

(50 mM Tris 7.5, 1 M NaCl, 1% NP-40.)
500 ml
25 ml 1M Tris HCl 7.5;
125 ml 4 M NaCl;
50 ml 10% NP-40.
ddH2O to 500ml.
2. RIPA Buffer
(50 mM TrisHCl pH7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS)
1L
50 ml 1 M Tris 7.4,
37.5 ml 4 M NaCl,
4 ml 0.5 M EDTA,
10 ml NP-40.
10 ml 10% SDS.
3. Cell Lysis Buffer (Flag-IP buffer)
(50 mM TrisHCl pH7.4, 250 mM NaCl, 0.5% Triton X100, 10% glycerol, 1 mM
DTT, PMSF, PI (Roche))
1L
50 ml 1 M Tris 7.4,
62.5 ml 4 M NaCl,
5 ml Triton X-100,
1 ml 1 M DTT,
100 ml glycerol.
4. H-Lysis solution:
(0.25M sucrose (MW=342), 3 mM CaCl2, 1 mM Tris pH8.0, 0.5% NP-40)
500 ml
43 g sucrose
1.5 ml 1 M Cacl2
0.5 ml 1 M Tris pH8.0
25 ml 10% NP-40
add ddH2O to 100 ml
Filter sterilize, store at 4oC.
5. H-Wash solution:
(300 mM NaCl, 5 mM MgCl2, 5 mM DTT, 0.5% NP-40)
500 ml
37.5 ml 4 M NaCl
2.5 ml MgCl2
2.5 ml DTT
25 ml 10% NP-40
6. H-Extraction solution (Histones Extraction):

(0.5 M HCl, 10% glycerol, 0.1 M 2-mercaptoethylamine-HCl (MW: 113.6)).
50 ml
2.25 ml HCl (11.2 M)
10 ml 50% glycerol.
0.55 g 2-mercaptoethylamine-HCl (Sigma name: cysteamine hydrochloride)
Recipe of making SDS-PAGE
SDS-PAGE 12% resolve 10% resolve gel 4% stacking gel

gel 10 ml
4x buffer 10 5 2.5
20 ml 40% Acr-Bis 6 5 1
(For 4x1mm ddH2O 4 10 6.5
plate) 10% APS (ul) 100 100 100
TEMED (ul) 20 20 20
4x buffer 5 5 2.5
20 ml 30% Acr-Bis 8 6.6 1.3
(For 4x1mm ddH2O 7 8.4 6.2
plate) 10% APS (ul) 100 100 100
TEMED (ul) 20 20 20
Voltage Time Buffer Volume

Electrophoresis 150V/200V 1h Tris/Glycine/SDS 300 ml tank
(15/30 mA)
Semi-Dry 5V 1-2 h 20% Methanol Make 500 ml
Transfer (40-60 mA/gel) 1x SDS running for 4 gels
buffer
Agarose 100V 30min-1h 10 μl EB to 100 350 ml tank

ml agarose/TAE
Buffers
Buffer solutions are made up of either a weak acid and it’s conjugate base or a weak base
and its conjugate acid.
Examples of buffers:
HCN/CN-
H2CO3/HCO3-
NH3/NH4+
HLac/Lac-
NH2OH/HNH2OH+
Henderson – Hasselbalch Equation
pH = pKa + log
[base]
[acid ]
The first thing you need to do when reading a buffer problem is determine which
compound is the weak acid (or weak base) and which component is the conjugate base
(or acid). For example, HF is a weak acid whose conjugate base is F-. The F- is added in
the form of a salt NaF. In water 100% of the NaF separates into Na+ and F- ions. The Na+
ions are spectator ions and are not included in the equation.
If you are given a Ka then it is the easiest (but not required) to set up an equation with
acid on the left side (see example i-a) If you write the equation with base on the left you
will need to use the Kb (see example i-b). In many cases a buffer problem can be easily
solved using Henderson – Hasselbalch Equation (see example i-c).
Example:
i Calculate the pH of the buffer solution that is 0.15 M HF and 0.15 M F-. Ka for HF is
6.8 x 10-4.
We can solve this problem in 3 different ways
a Using Ka
Conc. (M) HF + H2O ! H3O+ + F-
Initial [ ] 0.15 0 0.15

Change [ ] -x +x +x
Equilibrium [ ] 0.15 – x x 0.15 + x
1
 H 3O +   F-  x (0.15 + x )
Ka =  = 6.8 x 10-4 =
[HF] 0.15- x
Solve the equation for x assuming that x is negligible compared to 0.15. (If you
have forgotten the rule for ignoring x see Chemical Equilibrium handout)
0.15x
= 6.8 x 10-4 ⇒ x = 6.8 x 10-4 ⇒  H 3O +  = 6.8 x 10-4
0.15
pH = -log  H 3O +  = -log (6.8 x 10-4)
pH = 3.17
or
b Using Kb
Conc. (M) F- + H2O ! HF + OH-
Initial [ ] 0.15 0.15 0

Change [ ] -x +x +x
Equilibrium [ ] 0.15 – x 0.15 + x x
Kw 1x10-14
Kb = = -4
= 1.5 x 10-11
Ka 6.8 x10
[HF] OH-  (0.15 + x ) x

Kb = = 1.5 x 10-11 =
 F 
-
0.15- x
Solve the equation for x assuming that x is negligible compared to 0.15
0.15x
= 1.5 x 10-11 ⇒ x = 1.5 x 10-11 ⇒ OH -  = 1.5 x 10-11
0.15
pOH = -log OH -  = -log (1.5 x 10-11) = 10.83
pH = 14.00 – pOH = 14.00 – 10.83
2
pH = 3.17
or
c Using the Henderson – Hasselbalch Equation
pH = pKa + log
[base]
[acid ]
 F- 
pH = - log Ka + log
[HF]
0.15
pH = - log(6.8 x 10-4) + log = 3.17 + log 1 = 3.17 + 0
0.15
pH = 3.17
Adding strong acid or strong base to the buffer solution
By adding a small amount of HCl (strong acid) to the buffer, the HCl will react with the
base part of the buffer. At the end of this irreversible reaction no HCl will be left. The
base will then convert to a weak acid, which then dissociates only slightly into H3O+.
Therefore, the pH drops only a little.
By adding a small amount of NaOH (strong base) to the buffer, the NaOH will react with
the acid part of the buffer. At the end of this irreversible reaction no NaOH will be left.
The acid will then convert to a weak base, which then dissociates only slightly into OH-.
Therefore, the pH goes up only a little.
Steps for calculating the pH of a buffer solution after acid or base has been added to it:
1. Write an equation for irreversible reaction of the strong acid with the base
component of the buffer or the irreversible reaction of the strong base and the acid
component of the buffer. Note that the ion associated with the strong acid/base
becomes a spectator ion and is NOT involved in the reaction.
2. Calculate the moles of the conjugate acid/base pair of the buffer and the moles of
strong acid or base added to the buffer.
3. Under the irreversible equation write a chart showing moles of all the reaction
components before the reaction starts, the change into moles of the reactants and
products and the moles of all components after the reaction is over. This is called
the BCA chart (see example below).
4. Find the limiting reagent.
5. Calculate the moles of all substances after the reaction is over.
6. At this point if the strong acid/base added to the buffer was the limiting reagent
we still have a buffer solution. Calculate the concentration off the buffer solution
by taking into consideration any volume changes in the solution.
7. Calculate the pH of the buffer solution in any of the 3 ways shown above.
3
Examples:
i Calculate the pH of the solution formed when 7.0 ml of 0.10 M NaOH is added to
100 ml of the 0.15 M HF/F- buffer solution (this is the buffer solution that we used to
calculate the pH in the example above). The Ka for HF is 6.8 x 10-4.
mol HF
mol HF = 0.15 x 0.100 L = 0.015 mol HF
L
- mol F-
mol F = 0.15 x 0.100 L = 0.015 mol F-
L
mol OH -
mol OH- = 0.10 x 0.0070 L = 0.00070 mol OH-
L
Irreversible Reaction. (Note: Chart done in moles NOT molarity)
mol HF + OH- → F- + H2O
Before 0.015 0.00070 0.015

Change - 0.00070 -0.00070 + 0.00070
After 0.014 0 0.016
Final volume = 107 ml
Now find the new molarities
0.014 mol
[HF] = = 0.13 M
0.107 L
0.016 mol
 F-  = = 0.15 M
0.107 L
Reversible reaction. (Note: Chart usually done in molarity so it can be directly used in Ka
equation)
Initial [ ] 0.13 0 0.15

Change [ ] -x +x +x
Now we can calculate the pH in any of the 3 ways (this example is done only in 2 ways)
4
Using Ka since HF is a weak acid and on the left side of the equation
 H 3O +   F-  x (0.15 + x )
Ka =  = 6.8 x 10-4 =
[HF] 0.13- x
Solve the equation for x assuming that x is negligible compared to 0.15 and 0.13
0.15x
= 6.8 x 10-4 ⇒ 1.2x = 6.8 x 10-4 ⇒ x = 5.7 x 10-4 ⇒  H 3O +  = 5.7 x 10-4
0.13
pH = -log  H 3O +  = -log (5.7 x 10-4)
pH = 3.24 (the pH went up when strong base was added)
or
Using Henderson – Hasselbalch Equation
pH = pKa + log
[base]
[acid ]
 F- 
pH = - log Ka + log
[HF]
0.15
pH = - log(6.8 x 10-4) + log = 3.17 + log 1.2 = 3.17 + 0.079
0.13
pH = 3.25 (the answers may differ a little because of the significant figure rule and
rounding of the numbers)
ii Calculate the pH of the solution when 5.0 ml of 0.10 M HCl is added to 100 ml of the
original 0.15M HF/F- buffer solution.
mol HF
mol HF = 0.15 x 0.100 L = 0.015 mol HF
L
mol F-
mol F- = 0.15 x 0.100 L = 0.015 mol F-
L
5
mol HCl
mol HCl = 0.10 x 0.0050 L = 0.00050 mol HCl (in H2O, HCl becomes
L
H3O+ and Cl-. Cl- is a spectator ion and not involved in the reaction.)
Irreversible Reaction
mol F- + H3O+ → HF + H2O
Before 0.015 0.00050 0.015

Change - 0.00050 -0.00050 + 0.00050
After 0.015 0 0.016
Final volume = 105 ml
0.016 mol
[HF] = = 0.15 M
0.105 L
0.015 mol
 F-  = = 0.14 M
0.105 L
Reversible Reaction
Initial [ ] 0.15 0 0.14

Change [ ] -x +x +x
Now we can calculate the pH in any of the 3 ways (this example is done only in 2 ways)
Using Ka since HF is a weak acid and on the left side of the equation
 H 3O +   F-  x ( 0.14 + x )
Ka = = 6.8 x 10-4 =
[HF] 0.15- x
Solve the equation for x assuming that x is negligible compared to 0.15 and 0.13
0.14x
= 6.8 x 10-4 ⇒ 0.93x = 6.8 x 10-4 ⇒ x = 7.3 x 10-4 ⇒  H 3O +  = 7.3 x 10-4
0.15
6
pH = -log  H 3O +  = -log (7.3 x 10-4)
pH = 3.14 (the pH went down when strong acid was added)
or
Using Henderson – Hasselbalch Equation
pH = pKa + log
[base]
[acid ]
 F- 
pH = - log Ka + log
[HF]
0.14
pH = - log(6.8 x 10-4) + log = 3.17 + log 0.93 = 3.17 - 0.032
0.15
pH = 3.14
7
Problems:
1. Calculate the pOH of the 0.250 M lactic acid, HLac, mixed with 0.150 M Lac-. The Ka
for HLac is 1.3 x 10-3. Solve this problem using the Henderson – Hasselbalch
Equation.
Solution
2. Solution contains 10.0 g of pyridine, C5H5N and 15.0 g of HC5H5N per one liter of
solution.
i Calculate the pH of the solution if Kb for C5H5N is 1.40 x 10-9. Solve this problem
using the ICE chart.
Solution
ii Calculate the pH of the solution when 10.00 ml of 2.00 M KOH is added to 1.00
L of the buffer solution. Solve this problem using the ICE chart.
Solution
iii Calculate the pH of the solution when 20.00 ml of 2.00M HCl is added to 1.00 L
of the buffer solution. Solve this problem using the Henderson – Hasselbalch
Equation.
Solution
8
Solutions:
1. Calculate the pOH of the 0.250 M lactic acid, HLac, mixed with 0.150 M Lac-. The Ka
for HLac is 1.30 x 10-3. Do this problem using the Henderson – Hasselbalch Equation.
pH = pKa + log
[base]
[acid ]
 Lac- 
pH = - log Ka + log
[HLac]
0.150
pH = - log(1.30 x 10-3) + log = 2.886 + log 0.600 = 2.886 – 0.222 = 2.664
0.250
pOH = 14.000 – pH
pOH = 14.000 – 2.664
pOH = 11.336
Return to Problems
9
2. Solution contains 10.0 g of pyridine, C5H5N and 15.0 g of HC5H5N per one liter of
solution.
i Calculate the pH of the solution if Kb for C5H5N is 1.40 x 10-9. Do this problem
using the ICE chart.
First convert grams to moles of C5H5N and HC5H5N, and then to molarity of the
solution
1mol C5 H 5 N
10.0 g C5H5N x = 0.126 mol C5 H5 N
79.1g C5 H 5 N
0.126 mol C5 H 5 N
[C5 H 5 N ] = = 0.126 M C5 H5 N
1L
1mol HC5 H 5 N
15.0 g HC5H5N x = 0.187 mol HC5 H 5 N
80.1g HC5 H 5 N
0.187 mol HC5 H 5 N

[HC5H5 N ] = = 0.187 M
1L
Now write the equation followed by an ICE chart
+
Conc. (M) C5H5N + H2O ! HC5H5N + OH-
Initial [ ] 0.126 0.187 0

Change [ ] -x +x +x
Equilibrium [ ] 0.126 – x 0.187 + x x
Now write the equilibrium equation and substitute the information from the chart
 HC5 H 5 N +  OH -  (0.187 + x) x

Kb = = = 1.40 x 10-9
[ 5 5 ]
C H N ( 0.126 - x )
Solve the equation for x assuming that x is negligible compared to 0.126 and
0.187
0.187 x
= 1.40 x 10-9 ⇒ 1.76 x 10-10 = 0.187 x ⇒ x = 9.43 x 10-10
0.126
OH -  = 9.43 x 10-10 M
10
pOH = - log OH -  = -log (9.43 x 10-10) = 9.025
pH = 14.000 – pOH = 14.000 – 9.025
pH = 4.975
Return to Problems
11
ii Calculate the pH of the solution when 10.00 ml of 2.00 M KOH is added to 1.00 L
of the buffer solution. Do this problem using the ICE chart.
First write the BCA chart using the moles that you calculated in part i) for C5H5N
and HC5H5N and find the moles of KOH.
mol
mol OH- = 2.00 x 0.01000 L = 0.0200 mol OH-
L
mol HC5H5N + + OH- → C5H5N + H2O
Before 0.187 0.0200 0.126

Change - 0.0200 -0.0200 + 0.0200
After 0.167 0 0.146
Final volume = 1.010 L
0.167 mol
 HC5 H 5 N +  = = 0.165 M
1.010 L
0.146 mol
[C5 H 5 N ] = = 0.145 M
1.010 L
Now write the equation followed by an ICE chart
+
Conc. (M) C5H5N + H2O ! HC5H5N + OH-
Initial [ ] 0.145 0.167 0

Change [ ] -x +x +x
Equilibrium [ ] 0.145 – x 0.167 + x x
Now write the equilibrium equation and substitute the information from the chart
 HC5 H 5 N +  OH -  (0.167 + x) x

Kb = = = 1.40 x 10-9
[C5 H 5 N ] (0.145- x )
Solve the equation for x assuming that x is negligible compared to 0.145 and
0.167
0.167 x
= 1.40 x 10-9 ⇒ 2.03 x 10-10 = 0.167 x ⇒ x = 1.00 x 10-9
0.145
12
OH -  = 1.00 x 10-9 M
pOH = - log OH -  = -log (1.00 x 10-9) = 9.000
pH = 14.000 – pOH = 14.000 – 9.000
pH = 5.000 (pH raised when strong base added; was 4.975)
Return to Problems
13
iii Calculate the pH of the solution when 20.00 ml of 2.00M HCl is added to 1.00 L of
the buffer solution. Do this problem using the Henderson – Hasselbalch Equation.
First write the BCA chart using the moles that you calculated in part i) for C5H5N
and HC5H5N and find the moles of HCl.
mol
mol HCl = 2.00 x 0.02000 L = 0.0400 mol OH-
L
+
mol C5H5N + HCl → HC5H5N + Cl-
Before 0.126 0.0400 0.187

Change - 0.0400 -0.0400 + 0.0400
After 0.086 0 0.227
Final volume = 1.020 L
0.227 mol
 HC5 H 5 N +  = = 0.223 M
1.020 L
0.086 mol
[C5 H 5 N ] = = 0.084 M
1.020 L
pH = pKa + log
[base]
[acid ]
Kw 1 x 10-14
Ka = = -9
= 7.14 x 10-6
Kb 1.40 x 10
pH = - log Ka + log
[C 5 H 5 N ]
[HC5H5 N ]
0.084
pH = - log(7.14 x 10-6) + log = 5.146 + log 0.38 = 5.146 – 0.42
0.223
pH = 4.73 (pH lowered when strong base added; was 4.975)
Return to Problems
14
3 The Preparation of Buffers at Desired pH
Objectives: To become familiar with operating a pH meter, and to learn how to use the
Henderson-Hasselbalch equation to make buffer solutions at a desired pH
value.
Introduction: A buffer system is a mixture of a weak acid or a weak base and its salt
(conjugate base or conjugate acid, respectively) that permits solutions to
resist large changes in pH upon the addition of small amounts of hydrogen
ions (H+) or hydroxide ions (OH-). What does this mean? It means a
buffer helps maintain a near constant pH upon the addition of small
amounts of H+ or OH- to a solution.
In the definition of a buffer system, it should be noted that reference
is made to weak acids and bases. What is the distinction between a weak
and a strong acid or base? Strong acids (e.g., HCl) and bases (e.g.,
NaOH) almost completely ionize in water:
HCl H+ + Cl- ; NaOH Na+ + OH-
Many acids and bases, however, do not undergo complete ionization

in water. These compounds, then, are called weak acids and bases. Many
organic acids are weak acids. Weak acids do not completely dissociate in
water. The dissociation of an organic acid is described by the following
reaction:
+ -
HA H + A
weak conjugate
acid base
Note that the unprotonated product of the acid dissociation reaction is

referred to as a conjugate base. Recall the Bronsted-Lowry definition of
acids and bases. An acid is defined as a proton (H+) donor, whereas the
base is the H+ acceptor. In a conjugate acid/base pair such as that
indicated above, the weak acid always has one more H+ than its conjugate
base. Likewise, a weak base will have one less H+ than its conjugate acid.
O O
CH3 C OH H+ + CH3 C O-
weak acid conjugate base
acetic acid acetate
H2PO4- + H+ H3PO4
weak base conjugate acid
dihydrogen phosphate phosphoric acid
One can analyze the strength of a weak acid. This means that the
amount of hydrogen ion released can be determined. To do this, one can
use the following expression:
+] [A-]
Ka = [H[HA]
where Ka is defined as the acid dissociation constant.

The larger the value of Ka, the stronger the acid is. Because Ka values
vary over a wide range, they are usually expressed using a logarithmic
scale:
pKa = - log Ka
The hydrogen ion is one of the most important ions in biological
systems. The concentration of this ion affects most cellular processes.
For example, the structure and function of most biological
macromolecules and the rates of most biochemical reactions are strongly
affected by [H+]. The pH scale has been devised as a convenient method
of expressing hydrogen ion concentration. pH has been defined as the
negative logarithm of the hydrogen ion concentration:
pH = - log [H+]
The Henderson-Hasselbalch equation provides a convenient way to think
about buffers and pH:
[A-]
pH = pKa + log
[HA]
If one were examining the dissociation of acetic acid, the Henderson-

Hasselbalch relationship, then, would be:
[CH3 COO-]
pH = pKa + log
[CH3 COOH]
The Henderson-Hasselbalch equation can be used to determine if an

aqueous solution of a conjugate acid/base pair is functioning as a buffer.
If the concentration of the weak acid is equal to that of its conjugate base,
the ratio of these two components is one. When this is the case, the
Henderson-Hasselbalch equation reduces to
pH = pKa
because the log(1) is equal to zero.
When the pH of the solution is equal to the pKa of the ionizing group,
the solution is functioning at maximum buffering capacity (best buffer).
An aqueous solution of a conjugate acid/base pair functions as a good
buffer when the ratio of the conjugate base to weak acid ranges from 1:9
to 9:1. Substituting these ratios into the Henderson-Hasselbalch equation,
one finds that this aqueous solution functions as a good buffer when the
pH of the solution is within approximately one pH unit of the ionizing
group’s pKa.
pH = pKa + 1
because the log (1/9) is -0.999 and the log of (9/1) is +0.999.
Using the Henderson-Hasselbalch Equation

Example A: This is a two-component buffer system meaning that the weak acid and
its conjugate base are added separately. How would you prepare 10mL of a 0.01M
phosphate buffer, pH 7.40, from stock solutions of 0.10M KH2PO4 and 0.25M
K2HPO4? pKa of KH2PO4 = 7.20. This problem is similar to Problem 1 in
Experimental Procedures. The following approach may be helpful in solving this
type of buffer problem in which both the conjugate acid and base are added
separately. Please note that the numbers are not rounded off until the very end and
are rounded based on the limits of the pipets, cylinders, etc. that are required to
accurately measure the calculated volumes.
1. Use the Henderson Hasselbalch equation to find the ratio of A- to HA.
pH = pKa + log [A-] / [HA]
7.40 = 7.20 + log [A-] / [HA]
0.20 = log [A-] / [HA]
1.584893192 = [A-] / [HA]*
*Since [A-] / [HA] = 1.584893192, we can say that [A-] / [HA] = 1.584893192/ 1. In this case [A-
] = 1.584893192; [HA] = 1.
2. Calculate the decimal fraction (part/whole) of each buffer component.
A- = 1.584893192 / (1.000 + 1.584893192) = 1.584893192 / 2.584893192=
0.61313682
HA = 1.000 / 2.584893192= 0.38686318
3. Find the molarity (M) of each component in the buffer by simply multiplying the
molarity of the buffer by the decimal fraction of each component.
MA- = 0.01M x 0.61313682 = 0.006131368M
MHA = 0.01M x 0.38686318 = 0.003868632M
4. Calculate the moles of each component in the buffer. Moles = Molarity x Liters
of buffer
molesA- = 0.006131368M x 0.01L = 6.131 x 10-5 moles
molesHA = 0.003868632M x 0.01L = 3.869 x 10-5 moles
5. Calculate the volume of each stock solution required to make the buffer
Liters of stock = moles of the buffer component / Molarity of the stock
LA- = 6.131 x 10-5 moles / 0.25 M = 2.452 x 10-4 L = 245µL
LHA = 3.869 x 10-5 moles / 0.10 M = 3.869 x 10-4 L = 387µL
6. To prepare this buffer, one would use appropriately-sized pipets to measure and
transfer each component to a 10mL volumetric flask and bring the solution to
volume with dH2O.
Example B: This is a single-component buffer system. This means that one
component of the conjugate acid/base pair will be generated in situ (in solution). In
the following example, the conjugate base will be generated by reaction between the
weak acid (acetic acid) and strong base (sodium hydroxide). How would you prepare
10mL of a 0.02M acetate buffer, pH 4.30, from stock solutions of 0.05M acetic acid
(HAc) and 0.05M NaOH? pKa acetic acid = 4.76. This problem is similar to Problem 2
in Experimental Procedures.
1. Use the Henderson Hasselbalch equation to find the ratio of A- to HA.
4.30 = 4.76 + log [A-] / [HA]
-0.46 = log [A-] / [HA]
0.34673685 = [A-] / [HA]
2. Calculate the decimal fraction (part/whole) of each buffer component.
A- = 0.34673685 / (1.00 + 0.34673685) = 0.34673685 / 1.34673685 =
0.257464441
HA = 1.00 / 1.34673685 = 0.742535559
3. Find the molarity (M) of each component in the buffer by simply multiplying the
molarity of the buffer by the decimal fraction of each component.
MA- = 0.02M x 0. 257464441= 0.005149289 = 5.15 x 10-3 M
MHA = 0.02M x 0. 742535559= 0.014850711 = 1.49 x 10-2 M
4. Calculate the moles of each component in the buffer. Moles = Molarity x Liters
of buffer
molesA- = 0.005149289M x 0.01L = 5.14929 x 10-5 moles
molesHA = 0.014850711M x 0.01L = 1.485071.47 x 10-4 moles
5. Note: Since this buffer is prepared by the reaction of a weak acid (HAc) with a
strong base (NaOH), you must determine the total moles of the weak acid
component needed because the conjugate base is made in situ.
Total moles = 5.15 x 10-5moles NaOH + 1.49 x 10-4 moles HAc = 2.00 x 10-4
moles HAc. This sum indicates that, although in the buffer one only needs 1.49 x 10-4 moles
HA, an additional 5.15 x 10-5moles is needed to generate the conjugate base in situ.
6. Calculate the volume of each stock solution required to make the buffer
Liters of stock = Moles of the buffer component / Molarity of the stock
LA- = 5.14929 x 10-5 moles / 0.05 M = 1.03 x 10-3L NaOH = 1.0mL
LHA = 2.00 x 10-4 moles / 0.05 M = 4.00 x 10-3 L CH3COOH = 4.0mL
7. To prepare this buffer, one would use appropriately sized pipets or cylinders to
measure and transfer each component to a 10 mL volumetric flask and bring the
solution to volume with dH2O.
Example C: What is the pH of a solution resulting from a mixture of 200 mL of 0.1
M NaOH and 100 mL of 0.3 M HAc? This problem is similar to Problem 3 in
Experimental Procedures. It is a limiting reagent type problem.
1. Calculate the initial moles of each reactant. Moles = Molarity x Liters
MolesNaOH = 0.1M x 0.2L = 0.02moles
MolesHAc = 0.3M x 0.1L = 0.03moles
2. Write the balanced equation for this reaction. Note: This is a neutralization
reaction.
NaOH + HAc Na+Ac- + H+OH-
3. Determine the moles of each reactant remaining after the reaction. Recall: This
is a limiting reagent type problem. Since NaOH is present in the least amount
and, according to the balanced equation the reactants react in a 1:1 ratio, the
NaOH is completely consumed (i.e., zero moles of NaOH remain). Because the
reactants react in a 1:1 ratio,
Moles HAc remaining = MolesHAc initial – MolesHAc reacted
= 0.03moles – 0.02moles = 0.01moles
4. Determine the molarity of the buffer components (i.e., the conjugate acid-base
pair).
MHAc = molesHAc remaining / Liters solution = 0.01moles/0.3L = 0.033M
The molarity of the conjugate base Ac- resulting from the dissolution of the
product NaAc is:
MAc- = moles formed / Liters solution = 0.02moles / 0.3L = 0.067M
5. Use the H-H equation to calculate the pH.
= 4.76 + log (0.067/0.033)
= 4.76 + log 2.02
= 4.76 + 0.31
= 5.07
Try this one: Determine the pH of a solution resulting from a mixture of
5mL of 0.1M KOH and 30mL of 0.1M HAc.
Experimental Procedures:
1. Prepare 10 mL of a 0.01 M phosphate buffer, pH 7.70, from stock
solutions of 0.1 M K2HPO4 and 0.2 M KH2PO4. (pKa for the weak acid
= 7.20).
A. Use the Henderson-Hasselbalch equation to calculate the volume
of each stock solution needed.
pH = pKa + log [conjugate base] / [weak acid]
B. Check your calculations with other students. See the instructor if
there is uncertainty.
C. Make the solution and check the pH of a portion of your buffer
solution using the pH meter. (Instructions are on page 33)
2. Prepare 10 mL of 0.01 M acetate buffer, pH 3.80, from stock solutions of
0.1 M acetic acid and 0.02 M sodium hydroxide. pKaacetic acid = 4.76.
A. – C. See above.
D. Calculate the exact volume of the 0.01 M acetate buffer required to
make 10 mL of a 0.0005 M acetate buffer.
1. Prepare this new buffer using the following equation to aid you in
your calculations.
V1 = (V2 x M2)
M1
where: V1 = the volume of the concentrated solution (liters, L)
V2 = the volume of the diluted solution (liters, L)
M1 = the molar concentration of the concentrated solution
(moles/L)
M2 = the molar concentration of the diluted solution
(moles/L)
2. Check the pH of this new buffer.
E. Calculate the exact volume of the 0.01 M acetate buffer required to
make 10 mL of a 0.001 M acetate buffer.
1. Prepare this new buffer.
2. Check its pH.
Does a 10-fold dilution of your

original buffer (0.01M Æ 0.001M)
affect its pH. Does the 20-fold
dilution affect the pH? Why?
3. What is the pH of a solution resulting from the addition of 2.5 mL of 0.02M
NaOH to 5.0 mL 0.25M acetic acid? Use the Henderson-Hasselbalch
equation to calculate the pH of the resulting solution, and then follow
directions B and C above. pKaacetic acid = 4.76.
Record the data in your notebook (you should have 5 pH values):
The Preparation of Buffer Solutions of Desired pH STUDY GUIDE
1. The hydrogen ion concentration of tomato juice (pH 4) is how many times higher
than the hydrogen ion concentration of 0.01 M NaOH (pH 12)?
2. Phosphoric acid H3PO4 has three dissociable protons (it is triprotic) thus three pKa
values; 2.12, 7.21, and 12.32). What volume of 200 mM phosphoric acid and what
volume of 500 mM NaOH are required to make 100 mL of 20 mM phosphate buffer
pH 12?
3. What must be done to the electrode of the pH meter before placing it into a solution
to measure its pH?
4. In the phosphate buffer system containing K2HPO4 and KH2PO4, what is the weak
acid? What is its conjugate base?
5. If the weak acid in a buffer system has pKa 10.5, in what pH range is the system a
most effective buffer?
6. On the laboratory shelf are 250mM solutions of both acetic acid and sodium
hydroxide. How would you make a 100 mL solution of 25mM acetate buffer of pH
5.50 using these stock solutions?
7. How would you prepare a liter of 25% NaCl (w:w) aqueous solution?
8. How would you prepare a 1.5% NaBr (w:v) aqueous solution?
9. How would you prepare a 65% acetone (v:v) aqueous solution?
10. How would you prepare 500 mL of 0.12µM aqueous solution of glucose (C6H12O6)?
11. What is the percent conjugate acid in an acetate buffer of pH 3.80? (pKa = 4.76)
12. How many µmoles are equivalent to 100 pmoles?
13.
14. What is the ratio of conjugate base to weak acid in a buffer when the pH drops to
two units below the pKa?
Buffers Booklet-2003 cover.qxd 4/8/2003 9:43 AM Page 1
Buffers
A guide for the preparation and use of
buffers in biological systems
EMD Biosciences, Inc.

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Buffers Booklet-2003.qxd 4/10/2003 2:04 PM Page i
Buffers
A guide for the preparation and use of
buffers in biological systems
By
Chandra Mohan, Ph.D.
A brand of EMD Biosciences, Inc.

Copyright © 2003 EMD Biosciences, Inc., An Affiliate of Merck KGaA, Darmstadt, Germany.
All Rights Reserved.
Buffers Booklet-2003.qxd 4/10/2003 2:04 PM Page ii
A Word to Our Customers
We are pleased to present to you the newest edition of Buffers: A Guide for the
Preparation and Use of Buffers in Biological Systems. This practical resource has
been especially revamped for use by researchers in the biological sciences. This
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ii
Buffers Booklet-2003.qxd 4/10/2003 2:04 PM Page iii
Table of Contents:
Why Does Calbiochem® Biochemicals Publish a Booklet on Buffers? . . . . . . . . . .1
Water, The Fluid of Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Ionization of Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Dissociation Constants of Weak Acids and Bases . . . . . . . . . . . . . . . . . . . . . . . . . .4
Henderson-Hasselbach Equation: pH and pKa . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Determination of pKa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
pKa Values for Commonly Used Biological Buffers . . . . . . . . . . . . . . . . . . . . . . . . .7
Buffers, Buffer Capacity, and Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Biological Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Buffering in Cells and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Effect of Temperature on pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Effect of Buffers on Factors Other than pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Use of Water-Miscible Organic Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Solubility Equilibrium: Effect of pH on Solubility . . . . . . . . . . . . . . . . . . . . . . . .14
pH Measurements: Some Useful Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Choosing a Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Preparation of Some Common Buffers for Use

in Biological Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Commonly Used Buffer Media in Biological Research . . . . . . . . . . . . . . . . . . . . .22
Isoelectric Point of Selected Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
Isoelectric Point of Selected Plasma Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Approximate pH and Bicarbonate Concentration in

Extracellular Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Ionization Constants K and pKa for Selected Acids and

Bases in Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Physical Properties of Some Commonly Used Acids . . . . . . . . . . . . . . . . . . . . . . .27
Some Useful Tips for Calculation of Concentrations and

Spectrophotometric Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
CALBIOCHEM® Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
iii
Buffers Booklet-2003.qxd 4/10/2003 2:04 PM Page iv
iv
Buffers Booklet-2003.qxd 4/10/2003 2:04 PM Page 1
Why Does Calbiochem® Biochemicals Publish a

Booklet on Buffers?
We are frequently asked questions on the use of buffers that we offer to research
laboratories. This booklet is designed to help answer several basic questions
about the use of buffers in biological systems. The discussion presented here is
by no means complete, but we hope it will help in the understanding of general
principles involved in the use of buffers.
Almost all biological processes are pH dependent. Even a slight change in pH can
result in metabolic acidosis or alkalosis, resulting in severe metabolic complica-
tions. The purpose of a buffer in biological system is to maintain intracellular
and extracellular pH within a very narrow range and resist changes in pH in the
presence of internal and external influences. Before we begin a discussion of
buffers and how they control hydrogen ion concentrations, a brief explanation
of the role of water and equilibrium constants of weak acids and bases is
necessary.
1
Buffers Booklet-2003.qxd 10/18/2005 11:36 AM Page 2
Water: The Fluid of Life

Water constitutes about 70% of the mass of most living creatures. All biological
reactions occur in an aqueous medium. All aspects of cell structure and function
are adapted to the physical and chemical properties of water. Hence, it is
essential to understand some basic properties of water and its ionization
products, i.e., H+ and OH¯. Both H+ and OH¯ influence the structure, assembly,
and properties of all macromolecules in the cell.
Water is a polar solvent that dissolves most charged molecules. Water dissolves
most salts by hydrating and stabilizing the cations and anions by weakening
their electrostatic interactions (Figure 1). Compounds that readily dissolve in
water are known as HYDROPHILIC compounds. Nonpolar compounds such as
chloroform and ether do not interact with water in any favorable manner and are
known as HYDROPHOBIC compounds. These compounds interfere with
hydrogen bonding among water molecules.
Figure 1: Electrostatic interaction of Na+ and Cl¯ ions and water molecules.
Several biological molecules, such as protein, certain vitamins, steroids, and

phospholipids contain both polar and nonpolar regions. They are known as
AMPHIPATHIC molecules. The hydrophilic region of these molecules are
arranged in a manner that permits maximum interaction with water molecules.
However, the hydrophobic regions assemble together exposing only the smallest
area to water.
2
Ionization of Water
Water molecules undergo reversible ionization to yield H+ and OH¯ as per the
following equation.
H2O →
← H+ + OH¯
The degree of ionization of water at equilibrium is fairly small and is given by

the following equation where Keq is the equilibrium constant.
[H+][OH¯]
______________
Keq =
[H2O]
At 25°C, the concentration of pure water is 55.5 M (1000 ÷ 18; M.W. 18.0).
Hence, we can rewrite the above equation as follows:
[H+][OH¯]
______________
Keq =
55.5 M
or
(55.5)(Keq) = [H+][OH¯]
For pure water electrical conductivity experiments give a Keq value of 1.8 x
10-16 M at 25°C.
Hence, (55.5 M)(1.8 x 10-16 M) = [H+][OH¯]

or
99.9 x 10-16 M2 = [H+][OH¯]
or
1.0 x 10 M2 = [H+][OH¯]
-14
[H+][OH¯], ion product of water, is always equal to 1.0 x 10-14 M2 at 25°C. When
[H+] and [OH¯] are present in equal amounts then the solution gives a neutral pH.
Here [H+][OH¯] = [H+]2

or
[H+] = 1 x 10-14 M2
and
[H ] = [OH¯] = 10-7 M
+
As the total concentration of H+ and OH¯ is constant, an increase in one ion is

compensated by a decrease in the concentration of other ion. This forms the
basis for the pH scale.
3
Dissociation Constants of Weak Acids and Bases

Strong acids (hydrochloric acid, sulfuric acid, etc.) and bases (sodium hydroxide,
potassium hydroxide, etc.) are those that are completely ionized in dilute
aqueous solutions.
In biological systems one generally encounters only weak acids and bases. Weak
acids and bases do not completely dissociate in solution. They exist instead as an
equilibrium mixture of undissociated and dissociated species. For example, in
aqueous solution, acetic acid is an equilibrium mixture of acetate ion, hydrogen
ion, and undissociated acetic acid. The equilibrium between these species can be
expressed as:
k1
CH3COOH →
← H+ + CH3COO¯
k2
where k1 represents the rate constant of dissociation of acetic acid to acetate and
hydrogen ions, and k2 represents the rate constant for the association of acetate
and hydrogen ions to form acetic acid. The rate of dissociation of acetic acid,
-d[CH3COOH ]/dt, is dependent on the rate constant of dissociation (k1) and the
concentration of acetic acid [CH3COOH] and can be expressed as:
d [CH3COOH]
____________________ = k1 [CH3COOH]
dt
Similarly, the rate of association to form acetic acid, d[HAc]/dt, is dependent on

the rate constant of association (k2) and the concentration of acetate and
hydrogen ions and can be expressed as:
d [CH3COOH ]
__________________ = k2 [H+] [CH3COO¯]
dt
Since the rates of dissociation and reassociation are equal under equilibrium
conditions:
k1 [CH3COOH ] = k2 [H+] [CH3COO¯]
or
k1
_______
[H+] [CH3COO¯]
____________________
=
k2 [CH3COOH]
and
[H+] [CH3COO¯]
___________________
Ka =
[CH3COOH]
where
k1
_______ = Ka (Equilibrium constant)
k2
4
This equilibrium expression can now be rearranged to
[CH3COOH]
[H+] = Ka _______________
[CH3COO¯]
where the hydrogen ion concentration is expressed in terms of the equilibrium

constant and the concentrations of undissociated acetic acid and acetate ion. The
equilibrium constant for ionization reactions is called the ionization constant or
dissociation constant.
Henderson-Hasselbach Equation: pH and pKa

The relationship between pH, pKa, and the buffering action of any weak acid and
its conjugate base is best explained by the Henderson-Hasselbach equation. In
biological experiments, [H+] varies from 10-1 M to about 10-10 M. S.P.L.
Sorenson, a Danish chemist, coined the “p” value of any quantity as the negative
logarithm of the hydrogen ion concentration. Hence, for [H+] one can write the
following equation:
pH = – log [H+]
Similarly pKa can be defined as – log Ka. If the equilibrium expression is

converted to – log then
[CH3COOH]
– log [H+] = – log Ka – log ______________
[CH3COO¯]
and pH and pKa substituted:
[CH3COOH]
________________
pH = pKa – log
[CH3COO¯]
or
[CH3COO¯]
_______________
pH = pKa + log
[CH3COOH]
When the concentration of acetate ions equals the concentration of acetic acid,
log [CH3COO¯]/[CH3COOH] approaches zero (the log of 1) and pH equals pKa (the
pKa of acetic acid is 4.745). Acetic acid and acetate ion form an effective
buffering system centered around pH 4.75. Generally, the pKa of a weak acid or
base indicates the pH of the center of the buffering region.
The terms pK and pKa are frequently used interchangeably in the literature. The
term pKa (“a” refers to acid) is used in circumstances where the system is being
considered as an acid and in which hydrogen ion concentration or pH is of
5
interest. Sometimes the term pKb is used. pKb (“b” refers to base) is used when the
system is being considered as a base and the hydroxide ion concentration or pOH
is of greater interest.
Determination of pKa
pKa values are generally determined by titration. A carefully calibrated,
automated, recording titrator is used, the free acid of the material to be measured
is titrated with a suitable base, and the titration curve is recorded. The pH of the
solution is monitored as increasing quantities of base are added to the solution.
Figure 2 shows the titration curve for acetic acid. The point of inflection
indicates the pKa value. Frequently, automatic titrators record the first derivative
of the titration curve, giving more accurate pKa values.
Polybasic buffer systems can have more than one useful pKa value. Figure 3
shows the titration curve for phosphoric acid, a tribasic acid. Note that the curve
has five points of inflection. Three indicate pKa1, pKa2 and pKa3, and two
additional points indicate where H2PO4– and HPO4– exist as the sole species.
6 pKa = 4.76
pH
0
NaOH
Figure 2: Titration Curve for Acetic Acid
12
pKa3 = 12.32
10
8
pH
6
pKa2 = 7.21
2 pKa1 = 2.12
NaOH
Figure 3: Titration Curve for Phosphoric Acid
6
Table 1: pKa Values for Commonly Used Biological Buffers and Buffer Constituents
pKa
Product Cat. No. M.W. at 20°C
ADA, Sodium Salt 114801 212.2 6.60
2-Amino-2-methyl-1,3-propanediol 164548 105.1 8.83
BES, ULTROL® Grade 391334 213.2 7.15
Bicine, ULTROL® Grade 391336 163.2 8.35
BIS-Tris, ULTROL® Grade 391335 209.2 6.50
BIS-Tris Propane, ULTROL® Grade 394111 282.4 6.80
Boric Acid, Molecular Biology Grade 203667 61.8 9.24
Cacodylic Acid 205541 214.0 6.27
CAPS, ULTROL® Grade 239782 221.3 10.40
CHES, ULTROL® Grade 239779 207.3 9.50
Citric Acid, Monohydrate, Molecular Biology Grade 231211 210.1 4.76
Glycine 3570 75.1 2.341
Glycine, Molecular Biology Grade 357002 75.1 2.341
Glycylglycine, Free Base 3630 132.1 8.40
HEPES, Free Acid, Molecular Biology Grade 391340 238.3 7.55
HEPES, Free Acid, ULTROL® Grade 391338 238.3 7.55
HEPES, Free Acid Solution 375368 238.3 7.55
HEPES, Sodium Salt, ULTROL® Grade 391333 260.3 7.55
HEPPS, ULTROL® Grade 391339 252.3 8.00
Imidazole, ULTROL® Grade 4015 68.1 7.00
MES, Free Acid, ULTROL® Grade 475893 195.2 6.15
MES, Sodium Salt, ULTROL® Grade 475894 217.2 6.15
MOPS, Free Acid, ULTROL® Grade 475898 209.3 7.20
MOPS, Sodium Salt, ULTROL® Grade 475899 231.2 7.20
PIPES, Free Acid, Molecular Biology Grade 528133 302.4 6.80
PIPES, Free Acid, ULTROL® Grade 528131 302.4 6.80
PIPES, Sodium Salt, ULTROL® Grade 528132 325.3 6.80
PIPPS 528315 330.4 3.732
Potassium Phosphate, Dibasic, Trihydrate, Molecular Biology Grade 529567 228.2 7.213
Potassium Phosphate, Monobasic 529565 136.1 7.213
Potassium Phosphate, Monobasic, Molecular Biology Grade 529568 136.1 7.213
Sodium Phosphate, Dibasic 567550 142.0 7.213
Sodium Phosphate, Dibasic, Molecular Biology Grade 567547 142.0 7.213
Sodium Phosphate, Monobasic 567545 120.0 7.213
Sodium Phosphate, Monobasic, Monohydrate, Molecular Biology Grade 567549 138.0 7.213
TAPS, ULTROL® Grade 394675 243.2 8.40
TES, Free Acid, ULTROL® Grade 39465 229.3 7.50
TES, Sodium Salt, ULTROL® Grade 394651 251.2 7.50
Tricine, ULTROL® Grade 39468 179.2 8.15
Triethanolamine, HCl 641752 185.7 7.66
Tris Base, Molecular Biology Grade 648310 121.1 8.30
Tris Base, ULTROL® Grade 648311 121.1 8.30
Tris, HCl, Molecular Biology Grade 648317 157.6 8.30
Tris, HCl, ULTROL® Grade 648313 157.6 8.30
Trisodium Citrate, Dihydrate 567444 294.1 —
Trisodium Citrate, Dihydrate, Molecular Biology Grade 567446 294.1 —
1. pKa1 = 2.34; pKa2 = 9.60
2. pKa1 = 3.73; pKa2 = 7.96 (100 mM aqueous solution, 25°C).
3. Phosphate buffers are normally prepared from a combination of the monobasic and dibasic salts, titrated against
each other to the correct pH. Phosphoric acid has three pKa values: pKa1 = 2.12; pKa2 = 7.21; pKa3 = 12.32
7
Buffers, Buffer Capacity and Range

Buffers are aqueous systems that resist changes in pH when small amounts of
acid or base are added. Buffer solutions are composed of a weak acid (the proton
donor) and its conjugate base (the proton acceptor). Buffering results from two
reversible reaction equilibria in a solution wherein the concentration of proton
donor and its conjugate proton acceptor are equal. For example, in a buffer
system when the concentration of acetic acid and acetate ions are equal, addition
of small amounts of acid or base do not have any detectable influence on the pH.
This point is commonly known as the isoelectric point. At this point there is no
net charge and pH at this point is equal to pKa.
[CH3COO¯]
pH = pKa + log ________________
[CH3COOH]
At isoelectric point [CH3COO¯] = [CH3COOH] hence, pH = pKa
Buffer capacity is a term used to describe the ability of a given buffer to resist
changes in pH on addition of acid or base. A buffer capacity of 1 is when 1 mol
of acid or alkali is added to 1 liter of buffer and pH changes by 1 unit. The buffer
capacity of a mixed weak acid-base buffer is much greater when the individual
pKa values are in close proximity with each other. It is important to note that the
buffer capacity of a mixture of buffers is additive.
Buffers have both intensive and extensive properties. The intensive property is a
function of the pKa value of the buffer acid or base. Most simple buffers work
effectively in the pH scale of pKa ± 1.0. The extensive property of the buffers is
also known as the buffer capacity. It is a measure of the protection a buffer offers
against changes in pH. Buffer capacity generally depends on the concentration
of buffer solution. Buffers with higher concentrations offer higher buffering
capacity. On the other hand, pH is dependent not on the absolute concentrations
of buffer components but on their ratio.
Using the above equation we know that when pH = pKa the concentrations of
acetic acid and acetate ion are equal. Using a hypothetical buffer system of HA
(pKa = 7.0) and [A–], we can demonstrate how the hydrogen ion concentration,
[H+], is relatively insensitive to external influence because of the buffering
action.
For example:
If 100 ml of 10 mM (1x 10-2 M) HCl are added to 1.0 liter of 1.0 M NaCl at pH 7.0,
the hydrogen ion concentration, [H+], of the resulting 1.1 liter of solution can be
calculated by using the following equation:
8
[H+] x Vol = [H+]o x Volo

where
Volo = initial volume of HCl solution (in liters)
[H+]o = initial hydrogen ion concentration (M)
Vol = final volume of HCl + NaCl solutions (in liters)
[H+] = final hydrogen ion concentration of HCl + NaCl solution (M)
Solving for [H+]:
[H+] x 1.1 liter = 1.0 x 10-2 x 0.1 = 1 x 10-3

[H+] = 9.09 x 10-4
or pH = 3.04
Thus, the addition of 1.0 x 10-3 mol of hydrogen ion resulted in a pH change of
approximately 4 pH units (from 7.0 to 3.04).
If a buffer is used instead of sodium chloride, a 1.0 M solution of HA at pH 7.0

will initially have:
[HA] = [A] = 0.5 M
[A]
______
pH = pK + log
[HA]
0.5
______
pH = 7.0 + log or pH = 7.0
0.5
When 100 ml of 1.0 x 10-2 M (10 mM) HCl is added to this system, 1.0 x 10-3 mol
of A– is converted to 1.0 x 10-3 mol of HA, with the following result:
0.499/1.1
_______________
pH = 7.0 + log
0.501/1.1
pH = 7.0 - 0.002 or pH = 6.998
Hence, it is clear that in the absence of a suitable buffer system there was a pH
change of 4 pH units, whereas in a buffer system only a trivial change in pH was
observed indicating that the buffer system had successfully resisted a change in
pH. Generally, in the range from [A]/[HA] = 0.1 to [A]/[HA] = 10.0, effective
buffering exists. However, beyond this range, the buffering capacity may be
significantly reduced.
9
Biological Buffers
Biological buffers should meet the following general criteria:
• Their pKa should reside between 6.0 to 8.0.

• They should exhibit high water solubility and minimal solubility in organic
solvents.
• They should not permeate cell membranes.
• They should not exhibit any toxicity towards cells.
• The salt effect should be minimum, however, salts can be added as required.
• Ionic composition of the medium and temperature should have minimal
effect of buffering capacity.
• Buffers should be stable and resistant to enzymatic degradation.
• Buffer should not absorb either in the visible or in the UV region.
Most of the buffers used in cell cultures, isolation of cells, enzyme assays, and
other biological applications must possess these distinctive characteristics.
Good's zwitterionic buffers meet these criteria. They exhibit pKa values at or near
physiological pH. They exhibit low interference with biological processes due to
the fact that their anionic and cationic sites are present as non-interacting
carboxylate or sulfonate and cationic ammonium groups respectively.
Buffering in Cells and Tissues

A brief discussion of hydrogen ion regulation in biological systems highlights
the importance of buffering systems. Amino acids present in proteins in cells and
tissues contain functional groups that act as weak acid and bases. Nucleotides
and several other low molecular weight metabolites that undergo ionization also
contribute effectively to buffering in the cell. However, phosphate and bicarbon-
ate buffer systems are most predominant in biological systems.
The phosphate buffer system has a pKa of 6.86. Hence, it provides effective
buffering in the pH range of 6.4 to 7.4. The bicarbonate buffer system plays an
important role in buffering the blood system where in carbonic acid acts as a
weak acid (proton donor) and bicarbonate acts as the conjugate base (proton
acceptor). Their relationship can be expressed as follows:
[H+][HCO3¯]
______________
K1 =
[H2CO3 ]
In this system carbonic acid (H2CO3) is formed from dissolved carbon dioxide
and water in a reversible manner. The pH of the bicarbonate system is dependent
on the concentration of carbonic acid and bicarbonate ion. Since carbonic acid
10
concentration is dependent upon the amount of dissolved carbon dioxide the

ultimate buffering capacity is dependent upon the amount of bicarbonate and
the partial pressure of carbon dioxide.
+ _ H2CO2
H + HCO3
H2O H2O CO2
CO2
Blood Lung
Air Space
Figure 4: Relationship between bicarbonate buffer system and carbon dioxide.
In air breathing animals, the bicarbonate buffer system maintains pH near 7.4.
This is possible due to the fact that carbonic acid in the blood is in equilibrium
with the carbon dioxide present in the air. Figure 4 highlights the mechanism
involved in blood pH regulation by the bicarbonate buffer system. Any increase
in partial pressure of carbon dioxide (as in case of impaired ventilation) lowers
the ratio of bicarbonate to pCO2 resulting in a decrease in pH (acidosis). The
acidosis is reversed gradually when kidneys increase the absorption of bicarbon-
ate at the expense of chloride. Metabolic acidosis resulting from the loss of
bicarbonate ions (such as in severe diarrhea or due to increased keto acid
formation) leads to severe metabolic complications warranting intravenous
bicarbonate therapy.
During hyperventilation, when excessive amounts of carbon dioxide are

eliminated from the system (thereby lowering the pCO2), pH of the blood
increases resulting in alkalosis. This is commonly seen in conditions such as
pulmonary embolism and hepatic failure. Metabolic alkalosis generally results
when bicarbonate levels are higher in the blood. This is commonly observed after
vomiting of acidic gastric secretions. Kidneys compensate for alkalosis by
increasing the excretion of bicarbonate ions. However, an obligatory loss of
sodium occurs under these circumstances.
In case of severe alkalosis the body is depleted of water, H+, Cl¯ and to some
extent Na+. A detailed account of metabolic acidosis and alkalosis is beyond the
scope of this booklet. Readers are advised to consult a suitable text book of
physiology for more detailed information on the mechanisms involved.
11
Effect of Temperature on pH
Generally when we consider the use of buffers we make following two assump-
tions.
(a) The activity coefficients of the buffer ions is approximately equal to 1

over the useful range of buffer concentrations
(b) The value of Ka is constant over the working range of temperature.
However, in real practice one observes that pH changes slightly with change in
temperature. This might be very critical in biological systems where a precise
hydrogen ion concentration is required for reaction systems to operate with
maximum efficiency. Figure 5 presents the effect of temperature on the pH of
phosphate buffer. The difference might appear to be slight but it has significant
biological importance. Although the mathematical relationship of activity and
temperature may be complicated, the actual change of pKa with temperature
(∆pKa/°C) is approximately linear. Table 2 presents the pKa and ∆pKa/°C for several
selected zwitterionic buffers commonly used in biological experimentation.
6.7
6.8
pH
6.9
7.0
0 10 20 30 40
Temperature, ºC
Figure 5: Effect of Temperature on pH of Phosphate Buffer
12
Table 2: pKa and DpKa/°C of Selected Buffers

pKa pKa DpKa/°C Binding to
Buffer M.W.
(20°C) (37°C) Metal Ions
MES 195.2 6.15 5.97 -0.011 Negligible metal ion binding
ADA 212.2 6.60 6.43 -0.011 Cu2+, Ca2+, Mn2+. Weaker
binding with Mg2+.
BIS-Tris Propane* 282.4 6.80 — -0.016 —
PIPES 302.4 6.80 6.66 -0.009 Negligible metal ion binding
ACES 182.2 6.90 6.56 -0.020 Cu2+. Does not bind Mg2+,
Ca2+, or Mn2+.
BES 213.3 7.15 6.88 -0.016 Cu2+. Does not bind
Mg2+, Ca2+, or Mn2+.
MOPS 209.3 7.20 6.98 -0.006 Negligible metal ion binding
TES 229.3 7.50 7.16 -0.020 Slightly to Cu2+. Does not bind
Mg2+, Ca2+, or Mn2+.
HEPES 238.3 7.55 7.30 -0.014 None
HEPPS 252.3 8.00 7.80 -0.007 None
Tricine 179.2 8.15 7.79 -0.021 Cu2+. Weaker binding
with Ca2+, Mg2+, and Mn2+.
Tris* 121.1 8.30 7.82 -0.031 Negligible metal ion binding
Bicine 163.2 8.35 8.04 -0.018 Cu2+. Weaker binding
with Ca2+, Mg2+, and Mn2+.
Glycylglycine 132.1 8.40 7.95 -0.028 Cu2+. Weaker
binding with Mn2+.
CHES 207.3 9.50 9.36 -0.009 —
CAPS 221.32 10.40 10.08 -0.009 —
* Not a zwitterionic buffer
Effects of Buffers on Factors Other than pH

It is of utmost importance that researchers establish the criteria and determine
the suitability of a particular buffer system. Some weak acids and bases may
interfere with the reaction system. For example, citrate and phosphate buffers are
not recommended for systems that are highly calcium-dependent. Citric acid and
its salts are powerful calcium chelators. Phosphates react with calcium producing
insoluble calcium phosphate that precipitates out of the system. Phosphate ions
in buffers can inhibit the activity of some enzymes, such as carboxypeptidase,
fumarease, carboxylase, and phosphoglucomutase.
Tris(hydroxy-methyl)aminomethane can chelate copper and also acts as a

competitive inhibitor of some enzymes. Other buffers such as ACES, BES, and
TES, have a tendency to bind copper. Tris-based buffers are not recommended
when studying the metabolic effects of insulin. Buffers such as HEPES and
HEPPS are not suitable when a protein assay is performed by using Folin
reagent. Buffers with primary amine groups, such as Tris, may interfere with the
Bradford dye-binding method of protein assay. Borate buffers are not suitable for
gel electrophoresis of protein, they can cause spreading of the zones if polyols
are present in the medium.
13
Use of Water-Miscible Organic Solvents

Most pH measurements in biological systems are performed in the aqueous
phase. However, sometimes mixed aqueous-water-miscible solvents, such as
methanol or ethanol, are used for dissolving compounds of biological impor-
tance. These organic solvents have dissociation constants that are very low
compared to that of pure water or of aqueous buffers (for example, the dissocia-
tion constant of methanol at 25°C is 1.45 x 10-17, compared to 1.0 x 10-14 for
water). Small amounts of methanol or ethanol added to the aqueous medium will
not affect the pH of the buffer. However, even small traces of water in methanol
or DMSO can significantly change the pH of these organic solvents.
Solubility Equilibrium: Effect of pH on Solubility

A brief discussion of the effect of pH on solubility is of significant importance
when dissolution of compounds into solvents is under consideration. Changes in
pH can affect the solubility of partially soluble ionic compounds.
Example:
Mg(OH)2 →
← Mg2+ + 2OH¯
[Mg2+] [OH¯ ]2
________________
Here K =
[Mg(OH)2]
As a result of the common ion effect, the solubility of Mg(OH)2 can be increased
or decreased. When a base is added the concentration of OH¯ increases and shifts
the solubility equilibrium to the left causing a diminution in the solubility of
Mg(OH)2. When an acid is added to the solution, it neutralizes the OH¯ and shifts
the solubility equilibrium to the right. This results in increased dissolution of
Mg(OH)2.
14
pH Measurements: Some Useful Tips

1. A pH meter may require a warm up time of several minutes. When a pH
meter is routinely used in the laboratory, it is better to leave it “ON” with the
function switch at “standby.”
2. Set the temperature control knob to the temperature of your buffer solution.
Always warm or cool your buffer to the desired temperature before checking
final pH.
3. Before you begin make sure the electrode is well rinsed with deionized water
and wiped off with a clean absorbent paper.
4. Always rinse and wipe the electrode when switching from one solution to
another.
5. Calibrate your pH meter by using at least two standard buffer solutions.
6. Do not allow the electrode to touch the sides or bottom of your container.
When using a magnetic bar to stir the solution make sure the electrode tip is
high enough to prevent any damage.
7. Do not stir the solution while taking the reading.
8. Inspect your electrode periodically. The liquid level should be maintained as

per the specification provided with the instrument .
9. Glass electrodes should not be left immersed in solution any longer than
necessary. This is important especially when using a solution containing
proteins. After several pH measurements of solutions containing proteins,
rinse the electrode in a mild alkali solution and then wash several times with
deionized water.
10. Water used for preparation of buffers should be of the highest possible purity.
Water obtained by a method combining deionzation and distillation is highly
recommended.
11. To avoid any contamination do not store water for longer than necessary. Store
water in tightly sealed containers to minimize the amount of dissolved gases.
12. One may sterile-filter the buffer solution to prevent any bacterial or fungal
growth. This is important when large quantities of buffers are prepared and
stored over a long period of time.
15
CHOOSING A BUFFER
1. Recognize the importance of the pKa. Select a buffer that has a pKa value
close to the middle of the range required. If you expect the pH to drop during
the experiment, choose a buffer with a pKa slightly lower than the working
pH. This will permit the buffering action to become more resistant to changes
in hydrogen ion concentration as hydrogen ions are liberated. Conversely, if
you expect the pH to rise during the experiment, choose a buffer with a pKa
slightly higher than the working pH. For best results, the pKa of the buffer
should not be affected significantly by buffer concentration, temperature,
and the ionic constitution of the medium.
2. Adjust pH at desired temperature. The pKa of a buffer, and hence the pH,
changes slightly with temperature. It is best to adjust the final pH at the
desired temperature.
3. Prepare buffers at working conditions. Always try to prepare your buffer

solution at the temperature and concentration you plan to use during the
experiment. If you prepare stock solutions make dilutions just prior to use.
4. Purity and cost. Compounds used should be stable and be available in high
purity and at moderate cost.
5. Spectral properties: Buffer materials should have no significant absorbance

between 240 to 700 nm range.
6. Some weak acids (or bases) are unsuitable for use as buffers in certain
cases. Citrate and phosphate buffers are not suitable for systems that are
highly calcium-dependent. Citric acid and its salts are chelators of calcium
and calcium phosphates are insoluble and will precipitate out. Use of these
buffers may lower the calcium levels required for optimum reaction. Tris
(hydroxymethyl) aminomethane is known to chelate calcium and other
essential metals.
7. Buffer materials and their salts can be used together for convenient
buffer preparation. Many buffer materials are supplied both as a free acid
(or base) and its corresponding salt. This is convenient when making a series
of buffers with different pH’s. For example, solutions of 0.1 M HEPES and 0.1
M HEPES, sodium salt, can be mixed in an infinite number of ratios between
10:1 and 1:0 to provide 0.1 M HEPES buffer with pH values ranging from
6.55 to 8.55.
16
8. Use stock solutions to prepare phosphate buffers. Mixing precalculated

amounts of monobasic and dibasic sodium phosphates has long been
established as the method of choice for preparing phosphate buffer. By
mixing the appropriate amounts of monobasic and dibasic sodium phosphate
solutions buffers in the desired pH range can be prepared (see examples on
page 17).
9. Adjust buffer materials to the working pH. Many buffers are supplied as
crystalline acids or bases. The pH of these buffer materials in solution will
not be near the pKa, and the materials will not exhibit any buffering
capacity until the pH is adjusted. In practice, a buffer material with a pKa
near the desired working pH is selected. If this buffer material is a free acid,
pH is adjusted to desired working pH level by using a base such as sodium
hydroxide, potassium hydroxide, or tetramethyl-ammonium hydroxide.
Alternatively, pH for buffer materials obtained as free bases must be adjusted
by adding a suitable acid.
10. Use buffers without mineral cations when appropriate. Frequently,

buffers without mineral cations are appropriate. Tetramethylammonium
hydroxide fits this criterion. The basicity of this organic quaternary amine is
equivalent to that of sodium or potassium hydroxide. Buffers prepared with
this base can be supplemented at will with various inorganic cations during
the evaluation of mineral ion effects on enzymes or other bioparticulate
activities.
11. Use a graph to calculate buffer composition. Figure 6 shows the

theoretical plot of ∆pH versus [A-]/[HA] on two-cycle semilog paper. As most
commonly used buffers exhibit only trivial deviations from theoretical value
in the pH range, this plot can be of immense value in calculating the relative
amounts of buffer components required for a particular pH.
For example, suppose one needs 0.1 M MOPS buffer, pH 7.6 at 20°C. At
20°C, the pKa for MOPS is 7.2. Thus, the working pH is about 0.4 pH units
above the reported pKa. According to the chart presented, this pH corre-
sponds to a MOPS sodium/MOPS ratio of 2.5, and 0.1 M solutions of MOPS
and MOPS sodium mixed in this ratio will give the required pH. If any
significant deviations from theoretical values are observed one should check
the proper working conditions and specifications of their pH meter. The
graph can also be used to calculate the amount of acid (or base) required to
adjust a free base buffer material (or free acid buffer material) to the desired
working pH.
17
10
9
8
7
6
5
4
3
[A-]/[HA]
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.1 -0.8 -0.6 -0.4 -0.2 pKa 0.2 0.4 0.6 0.8 1.0
∆ pH from pKa
Figure 6: Theoretical plot of DpH versus [A-]/[HA] on two-cycle semilog paper.
Preparation of Some Common Buffers for Use in Biological

Systems
The information provided below is intended only as a general guideline. We
strongly recommend the use of a sensitive pH meter with appropriate tempera-
ture setting for final pH adjustment. Addition of other chemicals, after adjusting
the pH, may change the final pH value to some extent. The buffer concentra-
tions in the tables below are used only as examples. You may select higher or
lower concentrations depending upon your experimental needs.
1. Hydrochloric Acid-Potassium Chloride Buffer (HCl-KCl); pH Range 1.0

to 2.2
(a) 0.1 M Potassium chloride : 7.45 g/l (M.W.: 74.5)
(b) 0.1 M Hydrochloric acid
Mix 50 ml of potassium chloride and indicated volume of hydrochloric acid.

Mix and adjust the final volume to 100 ml with deionized water. Adjust the final
pH using a sensitive pH meter.
ml of HCl 97 64.5 41.5 26.3 16.6 10.6 6.7

pH 1.0 1.2 1.4 1.6 1.8 2.0 2.2
18
2. Glycine-HCl Buffer; pH range 2.2 to 3.6

(a) 0.1 M Glycine: 7.5 g/l (M.W.: 75.0)
Mix 50 ml of glycine and indicated volume of hydrochloric acid. Mix and adjust
the final volume to 100 ml with deionized water. Adjust the final pH using a
sensitive pH meter.
ml of HCl 44.0 32.4 24.2 16.8 11.4 8.2 6.4 5.0

pH 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6
3. Citrate Buffer; pH range 3.0 to 6.2

(a) 0.1 M Citric acid: 19.21 g/l (M.W.: 192.1)
(b) 0.1 M Sodium citrate dihydrate: 29.4 g/l (M.W.: 294.0)
Mix citric acid and sodium citrate solutions in the proportions indicated and
adjust the final volume to 100 ml with deionized water. Adjust the final pH using
a sensitive pH meter. The use of pentahydrate salt of sodium citrate is not
recommended.
ml of Citric acid 46.5 40.0 35.0 31.5 25.5 20.5 16.0 11.8 7.2
ml of Sodium citrate 3.5 10.0 15.0 18.5 24.5 29.5 34.0 38.2 42.8
pH 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2
4. Acetate Buffer; pH range 3.6 to 5.6

(a) 0.1 M Acetic acid (5.8 ml made to 1000 ml)
(b) 0.1 M Sodium acetate; 8.2 g/l (anhydrous; M.W. 82.0) or 13.6 g/l
(trihydrate; M.W. 136.0)
Mix acetic acid and sodium acetate solutions in the proportions indicated and
a sensitive pH meter.
ml of Acetic acid 46.3 41.0 30.5 20.0 14.8 10.5 4.8

ml of Sodium acetate 3.7 9.0 19.5 30.0 35.2 39.5 45.2
pH 3.6 4.0 4.4 4.8 5.0 5.2 5.6
19
5. Citrate-Phosphate Buffer; pH range 2.6 to 7.0

(a) 0.1 M Citric acid; 19.21 g/l (M.W. 192.1)
(b) 0.2 M Dibasic sodium phosphate; 35.6 g/l (dihydrate; M.W. 178.0)
or 53.6 g/l (heptahydrate; M.W. 268.0)
Mix citric acid and sodium phosphate solutions in the proportions indicated and
ml of Citric acid 44.6 39.8 35.9 32.3 29.4 26.7 24.3 22.2 19.7 16.9 13.6 6.5
ml of Sodium
5.4 10.2 14.1 17.7 20.6 23.3 25.7 27.8 30.3 33.1 36.4 43.6
phosphate
pH 2.6 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2 6.6 7.0
6. Phosphate Buffer; pH range 5.8 to 8.0

(a) 0.1 M Sodium phosphate monobasic; 13.8 g/l (monohydrate, M.W. 138.0)
(b) 0.1 M Sodium phosphate dibasic; 26.8 g/l (heptahydrate, M.W. 268.0)
Mix Sodium phosphate monobasic and dibasic solutions in the proportions

indicated and adjust the final volume to 200 ml with deionized water. Adjust the
final pH using a sensitive pH meter.
ml of Sodium
92.0 81.5 73.5 62.5 51.0 39.0 28.0 19.0 13.0 8.5 5.3
phosphate, Monobasic
ml of Sodium 8.0 18.5 26.5 37.5 49.0 61.0 72.0 81.0 87.0 91.5 94.7
phosphate, Dibasic
pH 5.8 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0
7. Tris-HCl Buffer, pH range 7.2 to 9.0

(a) 0.1 M Tris(hydroxymethyl)aminomethane; 12.1 g/l (M.W.: 121.0)
Mix 50 ml of Tris(hydroxymethyl)aminomethane and indicated volume of

hydrochloric acid and adjust the final volume to 200 ml with deionized water.
Adjust the final pH using a sensitive pH meter.
ml of HCl 44.2 41.4 38.4 32.5 21.9 12.2 5.0

pH 7.2 7.4 7.6 7.8 8.2 8.6 9.0
20
8. Glycine-Sodium Hydroxide, pH 8.6 to 10.6

(a) 0.1 M Glycine; 7.5 g/l (M.W.: 75.0)
(b) 0.1 M Sodium hydroxide; 4.0 g/l (M.W.: 40.0)
Mix 50 ml of glycine and indicated volume of sodium hydroxide solutions and

ml Sodium hydroxide 4.0 8.8 16.8 27.2 32.0 38.6 45.5

pH 8.6 9.0 9.4 9.8 10.0 10.4 10.6
9. Carbonate-Bicarbonate Buffer, pH range 9.2 to 10.6

(a) 0.1 M Sodium carbonate (anhydrous), 10.6 g/l (M.W.: 106.0)
(b) 0.1 M Sodium bicarbonate, 8.4 g/l (M.W.: 84.0)
Mix sodium carbonate and sodium bicarbonate solutions in the proportions

indicated and adjust the final volume to 200 ml with deionized water. Adjust the
final pH using a sensitive pH meter.
ml of Sodium carbonate 4.0 9.5 16.0 22.0 27.5 33.0 38.5 42.5
ml of Sodium bicarbonate 46.0 40.5 34.0 28.0 22.5 17.0 11.5 7.5
pH 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6
CALBIOCHEM®
Your Source for High Quality
PROTEIN GRADE®
and
ULTROL® GRADE
Detergents for Over 50 Years.
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21
Commonly Used Buffer Media in Biological Research
Krebs-Henseleit bicarbonate buffer, pH 7.4

119 mM NaCl
4.7 mM KCl
2.5 mM CaCl2
1.2 mM MgSO4
1.2 mM KH2PO4
25 mM NaHCO3
pH 7.4 (at 37°C) when equilibrated with 95% O2 and 5% CO2. Adjust the pH
before use.
Hank’s Biocarbonate Buffer, pH 7.4

137 mM NaCl
5.4 mM KCl
0.25 mM Na2HPO4
0.44 mM KH2PO4
1.3 mM CaCl2
1.0 mM MgSO4
4.2 mM NaHCO3
pH 7.4 (at 37°C) when equilibrated with 95% O2 and 5% CO2. Adjust the pH
before use.
Phosphate Buffered Saline (PBS), pH 7.4

150 mM NaCl
10 mM Potassium Phosphate buffer
(1 liter PBS can be prepared by dissolving 8.7 g NaCl, 1.82 g
K2HPO4.3H2O, and 0.23 g KH2PO4 in 1 liter of distilled water.
Adjust the pH before use).
A variation of PBS can also be prepared as follows:

137 mM NaCl
2.7 mM KCl
10 mM Na2HPO4
1.76 mM KH2PO4
22
Tris Buffered Saline (TBS), pH 7.4

10 mM Tris
150 mM NaCl
(1 liter of TBS can be prepared by dissolving 1.21 g of Tris base and
8.7 g of NaCl in 1 liter of distilled water. Adjust the pH before use.
Note: Tris has a pKa of 8.3. Hence, the buffering capacity at pH 7.4
is minimal compared to phosphate buffer (pKa = 7.21).
TBST (Tris Buffered saline and TWEEN®-20)

10 mM Tris-HCl, pH 8.0
150 mM NaCl
0.1% TWEEN®-20
Stripping Buffer for Western Blotting Applications

62.5 mM Tris buffer, pH 6.7 to 6.8
2% Sodium dodecyl sulfate (SDS)
100 mM β-Mercaptoethanol
Cell Lysis Buffer

20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Sodium EDTA
1 mM EGTA
1% TRITON® X-100
2.5 mM Sodium pyrophosphate
1 mM β-Glycerophosphate
1 mM Sodium orthovanadate
1µg/ml Leupeptin
23
Isoelectric Point of Selected Proteins

Protein Organism/Tissue Isoelectric Point
Acetylcholinesterase Electric eel, Electric organ 4.5
α1-Acid Glycoprotein Human serum 1.8
Acid Protease Penicillium duponti 3.9
Aconitase Porcine heart 8.5
Adenosine deaminase Human erythrocytes 4.7 - 5.1
Adenylate cyclase Mouse brain 5.9 - 6.1
Adenylate kinase Rat liver 7.5 - 8.0
Adenylate Kinase Human erythrocytes 8.5 - 9.0
Albumin Human serum 4.6 - 5.3
Alcohol dehydrogenase Horse liver 8.7 - 9.3
Aldehyde dehydrogenase Rat Liver (cytosol) 8.5
Aldolase Rabbit muscle 8.2 - 8.6
Alkaline phosphatase Bovine intestine 4.4
Alkaline phosphatase Human liver 3.9
cAMP-phosphodiesterase Rat brain 6.1
Amylase Guinea Pig pancreas 8.4
Amylase Human saliva 6.2 - 6.5
Arginase Rat liver 9.4
Arginase Human liver 9.2
ATPase (Na+-K+) Dog heart 5.1
Carbonic Anhydrase Porcine intestine 7.3
Carboxypeptidase B Human pancreas 6.9
Carnitine acetyltransferase Calf liver 6.0
Catalase Mouse liver (particulate) 6.7
Cathepsin B Human liver 5.1
Cathepsin D Bovine spleen 6.7
Choline acetyltransferase Human brain 7.8
α-Chymotrypsin Bovine pancreas 8.8
Collagenase Clostridium 5.5
C-Reactive protein Human 7.4
DNA polymerase Human lymphocytes 4.7
DNase I Bovine 4.7
Dipeptidase Porcine kidney 4.9
Enolase Rat liver 5.9
Epidermal Growth Factor Mouse submaxillary glands 4.6
Erythropoietin Rabbit plasma 4.8 - 5.0
Ferritin Human liver 5.0 - 5.6
α-Fetoprotein Human serum 4.8
Follicle stimulating hormone Sheep pituitary 4.6
Fructose 1,6-diphosphatase Crab muscle 5.9
Galactokinase Human placenta 5.8
β-Galactosidase Rabbit brain 6.3
Glucose-6-phosphate dehydrogenase Human erythrocytes 5.8 - 7.0
β-Glucuronidase Rat liver microsomes 6.7
24
Isoelectric Point of Selected Proteins, cont.

Protein Organism/Tissue Isoelectric Point
γ-Glutamyl transpeptidase Rat hepatoma 3.0
Glutathione S-transferase Rat liver 6.9, 8.1
D-Glyceraldehyde 3-phosphate dehydrogenase Rabbit muscle 8.3
L-Glycerol-3-phosphate dehydrogenase Rabbit kidney 6.4
Glycogen phosphorylase b Human muscle 6.3
Growth hormone Horse pituitary 7.3
Guanylate kinase Human erythrocytes 5.1
Hemoglobin Rabbit erythrocyte 7.0
Hemoglobin A Human erythrocytes 7.0
Hexokinase Yeast 5.3
Insulin Bovine pancreas 5.7
Lactate dehydrogenase Rabbit muscle 8.5
Leucine aminopeptidase Porcine kidney 4.5
Lipase Human pancreas 4.7
Malate dehydrogenase Rabbit heart (cytosol) 5.1
Malic Enzyme Rabbit heart mitochondria 5.4
Myoglobin Horse muscle 6.8, 7.3
Ornithine decarboxylase Rat liver 4.1
Phosphoenolpyruvate carboxykinase Mouse liver 6.1
Phosphofructokinase Porcine liver 5.0
3-Phosphoglycerate kinase Bovine liver 6.4
Phospholipase A Bee venom 10.5
Phospholipase C C. perfringens 5.3
Phosphorylase kinase Rabbit muscle 5.8
Pepsin Porcine stomach 2.2
Plasmin Human plasma 7.0 - 8.5
Plasminogen Human plasma 6.4 - 8.5
Plasminogen proactivator Human plasma 8.9
Prolactin Human pituitary 6.5
Protein kinase A Bovine brain catalytic subunit 7.8
Prothrombin Human plasma 4.6 - 4.7
Pyruvate kinase Rat liver 5.7
Pyruvate kinase Rat muscle 7.5
Renin Human kidney 5.3
Ribonuclease Bovine pancreas 9.3
RNA polymerase II Human HeLa, KB cells 4.8
Superoxide dismutase Pleurotus olearius 7.0
Thrombin Human plasma 7.1
Transferrin Human plasma 5.9
Trypsin inhibitor Soybean 4.5
Trypsinogen Guinea Porcine pancreas 8.7
Tubulin Porcine brain 5.5
Urease Jack bean 4.9
25
Isoelectric Points of Selected Plasma/Serum Proteins

Protein M.W. Species Isoelectric Point
α1-Acid Glycoprotein 44,000 Human 2.7
Albumin 66,000 Human 5.2
α1-Antitrypsin 51,000 Human 4.2 - 4.7
Ceruloplasmin 135,000 Human 4.4
Cholinesterase 320,000 Human 4.0
Conalbumin — Human 5.9
C-Reactive Protein 110,000 Human 4.8
Erythropoietin — Rabbit 4.8 - 5.0
α-Fetoprotein 70,000 Human 4.8
Fibrinogen 340,000 Human 5.5
IgG 150,000 Human 5.8 - 7.3
IgD 172,000 Human 4.7 - 6.1
β-Lactoglobulin 44,000 Bovine 5.2
α2-Macroglobulin 725,000 Human 5.4
β2-Macroglobulin 11,800 Human 5.8
Plasmin — Human 7.0 - 8.5
Prealbumin 50,000 - 60,000 Human 4.7
Prothrombin — Bovine 4.6 - 4.9
Thrombin 37,000 Human 7.1
Thyroxine Binding Protein 63,000 Human 4.2 - 5.2
Transferrin 79,600 Human 5.9
Approximate pH and Bicarbonate Concentration in

Extracellular Fluids
Fluid pH meq HCO3¯/liter
Plasma 7.35 - 7.45 28
Cerebrospinal Fluid 7.4 25
Saliva 6.4 - 7.4 10 - 20
Gastric Secretions 1.0 - 2.0 0
Tears 7.0 - 7.4 5 - 25
Aqueous Humor 7.4 28
Pancreatic Juice 7.0 - 8.0 80
Sweat 4.5 - 7.5 0 - 10
26
Ionization Constants K and pKa for Selected Acids and Bases

in Water
Acids and Bases Ionization Constant (K) pKa
Acetic Acid 1.75 x 10-5 4.76
Citric Acid 7.4 x 10-4 3.13
1.7 x 10-5 4.77
4.0 x 10-7 6.40
Formic Acid 1.76 x 10-4 3.75
Glycine 4.5 x 10-3 2.35
1.7 x 10-10 9.77
Imidazole 1.01 x 10-7 6.95
Phosphoric Acid 7.5 x 10-3 2.12
6.2 x10-8 7.21
4.8 x 10-13 12.32
Pyruvic Acid 3.23 x 10-3 2.49
Tris(hydroxymethyl)aminomethane 8.32 x 10-9 8.08
Physical Properties of Some Commonly Used Acids

ml required to
Molecular Specific % Weight/ Approx.
Acid make 1 liter
Weight Gravity Weight Normality of 1 N solution
Acetic Acid 60.05 1.06 99.50 17.6 57
Hydrochloric Acid 36.46 1.19 37 12.1 83
Nitric Acid 63.02 1.42 70 15.7 64
Perchloric Acid (72%) 100.46 1.68 72 11.9 84
Phosphoric Acid 98.00 1.70 85 44.1 23
Sulfuric Acid 98.08 1.84 96 36.0 28
27
Some Useful Tips for Calculation of Concentrations and

Spectrophotometric Measurements
As per Beer’s law
A = abc
Where A = absorbance
a = proportionality constant defined as absorptivity
b = light path in cm
c = concentration of the absorbing compound
When b is 1 cm and c is moles/liter, the symbol a is substituted by a symbol e

(epsilon).
e is a constant for a given compound at a given wavelength under prescribed

conditions of solvent, temperature, pH and is called as molar absorptivity. e is
used to characterize compounds and establish their purity.
Example:
Bilirubin dissolved in chloroform at 25°C should have a molar absorptivity (e) of

60,700.
Molecular weight of bilirubin is 584.
Hence 5 mg/liter (0.005 g/l) read in 1 cm cuvette should have an absorbance of
A = (60,700)(1)(0.005/584) = 0.52 {A = abc}
Conversely, a solution of this concentration showing absorbance of 0.49 should

have a purity of 94% (0.49/0.52).
In most biochemical and toxicological work, it is customary to list constants

based on concentrations in g/dl rather than mol/liter. This is also common when
molecular weight of the substance is not precisely known.
Here for b = 1 cm; and c = 1 g/dl (1%), A can be written as A 1%

1cm
This constant is known as absorption coefficient.
The direct proportionality between absorbance and concentration must be

established experimentally for a given instrument under specified conditions.
28
Frequently there is a linear relationship up to a certain concentration. Within

these limitations, a calibration constant (K) may be derived as follows:
A = abc.
Therefore,
c = A/ab = A x 1/ab.
The absorptivity (a) and light path (b) remain constant in a given method of
analysis. Hence, 1/ab can be replaced by a constant (K).
Then,
c = A x K; where K = c/A. The value of the constant K is obtained by measuring

the absorbance (A) of a standard of known concentration (c).
29
CALBIOCHEM® Buffers
We offer an extensive line of buffer materials that meet the highest standards of
quality. We are continuing to broaden our line of ULTROL® Grade Buffer
materials, which are of superior quality and are manufactured to meet stringent
specifications. In addition, whenever possible, they are screened for uniform
particle size, giving uniform solubility characteristics.
ADA, Sodium Salt CAPS, ULTROL® Grade

(N-2-Acetamido-2-iminodiacetic Acid, Na) [3-(Cyclohexylamino)propanesulfonic Acid]
M.W. 212.2 100 g M.W. 221.3 100 g
Cat. No. 114801 Cat. No. 239782 1 kg
2-Amino-2-methyl-1,3-propanediol CHES, ULTROL® Grade

M.W. 105.1 50 g [2-(N-Cyclohexylamino)ethanesulfonic Acid]
Cat. No. 164548 500 g M.W. 207.3 100 g
Cat. No. 239779
BES, Free Acid, ULTROL® Grade
[N,N-bis-(2-Hydroxyethyl)-2-aminoethane- Citric Acid, Monohydrate, Molecular Biology
sulfonic Acid] Grade
M.W. 213.3 25 g M.W. 210.1 100 g
Cat. No. 391334 Cat. No. 231211 1 kg
Bicine, ULTROL® Grade Glycine, Free Base

[N,N-bis-(2-Hydroxyethyl)glycine] M.W. 75.1 500 g
M.W. 163.2 100 g Cat. No. 3570
Cat. No. 391336 1 kg
Glycine, Molecular Biology Grade
BIS-Tris, ULTROL® Grade M.W. 75.1 100 g
{bis(2-Hydroxyethyl)imino]-tris(hydrox- Cat. No. 357002 1 kg
ymethyl)methane}
M.W. 209.2 100 g Glycylglycine, Free Base
Cat. No. 391335 1 kg M.W. 132.1 25 g
Cat. No. 3630 100 g
BIS-Tris Propane, ULTROL® Grade
{1,3-bis[tris(Hydroxymethyl)methylamino]- HEPES, Free Acid, Molecular Biology Grade
propane} (N-2-Hydroxyethylpiperazine-N′-2-
M.W. 282.4 100 g ethanesulfonic Acid)
Cat. No. 394111 1 kg M.W. 238.3 25 g
Cat. No. 391340 250 g
Boric Acid, Molecular Biology Grade
M.W. 61.8 500 g HEPES, Free Acid, ULTROL® Grade
Cat. No. 203667 1 kg (N-2-Hydroxyethylpiperazine-N′-2-
5 kg ethanesulfonic Acid)
M.W. 238.3 25 g
Cacodylic Acid, Sodium Salt Cat. No. 391338 100 g
(Sodium Dimethyl Arsenate) 500 g
M.W. 160.0 100 g 1 kg
Cat. No. 205541 5 kg
30
HEPES, Free Acid, ULTROL® Grade, 1 M PBS-TWEEN® Tablets

Solution (Phosphate Buffered Saline-TWEEN® 20 Tablets)
M.W. 238.3 100 ml Cat. No. 524653 1 each
Cat. No. 375368 500 ml
PIPES, Free Acid, Molecular Biology Grade
HEPES, Sodium Salt, ULTROL® Grade [Piperazine-N,N′-bis(2-ethanesulfonic Acid)]
(N-2-Hydroxyethylpiperazine-N′-2- M.W. 302.4 25 g
ethanesulfonic Acid, Na) Cat. No. 528133 250 g
M.W. 260.3 100 g
Cat. No. 391333 500 g PIPES, Free Acid, ULTROL® Grade
1 kg [Piperazine-N,N′-bis(2-ethanesulfonic Acid)]
M.W. 302.4 100 g
HEPPS, ULTROL® Grade Cat. No. 528131 1 kg
(EPPS; N-2-Hydroxyethylpiperazine-N′-3-
propane sulfonic acid) PIPES, Sesquisodium Salt, ULTROL® Grade
M.W. 252.3 100 g [Piperazine-N,N′-bis(2-ethanesulfonic Acid),
Cat. No. 391339 1.5Na]
M.W. 335.3 100 g
Imidazole, ULTROL® Grade Cat. No. 528132 1 kg
(1,3-Diaza-2,4-cyclopentadiene )
M.W. 68.1 25 g PIPPS
Cat. No. 4015 100 g [Piperazine-N,N′-bis(3-propanesulfonic Acid)]
M.W. 330.4 10 g
MES, Free Acid, ULTROL® Grade Cat. No. 528315
[2-(N-Morpholino)ethanesulfonic Acid]
M.W. 195.2 100 g Potassium Phosphate, Dibasic, Trihydrate,
Cat. No. 475893 500 g Molecular Biology Grade
1 kg M.W. 228.2 250 g
Cat. No. 529567 1 kg
MES, Sodium Salt, ULTROL® Grade
[2-(N-Morpholino)ethanesulfonic Acid, Na] Potassium Phosphate, Monobasic
M.W. 217.2 100 g M.W. 136.1 100 g
Cat. No. 475894 1 kg Cat. No. 529565 500 g
MOPS, Free Acid, ULTROL® Grade Potassium Phosphate, Monobasic, Molecular

[3-(N-Morpholino)propanesulfonic Acid] Biology Grade
M.W. 209.3 100 g M.W. 136.1 250 g
Cat. No. 475898 500 g Cat. No. 529568 1 kg
1 kg
Sodium Citrate, Dihydrate
MOPS, Sodium, ULTROL® Grade (Citric Acid, 3Na)
[3-(N-Morpholino)propanesulfonic Acid, Na] M.W. 294.1 1 kg
M.W. 231.2 100 g Cat. No. 567444
Cat. No. 475899 1 kg
Sodium Citrate, Dihydrate, Molecular
MOPS/EDTA Buffer, 10X Liquid Concentrate, Biology Grade
Molecular Biology Grade M.W. 294.1 100 g
M.W. 209.3 100 ml Cat. No. 567446 1 kg
Cat. No. 475916 5 kg
PBS Tablets Sodium Phosphate, Dibasic

(Phosphate Buffered Saline Tablets) M.W. 142.0 500 g
Cat. No. 524650 1 each Cat. No. 567550 1 kg
31
Sodium Phosphate, Dibasic, Molecular Triethanolamine, Hydrochloride*

Biology Grade M.W. 185.7 1 kg
M.W. 142.0 250 g Cat. No. 641752
Cat. No. 567547 1 kg
Triethylammonium Acetate, 1 M Solution
Sodium Phosphate, Monobasic M.W. 161.2 1 liter
M.W. 120.0 250 g Cat. No. 625718
Cat. No. 567545 500 g
1 kg Tris Base, Molecular Biology Grade
[tris(Hydroxymethyl)aminomethane]
Sodium Phosphate, Monobasic, Monohy- M.W. 121.1 100 g
drate, Molecular Biology Grade Cat. No. 648310 500 g
M.W. 138.0 250 g 1 kg
Cat. No. 567549 1 kg 2.5 kg
SSC Buffer, 20X Powder Pack, ULTROL® Tris Base, ULTROL® Grade
Grade [tris(Hydroxymethyl)aminomethane]
Cat. No. 567780 2 pack M.W. 121.1 100 g
Cat. No. 648311 500 g
SSPE Buffer, 20X Powder Pack, ULTROL® 1 kg
Grade 5 kg
Cat. No. 567784 2 pack 10 kg
TAPS, ULTROL® Grade Tris Buffer, 1.0 M, pH 8.0, Molecular Biology

(3-{[tris(Hydroxymethyl)methyl]amino}- Grade
propanesulfonic Acid) M.W. 121.1 100 ml
M.W. 243.2 100 g Cat. No. 648314
Cat. No. 394675 1 kg
Tris Buffer, 100 mM, pH 7.4, Molecular
TBE Buffer, 10X Powder Pack, ULTROL® Biology Grade
Grade M.W. 121.1 100 ml
(10X Tris-Borate-EDTA Buffer) Cat. No. 648315
Cat. No. 574796 2 pack
Tris, Hydrochloride, Molecular Biology
TES, Free Acid, ULTROL® Grade Grade
(2-{[tris(Hydroxymethyl)methyl]amino}- [tris(Hydroxymethyl)aminomethane, HCl]
ethanesulfonic Acid) M.W. 157.6 100 ml
M.W. 229.3 100 g Cat. No. 648317 1 kg
Cat. No. 39465 1 kg
Tris, Hydrochloride, ULTROL® Grade
TES, Sodium Salt, ULTROL® Grade [tris(Hydroxymethyl)aminomethane, HCl]
M.W. 251.2 100 g M.W. 157.6 250 g
Cat. No. 394651 Cat. No. 648313 500 g
1 kg
Tricine, ULTROL® Grade
{N-[tris(Hydroxymethyl)methyl]glycine}
M.W. 179.2 100 g
Cat. No. 39468 1 kg * Not for international sales outside the US.
Buy in Bulk and $ave!

To request a bulk quotation,
call our bulk department at
800-854-2855 or your local sales office.
32
Technical Note - Neubauer Chamber Cell Counting - Oscar Bastidas 1
Cell Counting with

Neubauer Chamber
Basic Hemocytometer Usage
Introduction Materials
Despite the fact of the recent technical The necessary elements to perform a cell count
development of scientific laboratories, the with Neubauer chamber are as follows:
Neubauer chamber remains the most common
method used for cell counting around the world. a) cellular dilution to measure
b) hemocytometer, or Neubabuer
This article has been written in order to help chamber
newbies and experimented researchers to c) optical microscope
perform a proper cell counting using a d) cover glass
Neubauer chamber or Hemocytometer. e) pippette / micropippete with
disposable tips.
The principles described in this article apply to f) Dilution buffer / PBS (if needed)
any cell counting chamber, although the
dimensions and volumes of each chamber may THE NEUBABUER CHAMBER, OR
differ. HEMOCYTOMETER
First, the parts and basic principle of the The Neubauer chamber is a thick crystal slide
Neubauer chamber are described with the size of a glass slide. (30 x 70 mm and
4 mm thickness)
Second, the article describes how to perform a
cell count step by step, in order to achieve In a simple counting chamber, the central area
reliable and reproducible results. The article is where cell counts are performed. The
describes best practices and recommendations chamber has three parts.
when performing a cell count.
c e f
a b
d
3
Fig 1. Necessary equipment to perform a cell count with hemocytometer.
www.celeromics.com
The central part, where the counting grid has Neubauer chamber’s counting grid is 3 mm x 3
been set on the glass. mm in size. The grid has 9 square subdivisions
of width 1mm. (See Fig. 4-1)
Double chambers are most common than
simple chamber. In this case, the chamber has In case of blood cell counting, the squares
two counting areas than can be loaded placed at the corners are used for white cell
independently. counting. Since their concentration is lower than
red blood cells a larger area is required to
perform the cell count.
Upper chamber The central square is used for platelets and red
cells. This square is split in 25 squares of width
0,2 mm ( 200µm). See Fig. 4-2. Each one of the
25 central squares is subdivided in 16 small
squares. Fig 4-3.
Therefore, the central square is made of 400

small squares.
GLASS COVER.
The glass cover is a squared glass of width 22

lower chamber mm. The glass cover is placed on the top of the
Neubabuer chamber, covering the central area.
The glass cover leaves room for the cell
concentration between the bottom of the
Fig 2. Neubauer comercial chamber chamber and the cover itself. The chamber is
designed so that the distance between the
bottom of the chamber and the cover is 0,1 mm.
It is not uncommon that the glass cover remains

slightly lifted when we introduce more liquid
than necessary in the chamber. To avoid this,
some counting chambers have two special
clamps to avoid the cover glass to avoid edge-
lift.
If the glass cover is lifted, the distance between
the chamber and the cover will be higher than
0,1 mm, and the cell counts will not be accurate.
PIPETTE
The pipette allows for the introduction of a

precise amount of liquid into the Neubauer
chamber. Historically, they have been
manufactured in glass. Nowadays, glass
pipettes have been replaced by micropipettes,
than can be calibrated with a maximum
Fig 3. Pile of glass covers, and box. capacity of 20, 200 and 2000 µl.
www.celeromics.com
1 1
1 1
3
2
3 3
2 2
1 1
2
1
2 2
1 1 1
1 1
Fig 4. Neubauer-improved chamber counting grid detail.
Cell count, step by step

With concentrations below 250.000 cells per
ml, (2,5 * 105 cells / ml) the amount of cells
STEP 1. Sample preparation. counted will not be enough to obtain a fair
estimation of the original concentration. 1
Depending on the type of sample, a
preparation of a dilution with a suitable
concentration should be prepared for cell Above 2,5 million cells / ml (2,5 * 106), the
counting. probability of counting errors increases as well
as the time and effort required to achieve a
Typically, the concentration range for a cell reliable cell count.
count with Neubauer chamber is between
250.000 cells / ml and 2,5 million cells / ml. Above 2,5 million, it is preferable diluting the
sample to obtain a final concentration closer to
It is recommended for the dillution the optimum 1 million per ml. It is important to
concentration to be around 106 cells / ml (1 write down the dilution performed to the
millón cells / ml) applying the required original sample.
dilutions.
1
See:
http://www.celeromics.com/conteo-celular-con-
concentraciones-bajas.htm, for an statistical explanation
www.celeromics.com
STEP 2. Introducing the sample into the STEP 3. Microscope set up and focus.
Neubauer chamber
1. Place the Neubauer chamber on the
Take 10 µl of dilution prepare in STEP 1 with the microscope stage. If the microscope
micropipette. has a fixing clamp, fix the Neubauer
chamber.
1) Put the glass cover on the Neubauer 2. Turn on the microscope light.
chamber central area. Use a flat 3. Focus the microscope until you can
surface to place the chamber, like a see a sharp image of the cells looking
table or a workbench. through the eyepiece and adjusting
2) Put a disposable tip at the end of the the stage.
micropipette. 4. Look for the first counting grid square
3) Adjust the micropipette to suck 10 µl. where the cell count will start. In this
You can adjust it by turning the upper example, 5 big squares from a
plunger roulette to select the required Neubauer-Improved chamber will be
pipetting volume. counted. See Fig. 6
4) Introduce the micropipette tip on the
dilution previously prepared (STEP 1) See http://www.celeromics.com/easy-
5) Push the pipette plunger slowly until formula-for-manual-cell-counting.htm,
you feel it has arrived to the end of its for the formulas to be applied for the most
travel. common counting chamber (Thoma,
6) Remove the pipette tip from the Fuchs-Rosenthal, Nageotte, etc)
dilution, and bring it to the Neubauer
chamber. When the pipette is loaded, 5. Start counting the cells in the first
it must always be held in vertical square.
position.
7) Place pipette tip close to the glass Different laboratories have different
cover edge, right at the centre of the counting protocols, but there is a
Neubauer chamber. popular unwritten rule that states:
8) Release the plunger slowly watching
how the liquid enters the chamber “Cells touching the upper and left
uniformly, being absorbed by limits should be counted, unlike cells
capillarity. See Fig. 5 touching the lower and right limits
9) In case of the appearance of bubbles, which should not be taken into
or that the glass cover has moved, account”
repeat the operation.
Congratulations!. The Neubabuer chamber

has been loaded, and it is ready to perform
the cell count.
.
Fig.5. Sample filling a Neubauer chamber.

Fig. 6. Count in a Neubauer chamber big
square.
www.celeromics.com
STEP 4: Concentration calculation
We apply the formula for the calculation of the

concentration
Number of cells
Concentration (cel / ml) = -------------------
Volume (in ml)
The number of cells will be the sum of all the

counted cells in all squares counted.
The volume will be the total volume of all the

Fig. 7. High cell concentration cell count. squares counted.
Since the volume of 1 big square is:

In case of high cell concentration, it will
become very easy to get lost when counting 0,1 cm x 0,1 cm = 0,01 cm2 of area counted.
cells. In this case, a counting technique in zig- Since the depth of the chamber is 0,1mm
zag is used. 0,1 mm = 0,01 cm
0,01 cm2*0,01 cm = 0,0001 cm2 = 0,0001ml =
6. Write down the amount of cells 0,1 µl
counted in the first square.
So, for the Neubauer chamber, the formula
7. Repeat the process for the remaining used when counting in the big squares.
squares, writing down the counting
results from all of them. The higher
the number of cells counted, the Number of cells x 10.000
higher the accuracy of the Concentration =
measurement. Number of squares
In case a dilution was applied, the

concentration obtained should be converted to
the original concentration before the dilution.
In this case, the concentration should be

divided by the dilution applied.
Fig. 6. Example of cell count in one of the 9 big

squares of a Neubauer chamber.
www.celeromics.com
The formula will be:
Number of Cells x 10.000

Concentration =
Number of square x dilution
Example:
For a 1 : 10 dilution. Dilution = 0,1
For a 1 : 100. Dilution = 0,01
Error
Errors in the range of 20%-30% are common

in this method due to pipetting errors,
statistical errors, chamber volume errors, and
errors from volume of simple introduced into
the chamber.
Even though, the Neubauer chamber remains

the most widely used cell counting method in
the world.
To obtain a detailed analysis of the error

introduced in a cell count, go to:
http://www.celeromics.com/cell-count-
error.htm
www.celeromics.com
Preparing Tissue Culture Cells for Paraffin-Embedding
University of Virginia Biorepository & Tissue Research Facility (BTRF)
Last update: 10/16/07 CAM
This protocol describes the initial steps in preparing cell culture for formalin-fixation and
paraffin-embedding. The end result is a cell culture preparation that mimics the histologic
processing of tissue samples, allowing the cell culture to be used as controls for tissue
immunohistochemistry and in situ hybridization.
The volume of the packed cell pellet ideally needs to be approximately 0.5 mL. This
requires approximately four 75 cm2 sized flasks, or two 150 cm2 flasks of near-confluent
cell culture. Less material will result in a size-limited preparation, but this may be
sufficient of only a few procedures are expected to be run.
For adherent monolayer cells, do not trypsinize, as this may destroy cell-surface protein
markers. Working quickly, pull the flasks from the incubator, and scrape the cells into the
media. Transfer to a sterile 50 mL polypropylene centrifuge tube. Spin at room
temperature for five minutes in swinging bucket centrifuge (setting 3 for 5 minutes in a
standard clinical centrifuge, or approximately 200 x g).
Aspirate media off cell pellet. Very slowly, add 20 ml neutral buffered formalin or zinc
formalin (4º C) (contact the BTRF if you require this reagent), letting it flow gently down
the side of the tube, in order not to disturb the pellet. You may re-centrifuge if the cell
pellet is disturbed.
The cells need to fix overnight at 4º C in formalin. You may bring the cell preparation the
day of preparation, or after overnight incubation. If delivery of the preparation cannot be
made within 24 hours of the start of fixation, remove the formalin and replace with 20 ml
of 70% EtOH WITHOUT resuspending the pellet. This will act as a non-crosslinking
preservative, and the cells can be kept this way indefinitely at 4 º C. Do NOT freeze the
cells.
The BTRF will process the cell pellet into a paraffin block that can ultimately be used for
making histologic sections.
Page 1 of 1
RNA extraction & QC:
Promega publications
− “Working with RNA” - http://promega.wordpress.com/2012/01/24/working-with-rna/

− “RNase Contamination Happens; Recombinant RNasin® Inhibitor Can Safeguard Your Samples.
http://www.promega.com/resources/articles/pubhub/rnase-contamination-happens-
recombinant-rnasin-inhibitor-can-safeguard-your-samples/
− “RNA Purification Kit Comparison: Yield, Quality and Real-Time RT-PCR Performance” -
http://www.promega.com/resources/articles/pubhub/promega-notes-2008/rna-purification-
kit-comparison-yield-quality-and-real-time-rt-pcr-performance/
RT-qPCR Resources (including those referenced in the webinar):
General
Gene Quantification - http://www.gene-quantification.info/
A-Z of quantitative PCR ( Editor: S.A. Bustin ), International University Line (IUL), La Jolla, CA,
USA
Design, validation, controls; normalization -
− MIQE Guidelines - Minimum information for publication of quantitative real-time PCR experiments -
http://www.rdml.org/miqe.php (Bustin et al., Clinical Chemistry, 2009)
− Sean Taylor, “A MIQE Case Study — Effect of RNA Sample Quality and Reference Gene Stability
on Gene Expression Data” 2011, Bio-Rad tech note 6245
− Garbarino-Pico, E., Rollag, M.D., Strayer, C.A., Niu, S., Besharse, J.C., and Green, C.B. (2007) Immediate
early response of the circadian polyA ribonuclease nocturnin to two extracellular stimuli. RNA 13: 745-
755. PMID:17400819
Pre-designed qPCR Primers:
− Primer Bank - http://pga.mgh.harvard.edu/primerbank/
− RTPrimerDB - http://www.rtprimerdb.org/
Primer design software:
− Primer3 - http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
− Primer-BLAST - http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Real-Time qPCR Analysis Software:
geNORM - http://medgen.ugent.be/~jvdesomp/genorm/
Reference gene normalization software
QPCR - https://rtpcr.genome.tugraz.at/rtpcr/
Web-based Real-Time PCR data management and analysis
Real-Time PCR Terms:
ΔR – baseline-corrected fluorescence - R minus average R over baseline region

Passive reference – free dye (unassociated with amplification product) used for normalization
(controls for experimental/optical variation)
Rn – normalized fluorescence (R divided by passive reference)
ΔRn – baseline-corrected, normalized fluorescence.
Experimental Target – target gene/transcript/channel/dye of interest
Control Target – normalizer, endogenous control, reference gene/transcript/channel/dye,
housekeeping gene
Experimental Sample – unknown, treated, e.g., tumor, mutant, +2 hours, etc.
Control Sample – calibrator, reference sample, e.g., untreated control, normal, time zero, wild
type, etc.
Cloning (using the Invitrogen TA Cloning Kit)
What’s needed:
• Good PCR products (e.g., bright, hopefully single bands as visualized on an agarose gel)
• Ligation reagents (H2O, 10X ligation buffer, PCR2.1 vector, ligase – from kit)
• 0.2 ml and 1.5-1.8 mL centrifuge tubes.
• PCR machine for incubating ligations at 14°C
• LB/amp plates (recipe at end of procedure)
• SOC (from kit) (Kept in freezer near gel bench).
• Ampicilin
• Competent cells (KEEP ON ICE PRIOR TO USE)
• Heating block set to 42°C
• Ice bucket with ice
• Shaking incubator set to 37°C
• Stationary incubator set to 37°C (should always be at this temperature)
• X-gal (recipe at end of procedure)
• 95-100% ethanol
• spreading stick
• Bunsen burner
Useful tips:
• Cloning involves bacteria. Wear gloves. Always dispose of any material (tips, tubes, plates, etc.) that
have come into contact with the bacteria into special cloning-waste containers that should be
autoclaved when full.
• Label all tubes and plates prior to use.
PROCEDURE
Ligation (Day 1)
1. Place 0.5 uL of PCR product into 0.2 ml tube. [IMPORTANT: Only use “post-PCR”
pipettes for transferring the PCR product or any solution containing a PCR
product.]
2. Add to tube containing PCR product:
• 1.25 uL H2O
• 0.25 uL 10X buffer
• 0.5 uL PCR2.1 vector
• 0.25 uL ligase (KEEP IN FREEZER OR ON ICE ALWAYS!)
The above reagents should be made as a cocktail for use with more than one sample,
particularly because the volumes of most are so small. When preparing the cocktail always add
the ligase right before aliquoting the cocktail to each of the PCR products. Make up cocktail for
N+0.5 (N<5) or N+1(N>5) for the number of samples to be ligated (=N).
3. Incubate ligations overnight at 14°C (in PCR machine that holds 0.2 mL tubes).
Transformation (Day 2)
1. Place tubes containing competent cells on ice. [The cells are kept in the –80°C freezer. Never
place thawed cells back into the freezer – they lose their competency with thawing!] Each tube
contains 50 uL of competent cells. Although Invitrogen recommends using the entire 50 uL,
transformations work just fine using only 16 (1/3 reaction) to 25 uL (1/2 reaction) of the cells.
Thus you will only need 1/2 or 1/3 as many tubes of cells for the number of ligations you want
to transform. [IMPORTANT: Only use “post-PCR” pipettes when working with
any solutions that contain cells/bacteria or ligation.]
2. Once cells are thawed, aliquot 25 uL (or 16 ul) of cells into a 1.5-1.8 mL centrifuge tube. You
can certainly leave 25 ul (or 16 ul) of cells in the tube the cells come in for use in the following.
3. Add 2 uL of the ligation to the cells. Tap tube gently to mix. Place tube on ice for 30 minutes.
4. While waiting for the 30 minute incubation to finish, place the SOC (kept in the –20°C freezer)
in the incubator at 37°C and verify that the heating block (with holes filled with water) is set to
42°C.
5. Heat shock: After the 30 minute incubation, place cells/ligation in the heating block at 42°C for
30-45 seconds. Then place on ice for 90 seconds.
6. Add 125 uL of SOC to cells/ligation mix. Place SOC tube back in freezer.
7. Incubate tubes in shaking incubator at 37°C for 1 hour at 225 rpm.
8. While cells are incubating, place LB/amp plates in the stationary incubator to warm.
9. A few minutes before the 1 hour incubation is complete, remove the X-gal from the –20°C
freezer and place on benchtop to thaw or warm up. Also, remove the LB plates from the
incubator, place on benchtop, and label.
10. After incubation, add 12.5 uL of X-gal to cell/ligation mix.
11. Add all of the cell/ligation/X-gal solution to the LB plate and spread evenly with sterile spreading
stick over plate.
12. Let plate set on benchtop for 10-15 minutes so that the solution absorbs into the agar.
13. Place plates upside down in incubator for 18 hours. After incubation there should be many
(>100) colonies evenly distributed on the surface of the agar in each plate. There should be blue
ones, white ones, white ones with blue dots in the middle, and whitish-blue ones. The white
colonies are usually the good ones (i.e., with an insert), but often so are the others except the
all-blue ones.
Screening of Colonies (Day 3)

1. Prepare PCR cocktail with M13 and M13R primers. Label colonies on plate to screen.
2. Make grid on warmed LB/amp plate (for the numbers of colonies to be screened).
3. Add cocktail to tubes/PCR plate.
4. The template for the amplification are the DNA present in the colonies (precisely, the vectors
in the bacteria). To add a small amount of the bacteria to the PCR reagents, barely touch the tip
of a toothpick (autoclaved or not, apparently it doesn’t matter) to a colony, touch the colony
end of the toothpick onto the gridded LB/amp plate, and then gently dab the toothpick into the
tube containing the PCR reagents. Be careful – too much of the cells and the PCR just won’t
work!
5. Run the PCR at 94C/30 seconds, 55C/30 seconds, and 72C/30 seconds for 30 cycles (extending
the annealing stage to 45-60 seconds if screening for large (>500 bp) inserts. It will be done in
about 1 hour and 15 minutes or so – you can get the agarose gels ready now!
6. After reaction is complete, run samples on 1.8% agarose/TBE gel. Amplifications of good inserts
will be approximately 180 bp larger than the insert size (due to the amplification of part of the
vector).
RECIPES
LB/amp plates (for 20-25 plates)
1. Into 500 mL flask add the following:

2.5 g tryptone -- or -- 5 g Bacto LB broth
1.25 g yeast extract 3.5 g agar
3.5 g agar
2.5 g NaCl [no need to pH]
2. Add H2O to a final volume of 250 mL
3. Add about 30-40 drops of 1 N NaOH until pH of solution is 7 (IF USING FIRST RECIPE – see above).
4. Cover top with foil and autoclave (with liquid/solutions setting) for 15 minutes.
5. Remove from autoclave and let cool on benchtop. You want the temperature of the solution to be high
enough so that the agar doesn’t solidify but low enough so that the ampicillin that you will add in the
subsequent step isn’t rendered inactive. If you can hold the flask on the palm of your hand for 15 seconds
or so it has reached the proper temperature (~55°C).
6. Dissolve 25 mg of ampicillin (kept in the refrigerator) in 1 mL of H2O and add to agar broth.
7. Pour LB/amp into plastic plates. If you are frugal (i.e., only pour enough LB/amp to fill about 2/3 of the
bottom of the plate), you will be able to prepare 20-25 plates. Gently swirl the plate to enable the
solution to completely cover the bottom of the plate.
8. Let plates set on benchtop until the agar solution has completely solidified (30-60 minutes)
9. Place LB/amp plates in bag and put so that plates are upside down into the refrigerator.
X-gal
Make up X-gal as 50 mg/mL stock solutions with dimethyl formamide (doesn’t freeze in freezer) or
DMSO (dimethyl sulfoxide) (freezes in freezer).
Aliquot only about 100-200 uL per tube of working solution. Frequent freeze/thaws may render the X-
gal inactive and so working stocks should be kept at small volumes.
STOCK SOLUTION RECIPIES:
Tris-HCl Buffer
10X Tris-HCl (0.5M Tris Base, pH7.6):

Trizma Base ---------------------------------- 61 g
Distilled water ------------------------------- 1000 ml
Adjust pH 7.6 using concentrated HCl
Store this solution at room temperature. Dilute 1:10 with distilled water before use
and adjust pH if necessary.
20X Tris-HCl (1M Tris Base, pH7.6):

Trizma Base ---------------------------------- 122 g
Adjust pH 7.6 using concentrated HCl
10X Tris-HCl-Tween 20 (0.5M Tris Base, 0.5% Tween 20, pH7.6):

Trizma Base ---------------------------------- 61 g
Adjust pH 7.6 using concentrated HCl and then add 5 ml of Tween 20.
Note: Tris-HCl Buffer is used for specific cases of immunohistochemical staining.
*** OR you can use Tris Base to make Tris-HCl (note that Tris base is different
from Trizma)
Tris is a chemical with basic properties, having a pKa of 8.1. It can be used to buffer
solutions from drastic pH changes, keeping them in the pH range of 7.0 to 9.0.
Make any Tris-HCl buffer in this pH range, at any molarity using these simple steps
1) Calculate Moles of Tris Base
mol/L * L = moles needed
2) Calculate Mass of Tris Base
Determine the mass of Tris base to weigh by multiplying the number of moles by
the molecular weight (121.14 g/mol) of Tris.
moles needed * g/mol = g
3) Dissolve Tris Base in Water
Dissolve the required mass of Tris into a volume of deionized water
approximately 1/3 of the desired volume of buffer to be made.
4) Adjust the pH
Using a pH meter, titrate the solution of Tris with 1M hydrochloric acid (HCl)
until the correct pH is reached.
5) Bring to Volume
Add the TrisHCl mixture to a volumetric flask of the desired volume and add
deionized water as required to complete the solution.
0.2M Phosphate Buffer-4 Liters (pH 7.4):
17.66g Sodium Phosphate Monobasic

90.03g Sodium Phosphate Dibasic Heptahydrate
4 Liters ddH2O
pH should be 7.4, if not adjust with 1.0N NaOH or 1.0N HCl
(3 Liters-13.25g Mono and 67.52g Dibasic)
0.1M Phosphate Buffer Saline (PBS)-8 Liters:
Prepare 4 liters of 0.2M phosphate buffer (see above recipe)

Add 72g NaCl (0.9% or 9g/liter)
Add 4 liters of ddH2O
pH=7.4
*For practical purposes, you can also make 16 liters of PBS by first preparing 4 liters of
0.4M Phosphate Buffer. This concentration uses twice as much Monobasic and Dibasic
since 0.4M versus 0.2M means the solution is twice as concentrated. But remember, you
must then dilute this solution by adding 12 liters of water to make a total of 16 liters at a
concentration of 0.1M. Also, since we are using 9gNaCl/liter, a total of 144g of NaCl
will be used.
0.1M Phosphate Buffer-1 liter:
0.5 Liter of 0.2 M Phophate Buffer Stock

0.5 Liter of ddH2O
0.1M Phosphate Buffered Saline with Azide-1 liter (PBS*):
Prepare one liter of 0.1M Phosphate Buffer (see recipe above)

Add 9g of NaCl
Add 200mg of sodium azide (contents of 1 eppendorf vial)
Add 0.3 ml Triton-X
*Be sure to use protective clothing and a mask when handling sodium azide under hood!!
1% Paraformaldehyde 2% Glutaraldehyde
0.2M Stock Phosphate Buffer pH 7.4 500ml

Paraformaldehyde 10g
Glutaraldehyde (25% in water) 80ml
Distilledwater QS to 1000ml
Dissolve the paraformaldehyde in about 400 ml of water. Heat this solution to 58 to 60

degrees C(do not allow the solution to get to hot!!). Add 1N NaOH drop by dropuntil the
solution turns clear. Add the phosphate buffer stock and allow to cool, and then add the
glutaraldehyde. Ph the solution to 7.4 and FILTER before use.
1% Paraformaldehyde 2% Glutaraldehyde (Hrp Fix)

Distilled water QS to 1000ml
Dissolve the paraformaldehyde in about 400ml of water. Heat the solutionto 58 to 60
degrees C (do not allow the solution to get too hot!!). Add 1N NaOH drop by dropuntil
the solution clears.Add phosphate buffer stock and allow the solution to cool. Add
theglutaraldehyde and FILTER.
2% PARAFORMALDEHYDE

Dissolve the paraformaldehyde in about 400ml of water. Heat the solutionto 58 to 60
degrees C(do not allow the solution to get too hot!!). Add 1N NaOH drop by dropuntil
the solution clears.Add phophate buffer stock and allow the solution to cool. pH to 7.4
and FILTER.
4% PARAFORMALDEHYDE
0.2 M Stock Phosphate Buffer pH 7.4 500ml

Dissolve the paraformaldehyde in about 400ml of water. Heat the solution to 58 to 60
degrees C(do not allow the solution to get too hot!!) Add 1N NaOH to the
paraformaldehyde solution drop by drop until the solution clears. Add phosphate buffer
stock and allow to cool. pH to 7.4 and FILTER.
4% PARAFORMALDEHYDE 0.2% GLUTARALDEHYDE

Dissolve the paraformaldehyde in about 400 ml of water. Heat the solutionto 58 to 60

C(do not allow the solution to get too hot!!). Add 1N NaOH drop by dropuntil the
solution clears. Add phosphate buffer stock and allow the solution to cool. Add the
glutaraldehyde. pH to 7.4 and FILTER.
Sucrose solution
10% 10gram in 90 ml 0.1 M PB
20% 20gram in 80 ml 0.1 M PB
30% 30gram in 70 ml 0.1 M PB
Acrylamide for separating gel (Acrylamide : BIS = 30 : 0.135)
Acrylamide 30.00 g
BIS 0.135 g
Make volume to 100 ml with MQ water. Keep in dark (Brown bottle)
Separating gel buffer (pH 8.8) (Final Conc.)

Tris 12.11 g 1M
SDS 0.27 g 0.27%
Dissolve in 80 ml MQ water, adjust pH to 8.8, make the vol. to 100 ml
Acrylamide for stacking gel (Acrylamide : BIS = 29.2 : 0.8)

Acrylamide 29.2 g
BIS 0.8 g
Make volume to 100 ml with MQ water. Keep in dark (Brown bottle)
Stacking gel buffer (pH 6.8) (Final Conc.)

Tris 3.03 0.25 M
SDS 0.20 g 0.2%
Dissolve in 80 ml MQ water, adjust pH to 6.8, make the vol. to 100 ml
SDS-PAGE running buffer

Tris 9.0 g
Glycine 43.2 g
SDS 3.0 g
Dissolve in 3 L MQ water
SDS-sample buffer
Glycerol 10 ml
Tris 0.757 g
SDS 2.5 g
2-Mercaptoethanol 5.0 ml
Dissolve in 100 ml MQ water
Bromophenol blue (BPB) solution

Dissolve 0.1 g BPB in 100 ml 10% glycerol
Blotting solution A (Final conc.)

Tris 36.33 g 0.3 M
Methanol 200 ml 20%
SDS 0.20 g 0.02%
Make vol to 1000 ml with MQ water, keep at 4 ºC
Blotting solution B (Final conc.)

Tris 3.03 g 25 mM
Methanol 200 ml 20%
SDS 0.20 g 0.02%
Blotting solution C (Final conc.)

Tris 3.03 g 25 mM
s-Amino-n-Caproic Acid 5.20 g 40 mM
Methanol 200 ml 20%
SDS 0.20 g 0.02%
Ponceau 3S staining solution

Dissolve 0.1 g Ponceau 3S in 100 ml 1% Acetic acid
Coomassie brilliant blue (CBB) solution

Methanol 1500 ml
Acetic acid 300 ml
Coomassie brilliant blue-R250 3 g
Destaining solution
Methanol 1100 ml
Acetic acid 300 ml
Acrylamide solution for 1D-PAGE(Acrylamide : BIS = 28.38 : 1.62)

Acrylamide(High grade) 28.38 g
BIS 1.62 g
Make vol to 1000 ml with MQ water, keep in dark
Lysis buffer for Western Blot

Lysis buffer stock
1 ml Tris HCl, pH 7.4 (pre-made stock)
0.584g NaCl
49 ml dH2O
This stock can be kept for up to 2 weeks at 4C
Lysis buffer for tissue prep

10 ml lysis buffer stock (see above)
1 protease inhibitor tablet
0.2 ml Phosphatase inhibitors (1 mM Na3VO4 and 5 mM NaF to block both tyrosine
and serine/threonine kinases, see below)
100 ml (50x Na3VO4 and NaF stock solution)

50 mM Na3VO4 0.92gl
500 mM NaF 2.0995g
100 ml dH2O
0.02 N H3PO4
Add 0.48 ml H3PO4 (42 N, specific weight 1.87, 72%) to 1000 ml MQ water
0.02 N NaOH
Dissolve 0.8 g NaOH to 1000 ml MQ water.
www.abcam.com/technical
RIPA buffer (RadioImmunoPrecipitation Assay) buffer:

RIPA buffer contains the ionic detergent sodium deoxycholate as an active constituent
and is particularly used for nuclear membrane disruption for nuclear extracts. A RIPA
buffer gives low background but can denature kinases. It can also disrupt protein-protein
interactions (and may therefore be problematic for
immunoprecipitations/pull down assays).
50mM Tris HCl pH 8

150 mM NaCl
1% NP-40
0.5% sodium Deoxycholate
0.1% SDS
The 10% sodium deoxycholate stock solution (5 g into 50 ml) must be protected from
light.
The 100 mM EDTA stock solution is made with 1.86 g into 40 ml H2O and then add
NaOH to dissolve and adjust pH to 7.4. Finally, adjust the total volume to 50 ml). Store
the buffer at 4°C.
Nonidet-P40 (NP-40) buffer:

20 mM Tris HCl pH 8
137 mM NaCl
10% glycerol
1% nonidet P-40
2 mM EDTA
Sodium orthovanadate preparation:
This needs to be done under the fume hood

• Prepare a 100 mM solution in double distilled water
• Set pH to 9.0 with HCl
• Boil until colorless
• Cool to room temperature
• Set pH to 9.0 again
• Boil again until colorless
• Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling
• Bring up to the initial volume with water
• Store in aliquots at -20°C
Note: do not permit great changes in volume during boiling; put a loose lid on the
container to protect from evaporation. Discard if the samples turn yellow.
TBS 10x (concentrated TBS)
24.23 g Trizma HCl
80.06 g NaCl
Mix in 800 ml ultra pure water.
pH to 7.6 with pure HCl.
Top up to 1 L.
TBST
For 1 L:
100 ml of TBS 10x
+ 900 ml ultra pure water
+ 1ml Tween20
Medium stripping buffer:

Make fresh stripping buffer:
15 g glycine
1 g SDS
10 ml Tween20
Set the pH to 2.2
make up to 1 L with ultrapure water
Harsh stripping buffer:

to be done under the fumehood
For 100 ml:
20 ml SDS 10%
12.5 ml Tris HCl pH 6.8 0.5M
67.5 ml ultra pure water
Add 0.8ml ß-mercaptoethanol under the fumehood.
Nuclear Fractionation Protocol Reagents

Buffer A –
10 mM HEPES,
1.5 mM MgCl2,
10 mM KCl,
0.5 mM DTT,
0.05% NP40 (or 0.05% Igepal or Tergitol)
pH 7.9
To prepare 250 ml stock of buffer A –

HEPES: 1M = 238.3 g/L, therefore 10 mM = 0.59 g/250 ml
MgCl2: 1M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 ml
KCl: 1M = 74.5 g/L, therefore 10 mM = 0.187 g/250 ml
DTT: 1M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 ml
NP40 = 0.05%
Buffer B –
5 mM HEPES,
1.5 mM MgCl2,
0.2 mM EDTA,
0.5 mM DTT,
26% glycerol (v/v)
pH 7.9
To prepare 250 ml stock of buffer B –

HEPES: 1M = 238.3 g/L, therefore 5 mM = 0.295 g/250 ml
MgCl2: 1M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 ml
EDTA: 1M = 372.2 g/L, therefore 0.2 mM = 0.0186 g/250 ml
DTT: 1M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 ml
26% Glycerol (v/v) = 65 ml
4.6 M NaCl
87.66 g/326 ml
TBS (Tris Buffered Saline) pH 7.6-7.8:

For 10 litres:
60.6 g TRIS HCl
13.9 g TRIS base
87.66 g NaCl
10 litres Ultra pure water (H2O)
TBS 0.025% Triton X-100:

For 1 litre:
250 μl Triton X-100
999.75 ml TBS
pH 7.6-7.8
1.6% H2O2 (Hydrogen Peroxide) in TBS:

For 400 ml:
6.4 ml H2O2 (GPR = 30% w/w)
393.6 ml TBS
pH 7.6-7.8
BS - Blocking serum in TBS (100ml):

NGS (10%) 10ml
BSA (2%) 2ml
Triton (0.4%) 4ml of 10% Triton
10% NS (Normal Serum) with 1% BSA (Bovine Serum Albumin, Fraction 5) in

TBS:
For 1 ml:
100 μl NS
10 mg BSA
900 μl TBS
pH 7.6-7.8
Primary antibody made up in TBS with 1% BSA:

(Example is of primary antibody used at a dilution of 1:10)
For 0.1 ml:
100 μl Primary antibody
10 mg BSA
900 μl TBS
pH 7.6-7.8
Secondary biotinylated antibody made up in TBS with 1% BSA:

(Example is of secondary biotinylated antibody used at a dilution of 1:200)
For 1 ml:
5 μl Secondary biotinylated antibody
995 μl TBS
pH 7.6-7.8
ABC (Avidin-Biotin) complex in TBS:

(Example is of ABC complex, each part used at a dilution of 1:100)
For 1 ml:
10 μl Streptavidin
10 μl HRP (or AP)-Biotin
980 μl TBS
pH 7.6-7.8
Bicarbonate/carbonate coating buffer (100 mM):

3.03 g Na2CO3,
6.0 g NaHCO3 (1 L distilled water)
pH 9.6,
PBS (500 ml):

1.16 g Na2HPO4,
0.1 g KCl,
0.1 g K3PO4,
4 g NaCl
(500 ml distilled water) pH 7.4
LABORATORY STOCK SOLUTIONS AND APPENDIX 2
EQUIPMENT
Common Stock Solutions, Buffers, and Media APPENDIX 2A
RECIPES
This section describes the preparation of buffers and reagents used in this manual for cell
culture, manipulation of tissue, and cell biological methods. When preparing solutions,
use deionized or distilled water and reagents of the highest available grade. Sterilization—
by filtration through a 0.22-µm filter or by autoclaving—is recommended for most
solutions stored at room temperature and is essential for cell culture applications. Where
storage conditions are not specified, store up to 6 months at room temperature. Discard
any reagent that shows evidence of contamination, precipitation, or discoloration.
Acid, concentrated stock solutions

See Table A.2A.1.
Acid precipitation solution
1 M HCl
0.1 M sodium pyrophosphate
Nucleic acids can also be precipitated with a 10% (w/v) solution of trichloroacetic acid
(TCA); however, this recipe is cheaper, easier to prepare, and just as efficacious.
Ammonium hydroxide, concentrated stock solution
See Table A.2A.1.
Ammonium acetate, 10 M
Dissolve 385.4 g ammonium acetate in 150 ml H2O
Add H2O to 500 ml
Ammonium sulfate, saturated
76 g ammonium sulfate
100 ml H2O
Heat with stirring to just below boiling point
Let stand overnight at room temperature
Table A.2A.1 Molarities and Specific Gravities of Concentrated Acids and Basesa
1M
Molecular Molarity Specific
Acid/base % by weight solution
weight (approx.) gravity
(ml/liter)
Acetic acid (glacial) 60.05 99.6 17.4 1.05 57.5
Ammonium hydroxide 35.0 28 14.8 0.90 67.6
Formic acid 46.03 90 23.6 1.205 42.4
98 25.9 1.22 38.5
Hydrochloric acid 36.46 36 11.6 1.18 85.9
Nitric acid 63.01 70 15.7 1.42 63.7
Perchloric acid 100.46 60 9.2 1.54 108.8
72 12.2 1.70 82.1
Phosphoric acid 98.00 85 14.7 1.70 67.8
Sulfuric acid 98.07 98 18.3 1.835 54.5
aCAUTION: Handle strong acids and bases carefully. Laboratory Stock
Solutions and
Equipment
Current Protocols in Cell Biology (1998) A.2A.1-A.2A.10 A.2A.1

Copyright © 1998 by John Wiley & Sons, Inc.
ATP, 100 mM
1 g ATP (adenosine triphosphate)
12 ml H2O
Adjust volume to 16.7 ml with H2O
Store in aliquots indefinitely at −20°C
Base, concentrated stock solutions
See Table A.2A.1.
BSA (bovine serum albumin), 10% (w/v)
Dissolve 10 g BSA (e.g., Sigma) in 100 ml H2O. Filter sterilize using a low-protein-
binding 0.22-µm filter. Store indefinitely at 4°C.
Lower-concentration stock solutions (e.g., 1%), which are useful for various applications,
can be made by diluting 10% stock appropriately with sterile water.
BSA is available in various forms that differ in fraction of origin, preparation, purity, pH,
and cost; the most commonly used is fraction V. Use the form that is appropriate for the
application; this may need to be optimized empirically.
CaCl2 , 1 M
147 g CaCl2⋅2H2O
H2O to 1 liter
Carbonate buffer
1.6 g Na2CO3 (15 mM final)
2.9 g NaHCO3 (35 mM final)
0.2 g NaN3 (3.1 mM final)
H2O to 1 liter
Adjust to pH 9.5
CAUTION: Sodium azide is poisonous; follow appropriate precautions for handling,
storage, and disposal.
CMF-DPBS (calcium- and magnesium-free Dulbecco’s phosphate-buffered saline)
8.00 g NaCl (0.137 M)
0.20 g KCl (2.7 mM)
2.16 g Na2HPO4⋅7H2O (8.1 mM)
0.20 g KH2PO4 (1.1 mM)
H2O to 1 liter
Store at room temperature
DEPC (diethylpyrocarbonate)-treated solutions
Add 0.2 ml DEPC to 100 ml of the solution to be treated. Shake vigorously to
dissolve the DEPC. Autoclave the solution to inactivate the remaining DEPC.
CAUTION: Wear gloves and use a fume hood when using DEPC, as it is a suspected
carcinogen.
Many investigators keep the solutions they use for RNA work separate to ensure that “dirty”
pipets do not go into them.
Do not treat solutions containing Tris with DEPC, as Tris inactivates the DEPC.
DMEM (Dulbecco’s modified Eagle medium), supplemented
Dulbecco’s modified Eagle medium, high-glucose formulation (see APPENDIX 2B;
e.g., Life Technologies), containing:
Common Stock
5%, 10%, or 20% (v/v) FBS, heat inactivated (optional; see recipe below)
Solutions, 1% (v/v) nonessential amino acids
Buffers, and 2 mM L-glutamine
Media continued
A.2A.2
Current Protocols in Cell Biology
100 U/ml penicillin
100 µg/ml streptomycin sulfate
Filter sterilize if anything nonsterile has been added
Store up to 1 month at 4°C
DMEM containing this set of additives is sometimes called “complete DMEM.” The
percentage of serum used is indicated after the medium name—e.g., “DMEM/5% FBS.”
Absence of a number indicates no serum is used. DMEM is also known as Dulbecco’s
minimum essential medium.
Ham’s F-12 nutrient mixture (APPENDIX 2B; available commercially, e.g., from Life Technolo-
gies), is sometimes added to DMEM; the resulting medium is known as DMEM/F-12.
Because of the higher bicarbonate content, DMEM requires ∼10% CO2 to maintain pH 7.4.
Culture media containing glutamine and penicillin should be warmed to 37°C as few times
as possible since components, especially glutamine, degrade rapidly at 37°C.
DPBS (Dulbecco’s phosphate-buffered saline)
8.00 g NaCl (0.137 M)
0.20 g KCl (2.7 mM)
0.20 g KH2PO4 (1.1 mM)
0.10 g MgCl2⋅6H2O (0.5 mM)
2.16 g Na2HPO4⋅7H2O (8.1 mM)
0.10 g anhydrous CaCl2 (0.9 mM)
H2O to 1 liter
DTT (dithiothreitol), 1 M
Dissolve 1.55 g DTT in 10 ml water and filter sterilize. Store in aliquots at −20°C.
EDTA (ethylenediaminetetraacetic acid), 0.5 M (pH 8.0)
Dissolve 186.1 g disodium EDTA dihydrate in 700 ml water. Adjust pH to 8.0 with
10 M NaOH (∼50 ml; add slowly). Add water to 1 liter and filter sterilize.
Begin titrating before the sample is completely dissolved. EDTA, even in the disodium salt
form, is difficult to dissolve at this concentration unless the pH is increased to between 7
and 8.
FBS (fetal bovine serum)
Thaw purchased fetal bovine serum (shipped on dry ice and kept frozen until
needed). Store 3 to 4 weeks at 4°C. If FBS is not to be used within this time,
aseptically divide into smaller aliquots and refreeze until used. Store ≤1 year at
−20°C. To heat inactivate FBS, heat serum 30 min to 1 hr in a 56°C water bath with
periodic gentle swirling during the first 10 to 15 min to ensure uniform heating.
Repeated thawing and refreezing should be avoided, as it may cause denaturation of the
serum.
Heat-inactivated FBS (FBS that has been treated with heat to inactivate complement protein
and thus prevent an immunological reaction against cultured cells) is useful for a variety of
purposes. It can be purchased commercially or made in the lab as described above.
HBSS (Hanks’ balanced salt solution)
0.40 g KCl (5.4 mM final)
0.09 g Na2HPO4⋅7H2O (0.3 mM final)
0.06 g KH2PO4 (0.4 mM final)
0.35 g NaHCO3 (4.2 mM final)
0.14 g CaCl2 (1.3 mM final)
0.10 g MgCl2⋅6H2O (0.5 mM final)
0.10 g MgSO4⋅7H2O (0.6 mM final) Laboratory Stock
8.0 g NaCl (137 mM final) Solutions and
Equipment
A.2A.3
1.0 g D-glucose (5.6 mM final)
0.2 g phenol red (0.02%; optional)
Add H2O to l liter and adjust pH to 7.4 with 1 M HCl or 1 M NaOH
Filter sterilize and store up to 1 month at 4°C
HBSS may be made or purchased without Ca2+ and Mg2+ (CMF-HBSS). These components
are optional and usually have no effect on an experiment; in a few cases, however, their
presence may be detrimental. Consult individual protocols to see if the presence or absence
of these components is recommended.
Bottles should be kept tightly closed to prevent CO2 loss and subsequent alkalinization.
HCl, 1 M
Mix in the following order:
913.8 ml H2O
86.2 ml concentrated HCl
HeBS (HEPES-buffered saline) solution, 2×
16.4 g NaCl
11.9 g HEPES acid
0.21 g Na2HPO4
800 ml H2O
Titrate to pH 7.05 with 5 M NaOH
Add H2O to 1 liter
Filter sterilize through a 0.45-µm nitrocellulose filter
Store in 50-ml aliquots at −20°C
If the solution is to be used for transfection, the pH should be between 7.05 and 7.12, and
should be tested for transfection efficiency.
KCl, 1 M
74.6 g KCl
H2O to 1 liter
LB medium
Per liter:
10 g tryptone
5 g yeast extract
5 g NaCl
1 ml 1 M NaOH
Autoclave 25 min
Although the pH is adjusted to near 7 with NaOH, the medium is not very highly buffered,
and the pH of a culture growing in the medium drops as the culture nears saturation.
The medium may also contain antibiotics (e.g., 50 µg/ml ampicillin, 12 µg/ml tetracycline),
galactosides (e.g., 20 µg/ml Xgal, 0.1 mM IPTG), or other nutritional supplements added
after the medium has been autoclaved.
To make LB agar for LB plates, add 5 g/liter agar or agarose.
MgCl2 , 1 M
20.3 g MgCl2⋅6H2O
H2O to 100 ml
MgSO4 , 1 M
24.6 g MgSO4⋅7H2O
H2O to 100 ml
Common Stock NaCl, 5 M
Solutions,
Buffers, and 292 g NaCl
Media H2O to 1 liter
A.2A.4
NaOH, 10 M
Dissolve 400 g NaOH in 450 ml H2O
Add H2O to 1 liter
PBS (phosphate-buffered saline)
8.00 g NaCl (0.137 M)
0.20 g KCl (2.7 mM)
0.24 g KH2PO4 (1.4 mM)
1.44 g Na2HPO4 (0.01 M)
H2O to 1 liter
PCR amplification buffer, 10×
500 mM KCl
100 mM Tris⋅Cl, pH 8.3 (see recipe below)
x mM MgCl2
0.1% (w/v) gelatin
Store in aliquots at −20°C
This solution can be sterilized by autoclaving. Alternatively, it can be made from sterile water
and stock solutions, and the sterilization omitted.
15 mM MgCl2 is the concentration (x) used for most PCR reactions. However, the optimal
concentration depends on the sequence and primer of interest and may have to be determined
experimentally (see APPENDIX 3).
PMSF (phenylmethylsulfonyl fluoride), 100 mM
Dissolve 0.174 g PMSF in 10 ml of 100% ethanol, isopropanol, or methanol. Store
in aliquots up to 2 years at −20°C.
CAUTION: Phenylmethylsulfonyl fluoride is toxic.
Make fresh dilutions from the alcohol stock for each use, because the half-life of PMSF in
aqueous solution is <30 min at room temperature and a few hours on ice.
If PMSF is being added to a solution without detergent, the solution should be stirred
vigorously during PMSF addition because PMSF has a tendency to form an insoluble
precipitate in aqueous solution.
Polylysine-coated tissue culture surfaces
Prepare a stock solution by dissolving 100 mg polylysine in 100 ml water (poly-L-
lysine or poly-D-lysine can be used; check specific protocol for choice of isomer)
and filter sterilize through a 0.22-µm filter. Store in 5-ml aliquots at −20°C. When
ready to use, dilute 1 part stock solution with 19 parts water to prepare a 50 µg/ml
working solution.
To coat culture dishes, multiwell plates, or chamber slides: Fill tissue culture dishes,
multiwell plates, or slide wells with the working solution and incubate 1 hr in a 37°C
incubator, then remove solution by vacuum aspiration and allow surface to dry.
To coat coverslips: Sterilize coverslips by autoclaving or by incubating them in 95%
ethanol and drying before coating. Place coverslips in a single layer in a petri dish
containing working solution and incubate 1 hr at 37°C. Remove coverslips using
sterile forceps and allow surface to dry.
Store coated tissue culture ware up to 3 months at 4°C. Use diluted solutions only once.
Potassium acetate buffer, 0.1 M
Solution A: 11.55 ml glacial acetic acid per liter (0.2 M) in water.
Solution B: 19.6 g potassium acetate (KC2H3O2) per liter (0.2 M) in water.
Laboratory Stock
continued Solutions and
Equipment
A.2A.5
Table A.2A.2 Preparation of 0.1 M Sodium
and Potassium Acetate Buffersa
Desired Solution A Solution B

pH (ml) (ml)
3.6 46.3 3.7
3.8 44.0 6.0
4.0 41.0 9.0
4.2 36.8 13.2
4.4 30.5 19.5
4.6 25.5 24.5
4.8 20.0 30.0
5.0 14.8 35.2
5.2 10.5 39.5
5.4 8.8 41.2
5.6 4.8 45.2
aAdapted by permission from CRC (1975).
Referring to Table A.2A.2 for desired pH, mix the indicated volumes of solutions A
and B, then dilute with water to 100 ml. Filter sterilize if necessary. Store up to 3
months at room temperature.
This may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium acetate
in the same volume. Acetate buffers show concentration-dependent pH changes, so check the
pH by diluting an aliquot of concentrate to the final concentration.
To prepare buffers with pH intermediate between the points listed in Table A.2A.2, prepare
closest higher pH, then titrate with solution A.
Potassium phosphate buffer, 0.1 M
Solution A: 27.2 g KH2PO4 per liter (0.2 M final) in water.
Solution B: 34.8 g K2HPO4 per liter (0.2 M final) in water.
This buffer may be made as a 5- or 10-fold concentrate simply by scaling up the amount of
potassium phosphate in the same final volume. Phosphate buffers show concentration-de-
pendent changes in pH, so check the pH of the concentrate by diluting an aliquot to the final
concentration.
Saponin, 10% (w/v)
Dissolve 1 g saponin in 10 ml PBS (see recipe above)
Store in 500-µl aliquots at −20°C
Once thawed, the 10% solution is stable for several months when stored at 4°C.
SDS, 20% (w/v)
Dissolve 20 g SDS (sodium dodecyl sulfate or sodium lauryl sulfate) in H2O to 100
ml total volume with stirring. Filter sterilize using a 0.45-µm filter.
It may be necessary to heat the solution slightly to fully dissolve the powder.
Common Stock
Solutions,
Buffers, and
Media
A.2A.6
Table A.2A.3 Preparation of 0.1 M Sodium and Potassium Phosphate Buffersa
Desired Solution A Solution B Desired Solution A Solution B

pH (ml) (ml) pH (ml) (ml)
5.7 93.5 6.5 6.9 45.0 55.0
5.8 92.0 8.0 7.0 39.0 61.0
5.9 90.0 10.0 7.1 33.0 67.0
6.0 87.7 12.3 7.2 28.0 72.0
6.1 85.0 15.0 7.3 23.0 77.0
6.2 81.5 18.5 7.4 19.0 81.0
6.3 77.5 22.5 7.5 16.0 84.0
6.4 73.5 26.5 7.6 13.0 87.0
6.5 68.5 31.5 7.7 10.5 90.5
6.6 62.5 37.5 7.8 8.5 91.5
6.7 56.5 43.5 7.9 7.0 93.0
6.8 51.0 49.0 8.0 5.3 94.7
aAdapted by permission from CRC (1975).
SDS electrophoresis buffer, 5×

15.1 g Tris base
72.0 g glycine
5.0 g SDS
Distilled, deionized H2O to 1 liter
Store up to 1 month at 0° to 4°C
Dilute to 1× before use
Do not adjust the pH of the stock solution; the pH is 8.3 when diluted to 1×.
Use purified SDS if appropriate.
SDS sample buffer
See Table A.2A.4.
Table A.2A.4 Preparation of SDS Sample Buffer
Final conc.
Ingredient 2× 4×
in 1× buffer
0.5 M Tris⋅Cl, pH 6.8a 2.5 ml 5.0 ml 62.5 mM
SDS 0.4 g 0.8 g 2% (w/v)
Glycerol 2.0 ml 4.0 ml 10% (v/v)
Bromphenol blue 20 mg 40 mg 0.1% (w/v)
2-Mercaptoethanolb,c 400 µl 800 µl ∼300 mM
H2O to 10 ml to 10 ml —
aSee recipe below.
bAlternatively, dithiothreitol (DTT), at a final concentration of 100 mM, can be substituted
for 2-mercaptoethanol.
cAdd just before use.
Sodium acetate, 3 M
Dissolve 408 g sodium acetate trihydrate (NaC2H3O2⋅3H2O) in 800 ml H2O
Adjust pH to 4.8, 5.0, or 5.2 (as desired) with 3 M acetic acid (see Table A.2A.1)
Add H2O to 1 liter Laboratory Stock
Solutions and
Filter sterilize Equipment
A.2A.7
Sodium acetate buffer, 0.1 M
Solution B: 27.2 g sodium acetate (NaC2H3O2⋅3H2O) per liter (0.2 M) in water.
Sodium phosphate buffer, 0.1 M
Solution A: 27.6 g NaH2PO4⋅H2O per liter (0.2 M final) in water.
Solution B: 53.65 g Na2HPO4⋅7H2O per liter (0.2 M) in water.
This buffer may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium
phosphate in the same final volume. Phosphate buffers show concentration-dependent
changes in pH, so check the pH by diluting an aliquot of the concentrate to the final
concentration.
TAE (Tris/acetate/EDTA) electrophoresis buffer, 10×
24.2 Tris base
3.72 g Na2EDTA⋅2H2O
H2O to 1 liter
TBE (Tris/borate/EDTA) electrophoresis buffer, 10×
108 g Tris base (890 mM)
55 g boric acid (890 mM)
960 ml H2O
40 ml 0.5 M EDTA, pH 8.0 (20 mM final; see recipe above)
TBS (Tris-buffered saline)
0.9% (w/v) NaCl
Store up to several months at 4°C
TE (Tris/EDTA) buffer
10 mM Tris⋅Cl, pH 7.4, 7.5, or 8.0 (or other pH; see recipe below)
1 mM EDTA, pH 8.0 (see recipe above)
TEA (triethanolamine) solution
50 mM triethanolamine, pH ∼11.5
0.1% (v/v) Triton X-100
0.15 M NaCl
Add Triton X-100 from a 10% stock (see recipe below).
Common Stock
Solutions,
Buffers, and
Media
A.2A.8
TEN (Tris/EDTA/NaCl) solution
150 mM NaCl
Tris⋅Cl, 1 M
Dissolve 121 g Tris base in 800 ml H2O
Adjust to desired pH with concentrated HCl
Adjust volume to 1 liter with H2O
Filter sterilize if necessary
Store up to 6 months at 4°C or room temperature
Approximately 70 ml HCl is needed to achieve a pH 7.4 solution, and ∼42 ml for a solution
that is pH 8.0.
IMPORTANT NOTE: The pH of Tris buffers changes significantly with temperature,
decreasing approximately 0.028 pH units per 1°C. Tris-buffered solutions should be adjusted
to the desired pH at the temperature at which they will be used. Because the pKa of Tris is
8.08, Tris should not be used as a buffer below pH ∼7.2 or above pH ∼9.0.
Triton X-100, 10% (w/v)
1 g Triton X-100
H2O to 10 ml
Stir to dissolve
Filter sterilize through a 0.45-µm filter
Store protected from light up to 6 months at room temperature
TTBS (Tween 20/TBS)
Dissolve 0.1% (w/v) polyoxyethylenesorbitan monolaurate (Tween 20) in TBS (see
recipe above). Store up to several months at 4°C.
SPECIAL CONSIDERATIONS FOR PCR EXPERIMENTS

Because the polymerase chain reaction (PCR) is designed to detect very small amounts
of DNA, only a few molecules of contaminating DNA will produce unwanted amplifica-
tion products. Ideally, PCR should not be carried out in the same room where large
quantities of DNA are handled. Even where such spatial separation is not practical, the
following housekeeping procedures will help avoid contamination with extraneous DNA
(H.D. Kay, pers. comm.).
1. Keep laboratory surfaces clean by swabbing with 5% to 10% chlorine bleach. Put
fresh absorbent paper bench protectors on bench before beginning PCR.
2. Wear disposable gloves and change them frequently while setting up PCRs.
3. Use only sterile disposable plasticware.
4. Keep a separate set of pipetting devices for setting up PCRs. If possible, use these
instruments only with cotton-plugged tips to minimize transfer of DNA by aerosol.
A separate microcentrifuge for PCR work is also desirable.
5. Whenever possible, set up PCRs in a laminar-flow hood or Class II biological safety
cabinet to help prevent contamination by airborne DNA particles. A UV light within
the hood or cabinet will help inactivate contaminating DNA.
6. Handle microcentrifuge tubes aseptically. Do not touch the interior of the hinged cap;
if this happens, discard the tube. Microcentrifuge tubes briefly before opening to Laboratory Stock
pellet drops around the cap and help keep reagents and reaction mixtures away from Solutions and
potentially contaminating fingers. Have only one tube open at a time, and open each Equipment
A.2A.9
tube away from the remaining tubes. Hand-held microcentrifuge tube openers (e.g.,
USA/Scientific Plastics) are available to facilitate aseptic technique.
7. Include negative controls (i.e., no primer and no template) in all PCRs.
SPECIAL CONSIDERATIONS FOR WORKING WITH RNA

RNA is susceptible to degradation by ribonucleases, which are ubiquitous, very stable,
and generally require no cofactors to function. Therefore, it is very important when
working with RNA to take precautions against RNase contamination.
1. Treat all water and salt solutions except those containing Tris with DEPC (diethyl-
pyrocarbonate; see recipe above). DEPC inactivates ribonucleases by covalent modi-
fication (however, it cannot be used with Tris solutions because Tris inactivates
DEPC).
CAUTION: DEPC is hazardous and a suspected carcinogen; follow appropriate precau-
tions for handling, storage, and disposal.
2. If possible, make separate stock solutions to use for working with RNA and keep
separate to ensure that “dirty” pipets do not come in contact with them.
3. Bake glassware 4 hr at 150°C. Rinse plasticware in chloroform or use directly out of
the package (when it is generally free from contamination). Autoclaving will not fully
inactivate many RNases.
4. Wear disposable gloves.
LITERATURE CITED
Chemical Rubber Company. 1975. CRC Handbook of Biochemistry and Molecular Biology, Physical and
Chemical Data, 3d ed., Vol. 1. CRC Press, Boca Raton, Fla.
Common Stock
Solutions,
Buffers, and
Media
A.2A.10
Commonly used lab solutions
CTAB/NaCl (directly from RVT protocol)

Add 1 g of NaCl to 15 ml of water.
Slowly add 2.5 g of CTAB. If necessary, heat to 65 °C until dissolved. Purify
through a 0.02-mm filter. Keep sterile and store at room temperature for a
maximum of 1 month.
2 M Tris/0.2 M EDTA, pH 8.5 (TE) (directly from RVT protocol) :

Add 24.228 g of Tris base, 40 ml of 0.5 M EDTA (pH 8.0) and 30 ml of H2O; adjust the pH to
8.5 and add water to make up a final volume of 100 ml.
Mountant (directly from RVT protocol)

Add 100 ml of 10 % ascorbic acid to 4.9 ml of 1_ PBS (pH 7.4).
Mix well and add 5 ml of 100 % glycerol and mix well. Filter the mountant
through a 0.02-mm syringe filter. Aliquot and store at _20 1C in the dark
3M Sodium Acetate ph 5.2 (note!! 0.3 M is used for some protocols- dilute x10):
Add 20.412 g Sodium acetate
Add approximately ~6 ml glacial acetic acid to pH 5.2
Add up to 50 ml with di H2O
“DNA B buffer”
11.69 g NaCl
50 ml of .5M EDTA
450 ml of H20
“DNA B+ SDS buffer”

Add 45 ml of DNA B buffer (shown above)
Add 5 ml of 10% SDS (SDS can be bought at 10% concentration).
Conversion Factors, Units, and Abbreviations
Conversion Factors
base ↔ mass
1 kb ds DNA (Na+) = 6.6 × 105 Da
1 kb ss DNA (Na+) = 3.3 × 105 Da
1 kb ss RNA (Na+) = 3.4 × 105 Da
1 MDa ds DNA (Na+) = 1.52 kb
average mass of dNMP = 330 Da
average mass of dNMP bp = 660 Da
base ↔ protein
1 kb coding capacity = 333 amino acids = 40,000 Da protein
10,000 Da protein = ~270 bp DNA
50,000 Da protein = ~1.35 kb DNA
100,000 Da protein = ~2.7 kb DNA
average mass of amino acid = 120 Da
mass of nucleic acid ↔ moles of nucleic acid

1 µg/ml of nucleic acid = 3.0 µM phosphate
1 µg of a 1-kb DNA fragment = 1.5 pmol; 3.0 pmol ends
1 pmol of a 1-kb DNA = 0.66 µg
1 µg pBR322 = 0.36 pmol DNA
1 pmol pBR322 = 2.8 µg
35 nmol of a 24-mer = 277 µg
absorbance of nucleic acid ↔ concentration of nucleic acid
Nucleic acid Concentration (µg/ml) for 1 A260 unit

ds DNA 50
ss DNA 33
ss RNA 40
mass of protein ↔ moles of protein
Molecular weight (Da) 1 µg 1 nmol

10,000 100 pmol or 6 × 1013 molecules 10 µg
50,000 20 pmol or 1.2 × 1013 molecules 50 µg
100,000 10 pmol or 6 × 1012 molecules 100 µg
150,000 6.7 pmol or 4 × 1012 molecules 150 µg
concentration of protein ↔ absorbance of protein
Protein A280 units for 1 mg/ml

IgG 1.35
IgM 1.2
IgA 1.3
protein A 0.17
avidin 1.5
streptavidin 3.4
bovine serum albumin 0.7
Important Licensing Information

These products may be covered by one or more Limited Use Label Licenses (See Catalog Number/Label License Index and Label Licenses in Appendix). By use of these products you accept
the terms and conditions of all applicable Limited Use Label Licenses. All products are for research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic use.
www.invitrogen.com
Conversion Factors, Units, and Abbreviations, continued
Calculations
g of solute
molarity of a solution =
[molecular mass of solute (g/mol)] × (L of solution)
molecular mass of a ds DNA fragment=(number of bp) × (660 Da/bp)

moles of ends of a ds DNA fragment = 2 × (g of DNA)/[molecular mass of DNA (Da)]
or = 2 × (g of DNA)/[(number of bp) × (660 Da/bp)]
moles of ends generated by a restriction endonuclease
circular DNA: 2 × (moles of DNA) × (number of sites)
linear DNA: 2 × (moles of DNA) × (number of sites) + 2 (original ends)
Units of Radioactivity
1 µCi = 2.2 × 106 disintegrations per minute = 3.7 × 104 becquerels
1 becquerel = 1 disintegration per second
Half-life of Common Radioisotopes
Radioisotope Half-life
Carbon-14 (14C) 5,730 years
Iodine-125 (125I) 60 days
Phosphorus-32 (32P) 14.3 days
Sulfur-35 (35S) 87.4 days
Tritium (3H) 12.4 years
Metric Prefixes
M = mega = 10 6 n= nano = 10 –9
k = kilo = 10 3 p= pico = 10 –12
m = milli = 10 –3 f = femto = 10 –15
µ = micro = 10 –6 a= atto = 10 –18
Abbreviations
ds double-stranded (as in ds DNA)
ss single-stranded (as in ss DNA)
bp basepair
kb kilobase: 1,000 bases or basepairs, as appropriate
Da Dalton, the unit of molecular mass
mol mole
M molarity, moles of solute per liter of solution

www.invitrogen.com
Conversion Factors, Units, and Abbreviations, continued
Genetic Code and Amino Acid Abbreviations
Second Position
U C A G
} } }
}
UUU Phenylalanine UCU UAU Tyrosine UGU Cysteine
UUC (Phe, F) UCC Serine UAC (Tyr, Y) UGC (Cys, C)
} }
UCA (Ser, S)
U UUA Leucine UCG UAA STOP UGA STOP
UUG (Leu, L) UAG
UGG Tryptophan
(Trp, W)
} } }
}
CUU CCU CAU Histidine CGU
CUC Leucine CCC Proline CAC (His, H) CGC Arginine
}
C CUA (Leu, L) CCA (Pro, P) CGA (Arg, R)
CUG CCG CAA Glutamine CGG
First Position
CAG (Gln, Q)
} } }
}
AUU Isoleucine ACU AAU Asparagine AGU Serine
AUC (Ile, I) ACC Threonine AAC (Asn, N) AGC (Ser, S)
} }
AUA ACA (Thr, T)
A AAA Lysine AGA Arginine
ACG
AUG Methionine AAG (Lys, K) AGG (Arg, R)
(Met, M)
} }
} }
GUU GCU GAU Aspartic acid GGU
GUC Valine GCC Alanine GAC (Asp, D) GGC Glycine
}
GUA (Val, V) GCA (Ala, A) GGA (Gly, G)
G
GUG GCG GAA Glutamic acid GGG
GAG (Glu, E)

www.invitrogen.com
Copy number calculation for QPCR:
A serial dilution of linearized plasmid DNA is used to generate a standard curve

for QPCR. Knowing the size of the plasmid that contains the gene of interest one
can calculate the number of grams/molecule also known as copy number as
follows:
Weight in Daltons (g/mol) = (bp size of plasmid+insert)(330 Da X 2 nucleotide/bp)
Ex. g/mol=(5950 bp)(330 Da X 2 nucleotide/bp)= 3927000 g/mol
Hence: (g/mol)/Avogadro’s number 6.02214199 × 1023)= g/molecule = copy

number
Ex. 3927000g/mol/Avogadro’s number 6.02214199 x 1023) = 6.52 x 10-18

g/molecule.
Knowing the copy number for a plasmid and the concentration of the plasmid that
is added to each PCR reaction, the precise number of molecule in that reaction
can be determined as follows:
Concentration of plasmid (g/µl)/copy number
Ex. (3 x 10-7 g/µl) / (6.52 x 10-18grams/molecules) = 4.6 x 1010 molecules/µl
Having calculated the number of molecules in a µl of linearized plasmid solution,

a series of dilutions can be made for subsequent amplification allowing one to
generate a standard curve. For the standard curve, the copy number of the
unknown samples can then be derived.
The copy numbers of the standards used to generate a curve are listed below.
4.60E+05
4.60E+04
4.60E+03
4.60E+02
4.60E+01
4.60E+00
Determination of Kinetics of glucose uptake by Erythrocytes
Aim: To determine the kinetics of glucose uptake by erythrocytes.
Introduction: RBCs are structurally and metabolically compared to other cells. Mature RBCs do
not possess nuclear and cytoplasmic subcellular structures. RBCs are entirely dependent on
glucose for its energy. Glucose is permeable in to erythrocytes. Glucose oxidation always ends in
the formation of pyruvic acid. Due to absence of enzyme PDC, pyruvate is not converted to
acetyl Co-A. In RBC glycolytic pathway takes diversion and forms 2,3 bisphosphoglycerate.
This diversion is called Rapport Luebering Cycle. In this assay known amount of glucose is
added to 50 µlof RBC and incubated. The glucose retained in the solution after the particular
time intervals is determined using DNS method.
Isolation of RBC:
1. To 9 ml of freshly drawn blood mix with 1 ml of 3.2% trisodium citrate, centrifuge at

1500 rpm for 15 min and separate supernatant plasma.
2. Then wash the packed erythrocytes with 10mM PBS, pH 7.4 thrice at 1500 rpm. After
washing use erythrocytes for assay.
Requirements:
1. Phosphate Buffered Saline (pH 7.4): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g
Na2HPO4.2H20 and 0.24 g KH2PO4 in 1 lt distilled water and set the pH 7.4.
2. Glucose Solution: Dissolve 90 mg glucose in 100 ml distilled water.
3. Dinitrosalicylic acid reagent: i. Suspend 1 gm of DNS powder in 10 ml distilled water, then
add 10 ml of 4 N NaOH and make a clear solution. ii. Dissolve 30 g potassium sodium
tartarate in sufficient water. Mix solutions i and ii and make up to 100 ml with water.
Procedure:
1. Pipette out 2 ml ofglucse solution in to the series of labeled 6 test tubes.
2. Add 50 µl of erythrocytes to all test tubes.
3. Now centrifuge the first test tube as soon as the RBCs are added and collect 1 ml of
supernatant. This tube serves as blank.
4. Then incubate the remaining test tubes for 5, 10, 15, 20, 25 min at room temperature.
5. After incubation centrifuge the tubes and collect 1 ml of supernatant as samples.
5. Now add 0.5 ml of DNS reagent, incubate on a boiling water bath for 20 min, cool to room
temperature, add 2.5 ml of water, mix and read at 540 nm.
7. Plot the curve by taking time of incubation along X-axis and absorbance at 540 nm along
Y - axis.
Sl. Volume Volume Incubati Volume Volume Volume A

No of of on time Centr of of DNS Keep of 540
Glucose RBC (min) ifuge supernat (ml) in distilled
(ml) (µl) at ant boilin water
(ml) g (ml)
1 2 50 0 3000 1 0.5 water 2 0.48
2 2 50 2 1 0.5 bath 2 0.34
3 2 50 4 rpm 1 0.5 for 20 2 0.27
4 2 50 6 for 1 0.5 min 2 0.25
5 2 50 8 5 1 0.5 2 0.23
6 2 50 10 min 1 0.5 2 0.21
7 2 50 12 1 0.5 2 0.18
0.6
0.5
0.4
A540
0.3
0.2
0.1
0
0 2 4 6 8 10 12 14
Incubation time in min
Result: Reduction of glucose concentration indicates the gradual uptake of glucose by

erythrocytes.
----------------
Molecular Biology of Life Laboratory BIOL 123
Dilutions
Occasionally a solution is too concentrated to be used as it is. For example, when one is
performing manual blood counts, the blood contains too many cells to be counted as such. Or
when performing a given test, it may be found that the concentration of the substance being
measured is too high for measurement with a certain instrument. In such cases, a dilution is
necessary.
Laboratory procedures in which an amount of one substance is added to another to

reduce the concentration of one of the substances are referred to as dilutions. A dilution usually
means the volume of original substance in the total volume of final solution.
In dilution statements, the smaller number is the number of parts of the substance that is
being diluted; the larger number refers to the total number of parts in the final solution unless
explicitly stated otherwise. All of the following statements mean the same thing:
1. Make a 1 to 10 dilution of serum in saline.

2. Make a 1 in 10 dilution of serum in saline.
3. Make a 1 to 10 dilution of serum with saline.
4. Make a 1/10 dilution of serum with saline.
5. Dilute the serum 10 times with saline
6. Make a dilution of 1 part serum and 9 parts saline.
7. Make a dilution of 1 part serum to 9 parts saline.
8. Make a 1:9 dilution of serum and saline.
The first five statements are symbolically shown as "1/10" dilutions and the last three are
shown as "1:9" dilutions. Terms such as "1/10", "1/100", "1/500", etc. are called dilution factors.
In each of the preceding dilutions, the numerator, 1, refers to the number of parts of serum and
the denominators, 10, 100, 500, refer to the number of total parts of each dilution. Saline is
called “diluent” in the above statements. Also note that in the preceding statements, the
directions can be followed by combining 1 ml of serum with 9 ml of saline to make 10 ml of the
final solution, by adding 2 oz. of serum to 18 oz. saline, etc. In all cases, there is 1 part of the
original substance in 10 parts of the final solution.
1 part of the concentrated material

+ 9 parts of the diluent____
10 parts of the final solution
Example: 5 ml of serum is diluted to 25 ml with saline. What is the serum dilution?
Set up the problem as 5 ml serum + X ml saline = 25 ml of the final solution.
X = 25 - 5
X = 20 ml of saline
The serum dilution is the amount of serum in the amount of total solution; hence, this is a
5/25 serum dilution which would equal a 1/5 dilution.
1
Dr. Eby Bassiri ebassiri@sas.upenn.edu
Series of Dilutions
Two important points should be considered in dilutions: how each dilution is made and
what each dilution contains. The important consideration is the concentration of the materials in
the dilution. However, it is first necessary to know how a dilution was made. A general rule to
use in calculating the concentration of solutions in a series is to multiply the original
concentration by the first dilution factor, this by the second dilution factor, this by the third
dilution factor, and so on until the final concentration is known.
Example: A 5M solution of HCl is diluted 1/5. The resulting solution is diluted 1/10. Determine
the concentration of each of the solutions.
In this example, one solution is made from a solution that was made from yet another.
To calculate the concentration of any of the solutions in such a series, first express all dilutions
as a fraction, then multiply the concentration of the beginning solution by the dilution factor used
in each succeeding step. The following steps are used in solving this example.
1. The concentration of the first solution is given as 5M HCl. This then is the first answer.
2. The second solution was made by a 1/5 dilution of the first solution. The concentration of HCl
in the second solution would be calculated by multiplying the concentration of the first solution
by the dilution factor used to produce the second solution. Hence, the second solution has an
HCl concentration of 1M.
5M HCl x 1/5 = 5M/5 =1M
3. To calculate the concentration of HCl in the third solution in the series, multiply the original
concentration of HCl by the value of each succeeding dilution factor.
5M x 1/5 x 1/10 = 5M/50 = 1M/10
Therefore, the concentration of HCl in the last solution is 0.1M.
Notice that in each solution of this problem the concentration is expressed in molarity.
This is because the concentration of the original solution was measured in molarity. In this type
of problem the concentration of each dilution will be expressed in the same unit as that used in
the original solution.
The calculations used to determine the concentration of each solution in a series may be
used in reverse to produce a dilution series having prescribed concentrations at each step.
Example: Make the following dilutions of serum in buffer: 1/10, 1/100, and 1/500.
Before attempting to solve such a problem, it should be mentioned that any one dilution
could usually be made by several procedures depending on how large a volume of the final
solution is needed and how much of the original solution (to be diluted) is available. For
example, in the problem above, we may need only 10 ml of the 1/500 dilution, so if we take 1 ml
of the original serum and add 499 ml of buffer, we will be left with a large volume of a solution,
most of which is of no use to us. Additionally, storage space is a precious commodity to many
research and health workers. Now back to solving our original problem.
2
1. To make the first dilution, place 1 part serum in a vessel and bring the total volume up to 10
parts total. This is a 1 to 10 dilution of serum in buffer. The "parts" can mean any multiple or
fraction of any unit of measurement. For example, let 1 part equal 1 ml. This means that 1 ml of
serum was brought up to 10 ml total with buffer. One could let 1 part equal 0.5 ml, 0.001 ml, 1
liter, etc., as long as all related calculations are based on the same thing.
2. To make the 1/100 serum in buffer dilution, use the first dilution as a starting point and repeat
the procedure in step 1 above; i.e., place 1 part of the 1/10 diluted serum in a vessel and bring
the volume up to 10 parts total. If a 1/10 dilution was not prepared ahead of time, one could
make the 1/100 dilution directly, provided sufficient buffer was available. In this case, 1 part of
the undiluted serum would need to be mixed with 99 parts of the buffer in the vessel.
3. To make the 1/500 dilution of serum in buffer, the easiest way is to make a 1/5 dilution of the
1/100 dilution that was already prepared; i.e., bring 1 part of the 1/100 dilution of serum in buffer
up to 5 parts total volume. Thus:
1/100 x 1/5 = 1/500
Serial Dilutions
Many procedures call for a dilution series in which all dilutions after the first one are the
same. This type of dilution series is referred to as a serial dilution. This method and calculations
discussed here are used in producing a series of solutions having equal increments of dilution.
Note that "serial dilution" is a special case of "series of dilutions".
Example: A serum sample is diluted twice with buffer. A series of five dilutions is made of this
first dilution by diluting it 1/10, re-diluting 1/10, and then three times more, each resulting
solution then being a 1/10 dilution of the previous one in the series. The concentration of serum
in each solution is as follows: 1/2, 1/20, 1/200, 1/2,000, 1/20,000, and 1/200,000.
Example: The instructions indicate that a 1/10,000 dilution of a stock solution is to be made in
water. This means 1 part of stock solution and 9,999 parts of water. If you equate 1 part as 1 ml,
this would mean that we would end up with 10,000 ml (= 10 liters) of the diluted solution which is
rather quite a large volume. Most probably, you may not have enough shelf space to store this
amount. Further, you may actually need only a few ml of the final diluted solution. So we have to
use some other way of doing the dilution without ending up with such surplus. One quick,
economical and efficient way is as follows:
1 ml stock/10 ml water x 1/10 x 1/10 x 1/10 = 1/10,000 dilution
This means taking only 1 ml of the original stock and diluting it 1/10 four times. This
would produce 10 ml of a 1/10,000 dilution of stock in water.
Another way is to dilute the stock 1/10 twice and then perform a further 1/100 dilution:
1/10 x 1/10 x 1/100 = 1/10,000 dilution
This would yield 100 ml of a 1/10,000 dilution of stock in water.
Yet another way is to make a 1/100 dilution twice:
1/100 x 1/100 = 1/10,000 dilution

3
This would produce 100 ml of a 1/10,000 dilution of stock in water.
Any combination of dilutions that will yield a final concentration of 1/10,000 may be used.
As mentioned earlier, the combination is determined in part by the glassware available and the
volume needed.
Determining Volumes and Concentrations
To decide what dilution to use, one needs to know several things:
1. Original concentration of the substance being diluted

2. Final volume desired
3. Final concentration desired
4. Number of dilutions to be made (at times)
Example: A 1/200 stock solution of boric acid is on hand. The procedure requires 50 ml of a
1/500 solution. How would the necessary amount be made without making excess?
Going from 1/200 to 1/500 produces a 2/5 ratio. This ratio is found by X below:
(1/200) X = (1/500)
X = (200/500) = 2/5
This means taking 2 parts of the original boric acid solution and bringing up the volume to 5
parts. So, if we need 50 parts (1 part = 1 ml), we should take 20 ml of the stock.
A quick way to do problems of this sort is to use the famous formula:
C1 V 1 = C2 V 2
where C1 and C2 are concentrations of solutions 1 and 2 and V1 and V2 are their respective
volumes. If we fill the parts for the problem at hand, we will have:
(1/200) V1 = (1/500) (50)
Solving the fractions for V1, we get V1 = 20 ml. Again this means that if we take 20 ml of the
stock boric acid and add 30 ml water, we would have 50 ml of the desired solution.
Some Final Notes
1. After doing your calculations, it is always a good idea to work backwards and see if you come
up with the original data given. This is a good check as to the correctness of your calculations.
2. When working with very large or very small numbers, it is customary to convert them to a
number between 1 and 9, times a power of base 10. For example:
32,000,000 = 3.2 x 107

0.000047 = 4.7 x 10-5
4
Note in the above examples that (a) the first number in the conversion is between 1 and 9 and
(b) the power of base 10 can be positive or negative. A positive power means the number is
large(r than 1) and a negative power means the number is small(er than 1).
Dilution factors are similarly converted to powers of 10. For example:
1/10 = 0.1 = 10-1 1/100 = 0.01 = 10-2

1/10,000 = 0.0001 = 10-4 1/1,000,000 = 0.000001 = 10-6
Similarly, reciprocals of dilution factors can be converted to powers of 10 as well. For example:
10 = 101 100 = 102
10,000 = 104 1,000,000 = 106
3. Units of measurements can be converted as follows:
1 Kg = 1000 g
1 g = 1000 mg
1 mg = 1000 µg
1 µg = 1000 ng
1 ng = 1000 pg
1 liter = 1000 ml
1 ml = 1000 µl
4. Although already mentioned at the beginning of this chapter, it is worth repeating that "the
original solution is always more concentrated (think big numbers, possibly with positive powers)
than the diluted samples (think small numbers, possibly with negative powers)”. Check these
rules of thumb whenever you are confronted with a dilution problem. Can you verify these rules
in the preceding examples?
5. The ratio of the numbers rather than the numbers themselves is important. For example, 1 ml
in 10 ml, 0.1 ml in 1.0 ml, 5 ml in 50 ml, etc. all give the same dilution (ratio). The only
differences are the amounts of the individual constituents as well as the final product.
6. Log on to “http://www.bio.upenn.edu/computing/media/Instructional.Dilution.php” to see a

video explaining dilutions.
Use of any section of this Lab Manual without the written consent of Dr. Eby Bassiri, Dept. of
Biology, University of Pennsylvania is strictly prohibited.
5
DNA Extraction
Center for Coastal Margin Observation & Prediction (www.stccmop.org)
Solutions:
DNA extraction buffer: Contains 0.1 M EDTA @ pH 8, 1% SDS and 200 µg/mL proteinase K. Make a stock of 50 mL
0.1 M EDTA-1% SDS by combining 10 mL EDTA pH8, 5 mL 10% SDS and 35 mL MilliQ water for a total volume of 50
mL. Mix well by vortexing. Before adding DNA extraction buffer to field sample make a DNA EXTRACTION BUFFER
WORKING SOLUTION. Add 10 µl of 20 mg/mL Proteinase K to 1 mL of 0.1M EDTA-1% SDS, mix by gently shaking
tube up and down and quickly centrifuge to bring down the liquid.
Notes: 1) DNA extraction buffer contains SDS, if the room temperature is lower than 25ºC, SDS will usually precipitate.
If precipitate forms, microwave the buffer for 10 seconds and mix to dissolve. Repeat one more time if needed.
2) Proteinase K can be purchased from Fisher Scientific as solution: cat. no. NC9499957, 20 mg/mL Proteinase K
(easiest to use)
CTAB (N-cetyl-N,N,N,-trimethyl ammonium bromide): CTAB is a light powder, somewhat similar to SDS, used
eliminate contaminants in field samples. Make a 10% CTAB working solution (50 mL): Combine 50 mL of 0.7 M NaCl
and 2.5 g of CTAB into a 50 mL polypropylene tube (Fisher Scientific cat no. 06-443-18). Rotate slowly at 60˚C for
several hours to dissolve powder completely. Store at Room Temperature for up to 6 months. Can also make by
dissolving 4.1 g NaCl in 80 mL MilliQ water. While stirring, add 10 g CTAB. To dissolve, heat the solution at 65ºC. Adjust
the volume to 100 mL with MilliQ water. Store at RT for no longer than 6 months.
Notes: 1) 10% CTAB is very sticky at RT and cannot accurately be pipetted. Therefore it is convenient to make 1 mL
aliquots. When you need to use 10% CTAB, pre-heat a 1 mL aliquot at 55-60ºC. When adding to DNA solution pipet
immediately, trying to keep the aliquot heated. Pipet up and down for several times to make sure all the CTAB in the
tip is washed out. You can also microwave the 10% CTAB solution for several seconds, but be careful not to overheat
the solution so that the liquid will boil out.
2) CTAB can be purchased from Fishier Scientific: cat. no. AC22716-1000, 100 g CTAB
DNA cleaning kit: Zymo DNA Clean & Concentrator kit- 25 columns (Cat. No. D4006, Zymo Research, Orange, CA)
Field sample DNA isolation:
1. Filter water sample through a 0.22 µm Sterivex filter

2. When ready to process sample, break Sterivex following DNA extraction protocol in blue notebook.
3. Add the cut up filter membrane into a 2 mL mircocentrifuge tube (with orange cap), trying to get the filter as close to
the bottom as possible
4. Add 0.5 mL DNA extraction buffer, making sure the filters are completely covered by the buffer
5. Incubate at 55ºC for 16 hours to 3 days
6. Add 165 µl 5M NaCl and 165 µl pre-warmed 10% CTAB, mix well by vortexing
7. Centrifuge briefly and incubate at 55ºC for 10 minutes
8. Pipette out the DNA solution, making sure to avoid taking any of the filter, into a 1.5 mL epindorf tube (~650 µl)
9. Add 600 µl chloroform, close the cap and vortex for 1 minute
10. Centrifuge at the highest setting (>13000 rpm) for 10 minutes
11. Transfer the supernatant (~600 µl) into a 2 mL collection tube, being careful to not suck up the interface
12. This is the crude DNA solution, purify DNA using DNA cleaning kit
13. To purify crude DNA, add 2 volumes of DNA binding buffer (1.2 mL) to DNA solution, mix well by vortexing
14. Transfer 700 µl of DNA/buffer mixture into a filter column and centrifuge at top speed (>13000 rpm) for 15 seconds
15. Discard the flow through and return the column back to the collection tube
16. Repeat steps 14-15 until all the DNA/buffer mixture has been transferred to the filter cartridge
17. Add 300 µl DNA wash buffer to the column (make sure you have added 100% ethanol into the concentrated washing
buffer), centrifuge at top speed (>13000 rpm) for 15 sec
18. Discard flow through and add another 200 µl DNA wash buffer, centrifuge at top speed for 1 min
19. Transfer the column into a clean 1.5 mL epindorf tube
20. Add 15-50 µl elution buffer (10mM Tris-Cl pH 8) to the white base of column and incubate at RT for 1 minute
21. Centrifuge at 10,000 rpm for 15 seconds
22. Add another 15-50 µl elution buffer to the base of the column, incubate at RT for 1 minute, centrifuge at 10000 rpm
for 15 seconds
23. Discard column and vortex elution briefly and store DNA at -20ºC
DNA Laddering
Hank Dudek 8/23/95
I. Protocol
1. Harvest cells
Optional: wash plate with 37°C PBS (gently, so as not to lose cells); check on microscope,
after aspirate PBS or media
Place plate on ice
Lyse with DNA ladder buffer 4°C
150 ul/60 mm plate
Scrape, transfer to eppendorf
Rotate 4°C 20 min
2. Microcentrifuge 15 min, 4°C
To pellet chromatin
Transfer sup
3. Phenol/CHCl3 extract
Add equal volume phenol/CHCl3
Mix by pipetting
Microfuge 2 min; transfer sup
Will often get a very large interface
4. Optional: re-extract organic
Add 300-500 ul TE8.0; mix; spin; pool sups
5. Ethanol Precipitate
In eppendorf tube or 15 ml tube, depending on total sup volume
Add 1/10 vol. 3 M NaOAcetate, 2 vol. ethOH
–20°C overnight
6. Recover DNA
Centifuge EtOH ppt:
If in: 15 ml tubes: Sorvall SA600, 8,000 RPM, 10 min, 4°C epp tubes: microfuge 15 min,
4°C
If was in 15 ml tube, probably best to resuspend in small volume (e.g., 500 ul TE), then re-
EtOH ppt in epp tube, so pellet will be tight
80% EtOH wash: add ≈500 ul; vortex; re-spin
Speed-vac dry
Resuspend in TE 8.0: ≈25 ul/eppendorf; room temp ≈30 min
7. RNase
Add 1ul 1 ug/ul RNase A; room temp 30 min
8. Gel electrophoresis
Add 5X loading buffer (*omit bromophenol blue: can interfere with band visibility; may
be OK to add xylene cyanol)
Run on 1.2% agarose gel, in TAE buffer
II. Reagents
DNA Laddering Buffer:
40 ml
0.5% Triton X-100 800 ul 25%
5 mM Tris pH 7.4 200 ul 1M
20 mM EDTA 1.6 ml 500 mM
37.4 ml dH2O
III. Notes
Reference: Hockenberry et al. Nature
DNA Isolation: TRIZOL Reagent
1. Tissues: KEEP FROZEN on dry ice, Homogenize in 1 mL Trizol, under Fume Hood
using tissue homogenizer.
a) Put probe on drill.
b) Set up epi tubes in rack.
c) Dislodge tissue form cryotube with forceps, tap into homogenizer tube, throw out cryotube.
d) As soon as you add Trizol, homogenize from low to high speed for one min.
e) Pipette into epi tube, discard probe and homogenizer tube (Can freeze at -20C) (Thaw on bench, at room temp
to continue)
2. Phase Separation:
a) Incubate cells/TRIZOL at 15-30°C for 5 minutes.
b) Add 200 ul of chloroform per 1 ml of TRIZOL used. Cap tubes and vortex for 15 seconds.
c) Incubate at 15-30°C (room temp) for 15 minutes.
d) Centrifuge samples at 12,000 RPM for 15 minutes at 2-8°C. (Cold room)
*after centrifugation: aqueous = RNA. interphase = protein/DNA. organic = DNA.
3. DNA Precipitation:
a) Remove aqueous (RNA) phase to waste, or save in cryovial.
b) Add 300 ul 100% Ethanol (200 proof EtOH) per 1 ml TRIZOL used to interphase/organic phase.
c) Vortex gently.
d) Incubate samples at 15-30°C for 2-3 minutes.
e) Centrifuge at 12,000 RPM for 5 minutes at room temp.
4. DNA Wash:
a) Remove phenol/ethanol sup (contains protein) to waste. (Pipet into 50ml conical & dispose in Trizol Waste
container)
b) Wash DNA pellet two times in 0.1M sodium citrate solution. Add 1 ml Na citrate per 1 ml Trizol used.
c) Gently vortex. Incubate samples in wash solution at 15-30°C for 30 minutes (periodic mixing).
d) Spin at 12,000 RPM for 5 minutes at room temp.
e) After washes resuspend DNA in 1 ml 75% Ethanol per 1 ml TRIZOL used. USE: 200 proof EtOH and PCR-
grade water. (eg. For 50ml, 37.5ml of ethanol and 12.5ml of water)
f) Incubate at 15-30°C for 10-20 minutes (periodic mixing).
g) Centrifuge at 12,000 RPM for 5 minutes at room temp. Remove sup using p200 pipettor.
h) Briefly dry pellet for 2-5 minutes under vacuum. (Can leave on bench)
*Add 100µl of water and freeze at –80 or continue to next step.
5. Redissolving the DNA:

a) Dissolve pellet in 100ul (or 200-500ul for thymus) of sterile water with a pipette. Can place sample at
55°C for 10 minutes to increase solubility.
b) *Solution may still contain insoluble fragments (membrane, protein, etc).
c) Centrifuge samples at 12,000 RPM for 10 minutes in cold room to remove insoluble material.
d) Transfer sup containing DNA to a new, autoclaved, labeled, flip-top eppendorf.
e) Spec. AMOUNT: 1ul of sample + 50ul of dH2O (198ul H2O+2ul sample)
f) Store at -80.
Solutions:
TRIZOL Reagent Life Technologies #15596 Ethanol: 100% & 70%
0.1M sodium citrate in 10% Ethanol: 8mM NaOH:
2.941 g sodium citrate .032 g NaOH
90 ml H2O 100 ml H2 O
10 ml 100 % Ethanol
RNA Isolation: TRIZOL Reagent
*If frozen, thaw quickly in 37°C bath, transfer to 15ml conical w/ 10ml of cold 1xPBS to wash freezing
media.
1. Homogenization:
Cells: Pellet at 1500 for 5 min. Aspirate dry. Add 1 ml of room temp TRIZOL (do not take directly from
stock) per 5-10x106 cells. Lyse cells by repetitive pipetting.
Tissues: Homogenize in 1 mL Trizol using tissue homogenizer (Omni International).
2. Phase Separation:
-Incubate cells/TRIZOL at 15-30°C for 5 minutes.
-Add 200 ul of chloroform per 1 ml of TRIZOL used. Cap tubes and vortex for 15 seconds.
-Incubate at 15-30°C for 10-15 minutes w/ a vortex half way through.
-Centrifuge samples at 12,000 RPM for 15 minutes at 2-8°C. (cold room)
*after centrifugation: aqueous = RNA. interphase = protein/DNA. organic = DNA.
3. RNA Precipitation:
-Transfer aqueous phase to an eppendorf tube CAREFULLY, using a P-200 pipettor. Discard lower,
DNA phase or freeze at -80.
-Add 500 ul Isopropyl Alcohol, mix gently, and incubate at room temp for 10 minutes.
-Centrifuge at 12,000 X g for 10 minutes at 4°C. The RNA precipitate will form a gel-like pellet at the
bottom of the tube.
4. RNA Wash:
-CAREFULLY pull off supernatant with a P-200.
-Wash the pellet once with 1ml of 75% Ethanol (ICE COLD). STOP HERE IF NO SPEC AT THIS
TIME. FREEZE AT -80C.
-Centrifuge at 12,000 X g for 15 minutes at 4°C. The RNA should form a visible, white pellet.
5. Dissolving RNA:
-CAREFULLY pull off supernatant with a P-200.
-Air dry pellet (do not dry under vacuum). Allow to dry completely so there is no residual ethanol.
-Dissolve RNA in Rnase-free water (DEPC WATER), volume dependent on size of tissue, number of
cells, or size of pellet. 10-100ul
-Check RNA concentration by spec.
Solutions:
TRIZOL Reagent Life Technologies #15596
Ethanol: 100% & 75%
Isopropyl
Chloroform
7/31/2018 Don’t throw leftover silica DNA purification columns/buffers away! Use them up! (DIY Silica Spin Kits) – Pipette Jockey
Pipette Jockey
I make, optimize and reverse engineer methods and reagents for the molecular biology lab. I also make
other cool stu like lab equipment. What follows are my adventures.
Don’t throw leftover silica DNA puri cation

columns/buffers away! Use them up! (DIY Silica Spin Kits)
Silica based nucleic acid puri cation columns and kits are a common consumable in
the lab. They’re used for cleaning up PCR reactions, purifying gel slices and plasmid
DNA minipreps. Inevitably, when you reach the bottom of the box you are usually
left with one of two scenarios: 1) You don’t have enough of a speci c bu er or 2) You
don’t have enough columns. Both of these scenarios usually result in the remaining
components of the kit being tossed out or forgotten in your cupboard for a decade
(that somehow make their way into my hands).
Why not source your own columns, mix your own bu ers to both reduce waste and
save money! Grab a snack, ease into your bunny slippers, this is a long one.
There are two major technologies when it comes to purifying DNA column-style;
silica based puri cation and anion-exchange resin puri cation. In this post I’ll be
talking about silica kits, but the anion exchange post is coming too.
Silica Based Kits
http://pipettejockey.com/2017/06/16/dont-throw-those-silica-dna-purification-columnsbuffers-away-use-them-up/ 1/13
Silica puri cation is by far the most common technology due to the low cost of
materials, and is generally found in kits for smaller scale puri cations, such as
miniprep kits, PCR cleanup kits and gel puri cation kits. Silica will bind DNA in the
presence of a high concentration of chaotropic salts such as guanadinium
hydrochloride (Gu-HCl) or sodium iodide (NaI). The silica is then washed a few
times with a high concentration of ethanol and nally eluted in a low volume of pH
8-8.5 Tris.
So, what if you run out of columns for a particular kit? Well, as long as it’s based on
silica/chaotropic salt technology, you can use them interchangeably. One caveat
here is that di erent manufacturers deposit di ering amounts of silica into their
columns, which translates to a higher or lower binding capacity of DNA. Also, silica
columns can dry out, signi cantly impacting binding capacity. Dried up columns
can be re-humidi ed and reused.
Don’t have old silica columns in a drawer somewhere? You can buy the silica spin
columns by themselves much cheaper than you could get from Qiagen/GE/etc.
Enzymax is the cheapest at 0.38$ per column, with Epoch Life Sciences at 0.64$ per
piece. Other manufacturers do exist but I have had good experiences with both
companies.
Now, what if you have the columns, but are missing a speci c bu er? Don’t be
scared! Since these silica kits all use the same underlying technology, the bu ers
themselves are interchangeable and vary little in their composition. Openwetware
has the compositions of the majority of Qiagen bu ers, taken straight from their
manuals and patents. I’ve also scoured patents from other manufacturers for their
recipes so that you can see how similar they are. The trick here is to use the
appropriate bu er for your speci c application.
For silica kits you will generally use three types of bu ers: binding bu ers that allow
your DNA to bind to the silica, wash bu ers which clean away unbound
DNA/RNA/protein/salt, and elution bu ers which dissolve your DNA that you can
then use.
Miniprep Bu ers:
Qiagen Bu er P1: 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 50 ug/mL RNase A
Qiagen Bu er P2: 200 mM NaOH, 1% SDS
Binding bu er for PCR product/enzymatic reaction cleanup:
Qiagen Bu er PB :5 M Gu-HCl, 30% isopropanol, add 5 volumes to 1 volume of

PCR/enzymatic reaction, load onto column.
Qiagen Bu er PB (Enzymax variant): 5 M Gu-HCl, 20 mM Tris-HCl pH 6.6, 30%

ethanol
The isopropanol/ethanol is likely added here to prevent smaller DNA fragments (i.e.
primers) from binding to the membrane. You could likely adjust the concentration
of isopropanol to exclude binding of larger pieces. Machery Nagel does an excellent
job demonstrating this in their PCR cleanup manual, although they dilute their PCR
binding bu er with water.
Binding bu er for agarose gel slice puri cation:
Qiagen Bu er QG: (5.5 M guanidine thiocyanate, 20 mM Tris HCl pH 6.6) Add 300 uL
of bu er to 100mg of gel slice, heat to solublize, load onto column. Enzymax
suggests same recipe for their columns.
Promega Gel Bu er: (6M guanidine thiocyanate, 10 mM EDTA) Recipe from their

patent. EDTA used to prevent discoloration of guanidine. Use ~333 uL of bu er per
100 mg of gel slice, heat to melt, load onto column.
Binding/neutralization bu ers for plasmid minipreps:
Qiagen Bu er N3: 4.2 M Gu-HCl, 0.9 M potassium acetate pH 4.8. This is an

interesting bu er which was gleaned from the patent, Qiagen calls it proprietary.
Essentially it is Solution 3 from your classic alkaline lysis miniprep with Gu-HCl
(1.73M nal conc.) thrown in to get the plasmid to bind to the silica. Resuspend cells
in 250 uL Bu er P1, lyse with 250 uL Bu er P2, neutralize with 350 uL of Bu er N3,
load unto column.
Qiagen Bu er N3 (Enzymax Variant): 4M Gu-HCl, 0.5M Potassium Acetate pH 4.2
Testing homemade Bu er N3

GE Neutralization bu er (4.4M Gu-HCl, 0.65M potassium Acetate

and 3.1M Glacial Acetic Acid). Got this from a GE healthcare patent, slightly higher
Gu-HCl (2.2M nal conc.) content. The acetate/acetic acid portion is very close too,
you’d be adding that much acetic acid to potassium acetate to make it pH 4.8 as in
the Qiagen bu er. GE uses 350 uL of this bu er for 175 uL of lysis bu er (200mM
NaOH, 1%SDS) + 175 uL resuspension bu er (100 mM Tris-Cl pH7.5; 10 mM EDTA;
400ug/ml RNase A). Slightly di erent resuspension bu er, but identical lysis bu er.
Wash bu ers for PCR/Enzymatic cleanup, gel puri cation/plasmid preps:
Qiagen Bu er PB: 5 M Gu-HCl, 30% isopropanol. This is an optional wash bu er

used to remove residual nucleases/carbohydrates that may be stuck to the silica,
recommended when isolating plasmids from endA+ strains. Wash once with 500 uL.
Proceed with regular washing steps.
Qiagen Bu er PE: (10 mM Tris-HCl pH 7.5, 80% ethanol) Wash twice with 750 uL.
5X Qiagen Bu er PE (Enzymax Variant): (80 mM NaCl, 8mM Tris-HCl pH 7.5) Dilute

to 1X with 100% ethanol, 1X conc: 16 mM NaCl, 1.6 mM Tris-HCl pH 7.5, 80% EtOH.
GE Wash bu er: (2mM Tris-Cl pH8; 0.2mM EDTA and 80% ethanol) Very similar to
Qiagen, would work interchangeably. GE uses 400 uL for two washes, but 750 uL like
Qiagen is probably better.
Promega Wash Bu er: 80% Isopropanol
Elution bu ers for PCR/Enzymatic cleanup, gel puri cation/plasmid preps:
Qiagen Bu er EB: (10 mM Tris·Cl, pH 8.5)
Qiagen Bu er EB (Enzymax Variant): (10 mM Tris-HCl, pH 8.0)
Machery Nagel AE Bu er: (10 mM Tris·Cl, pH 8.5)
GE Elution Bu er: (10 mM Tris-HCl, pH 8.0)
Any high pH (8-8.5) bu er will work, as will water at a lower e ciency. The
presence of EDTA can also lower recovery. The amount of bu er used to elute will
dictate recovery, more bu er will recover more DNA but at a lower concentration.
So, those are the bu ers you need to use any brand of silica spin kit. As you can see
most of the magic is in the binding bu ers, which when applied to your sample have
a nal Gu-HCl/thiocyanate concentration of ~1.5-4M. As long as your sample has
that concentration, the DNA will bind to the silica. Wash bu ers are 80%
ethanol/isopropanol, with Tris/EDTA added for avour. Elution bu ers are even
simpler, with basically all manufacturers disclosing it to be Tris pH 8-8.5.
21 thoughts on “Don’t throw leftover silica DNA

puri cation columns/buffers away! Use them up! (DIY
Silica Spin Kits)”
Dr. Atthaboon Watthammawut

June 16, 2017 at 9:53 am
You’re so right about these kits and solutions… It’s almost they “setup” the kit to
make one component to be short of another… A gel pcr puri cation kit is directly
related to the Plasmid prep kits… And like you pointed out…if we really spent the
time studying the solutions comprising the kit…gone would be those dreaded days of
running out of sometimes only “one” solution requiring us to buy a new kit. This
happened often when I used a Machery Nagel Maxi prep kit… And due to the large
reaction volumes always led to some overuse of some solutions… Especially the
annoying resuspension bu er which is essentially TE bu er plus RNase A… Bought a
cheap vial of RNase A and made some TE and saved 200 bucks lol
Poor scientist
September 12, 2017 at 1:14 pm
Could one reuse an already used column?
Pipette Jockey 
Yeah buddy, you would regenerate them akin to anion exchange columns where
you run 1M HCl through them several times, let them soak in HCl overnight, then
rinse with distilled water and run some guanidinium bu er to get the silica ready
to bind again.
http://www.biotechniques.com/BiotechniquesJournal/2007/February/Regeneratio
n-of-commercial-nucleic-acid-extraction-columns-without-the-risk-of-
carryover-contamination/biotechniques-41235.html
Poor scientist
Thanks. Should have looked into your site better before asking…You had the
information on another post. Great site and good detective work.
Two more questions and excuse my ignorance. You show two binding bu ers,
one for plasmids and one for PCR products. The third one “minipreps” is for
what? And related to this, can one of the above be used for genomic DNA from
bacteria.
Pipette Jockey 
The miniprep bu er is for extracting plasmid DNA from E.coli, agrobacterium,

yeast. It essentially combines the classic Bu er 3 of a plasmid prep, which
contains acetic acid to neutralize Bu er 2, as well as guanidinium to get that
plasmid DNA to bind to the silica.
Interesting question about the genomic DNA, hadn’t thought about it as I do

genomic extraction pretty infrequently with the CTAB/phenol based method.
Spin kits are also used for genomic DNA extraction, the magic comes in the
initial lysis bu er which releases the genomic DNA, it involves lysozyme and
proteinase K. After the genomic DNA has been liberated then it’s the usual fare
of adding a guanidinium bu er (5-6M) to the lysate, and put it through the
spin kit as usual. Just some quick reading from the Qaigen DNAeasy kits
suggests that ethanol is added at this step too, putting the composition close to
the PCR puri cation bu er listed. Give me a bit of time to dig you up a solid
recipe for a genomic DNA spin kit lysis and binding bu er.
Poor scientist
I also use the CTAB/phenol and would be nice to be able to clean any residual
phenol an salts this way. Thanks!
Helen
October 12, 2017 at 3:35 pm
Do you have any suggestions for a binding bu er for genomic DNA puri cation on
silica spin columns, or ny binding bu ers that don’t contain guanidine?
Pipette Jockey 
October 12, 2017 at 6:17 pm
Genomic DNA puri cation by spin kit would follow the same basic steps as the
other methods, the magic being in the way that you initially lyse your cells. I’m
trying to nd a decent recipe for you and another poster, but from my reading of
the DNeasy kit the composition is close to the PCR puri cation bu er but with
ethanol vs iso.
If for some reason you couldn’t use guanidine then Sodium Iodide would make a
reasonable substitute for it, check out this paper for DIY NaI based genomic DNA
extraction https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-
6-1
If you can’t use chaotropes like GuHCl or NaI, then the DEAE resin kits would be
the way to go, which purify DNA using salt gradients.
But yeah, the recipe is in the works, I’ll post it as soon as I can, been a bit busy
submitting a paper, but that’s done now, yay!
Orchid girl
November 23, 2017 at 2:23 pm
Hi there,
I’ve been searching for the recipe of Qiagen’s RW1 bu er from RNeasy plant kit.
Any ideas?? Thanx!

Pipette Jockey 
November 24, 2017 at 12:32 am
Unfortunately Qiagen has never released the recipe for RW1 either in their manuals
or patents for whatever reason. The best I can o er you is a look at one of their
patents (US 8,624,020 B2), where they looked for alternatives to ethanol in their
bu ers so they could avoid restrictions on shipping ethanol.
Their RW1 replacement is: 20% Tetraethylene glycol (or 20% 1,3 butanediol), 900
mM guanidine isothiocyanate and 10 mM Tris pH 7.5, Bu er RPE: 70%
Tetraethylene glycol (or 80% 1,3 butanediol), 100 mM NaCl, 10 mM Tris pH 7.5
Allegedly this washing combo worked better than the ethanol version. I don’t have
the RNAeasy kits to try the recipe out for myself, but depending on how bad you
want more RW1 (vs buying a bottle), you could try the ethanol free version, or
substitute 30-50% ethanol for the tetraethylene glycol in RW1
Orchid girl
November 24, 2017 at 2:09 pm
Thank you so much!
I’ll take a look and think about it. If I manage to test it, I’ll post the results here.
Psyonical
April 4, 2018 at 7:38 pm
Thank you for this information, I stumbled upon it a few days ago and I honestly
have to say it was enlightening to the processes I have been using.
Anyways, I had a question about the Agarose binding bu er recipes you had there.
So I don’t have the guanidine thiocynate but would guanidine HCl be a resonable
substitute? Also, since I already gave it a shot, I tried using 4M Guanidine HCl in
20mM Tris 6.6 pH and it took forever for the gel pieces to dissolve(almost two days
at 70C). Could my concentration of Guanidine HCl be too low ( I did see it was 6M
there but I remember seeing 4M somewhere else) or should there something I may
need to do to di erently? Just looking to troubleshoot my problem. Again thank you
for your information and thank you for any help.
Pipette Jockey 
April 4, 2018 at 11:19 pm
Hey Buddy, sorry you’re having trouble.
The Qiagen patent does mention using Guanidine-HCL, however they don’t
actually give a recipe of those bu ers, just the thiocyanate which I guess they
settled on. It’s possible the Gu-HCL will work, but perhaps at a lower e ciency of
binding to the silica. I’d bump the concentration up to 6M, but I’m not sure if that
will help with your dissolution problem. You mention you apply heat, but what
type of agarose do you use? We use low-melt grade, which is quite fragile but
melts very easily. I don’t think I ever tried a gel extraction from regular agarose.
Psyonical
April 5, 2018 at 2:05 pm
Thanks for the reply. It is the melting temperature. I thought the agarose was
low melting but I was wrong, I have to bump my temp up to 88 to break it down. I
will also bump the Gu-HCl concentration up and see what happens. Thanks
again.
Diana R
June 20, 2018 at 7:19 pm
Hey there. Useful stu you have around here, it’s great.
I have some questions. I’ve been using the NucleoSpin by Macherey Nagel columns
for RNA isolation. The kit consists in silica-membrane columns and the following
steps: cell lysis, RNA binding, DNAse treatment, RNA washing and elution.
Unfortunately, they were not very useful for the isolation (we obtained a ridiculously
low quantity of RNA, but that’s another story), but we decided to use the columns
for the DNAse treatment and washing after a phenol-cloroform extraction.
Can these columns be reused, if we intend to use them for DNAse treatment only?
How can they be washed?
Also, you’ve inspired me to search the components of the DNAse bu er from these
guys. I don’t live in the US so this stu takes forever to arrive down here. No way I’m
going to wait 2 months for a 7 mL solution.
Pipette Jockey 
June 20, 2018 at 9:01 pm
Hello, I’m glad you nd some of the stu useful!
Yeah, RNA extraction columns are pretty notoriously low yielding, we found that
too. We have regenerated Qiagen anion exchange (DEAE-silica) columns (mostly
maxi preps) with the following method https://www.future-
science.com/doi/10.2144/000112327 about 10 times per column. Basically let it
soak in 1M HCl for a while, wash it plenty with H2O and re-equilibriate with bu er
QBT. I haven’t applied this to RNA silica/guanidinium columns, but I would guess
that the method would work the same, except you would equilibriate with
whatever the binding bu er for the RNA is. Haven’t dug too deep into RNA kit
patents but I believe they are out there.
sam
July 17, 2018 at 6:16 am
i ran out of AW1 bu er of qiagen pe tissue dna extraction kit. can AW1 bu er of
other qiagen dna extraction be used?
Pipette Jockey 
July 18, 2018 at 2:20 am
Yup, should be cross compatible, assuming the AW1 from the other kit wasn’t
prepared too long ago, the ethanol can evaporate…
emma_belmont
July 18, 2018 at 10:14 pm
This is a really cool site. I’m glad the mega biotech corporations don’t quite have all
the power, haha.
I have just a quick question that could use your expertise: I do quite a few PCR
puri cations and recently our lab transferred from using machery-nagel kits to
qiagen kits. The binding bu er in macherey-nagel’s (Bu er NTI) has a feature
where by diluting it with h2o, smaller fragments can be selected for based on size,
and washed out.
Qiagen’s binding bu er (PB Bu er) which I now know is using gu-hcl, dosent seem
to have this feature.
I have an inkling macherey-nagel may use guanidium thiocyanate in their NTI
bu er rather than gu-hcl as well.
I would love to see some more information on how macherey-nagel’s NTI bu er can
do this. I can’t seem to nd their recipe anywhere.
Pipette Jockey 
July 19, 2018 at 12:20 am
Hey! Yeah, they don’t have all the power
As for Bu er NTI, I can’t seem to nd any patent information regarding the

formulation and you’re right, it is thiocyanate based. However with Qiagen’s PB
bu er the dilution trick should work in a similar way, you are essentially just
adjusting the binding conditions and the more concentrated the binding bu er is
the tighter the binding to the silica will be. So, a higher ratio of PB will mean
smaller pieces will bind. Unlike MN, Qiagen doesn’t describe this in their manual
but I’m con dent it would work the same way, you’ll just have to do a gradient to
gure it out, use MN’s manual as a guide.
I’ll let you know if I nd any literature as to the composition of NTI, this is the best
I could nd: https://patents.justia.com/patent/6177009
Good luck!
emma_belmont
July 19, 2018 at 12:53 pm
Thanks for the response!
I’ll de nitely begin some PB bu er gradients to test!
Pipette Jockey / Proudly powered by WordPress
EMBEDDING & SECTIONING TISSUE
FIXATION
1) When possible cardiac perfuse with 4% PFA (~7.4 pH, 310mOsm) in PBS,
heparinized saline may be used prior to PFA to increase perfusion efficiency
2) Post fix samples for 2-4 h in 4% PFA
3) Remove excess PFA with PBS rinse
4) Begin sucrose infiltration for cyroprotection (see below)
FROZENS:
OTC MEDIUM
1) Tissue ready for embedding should be fixed (usually in 4%PFA) and in stored in PBS
2) Sucrose infiltration (CRYOPROTECTION)
10% Sucrose (15 min or until sample drops to bottom of vial)
2:1 10% Sucrose:30% Sucrose (15 min or until sample drops to bottom of vial)
3) Partially fill dry ice container with dry ice and add methanol to create a cool bath, let
sit
4) Label Tissue Tek wells with each animal number and fill with OTC (TissueTek)
freezing compound
5) Remove excess sucrose from tissue by blotting on Kimwipes and place tissue in
center of well filled with OTC
6) Orient tissue into the bottom of the well and freeze by floating on methanol bath
CAUTION: do not get methanol on the OTC, it will not freeze correctly
7) Place frozen tissue blocks in –20 freezer after they are frozen
8) Ready to be sliced after they are frozen completely
9) Note: do not store slides in the cryostat over night, they will dry out and be no good.
It is also a good idea to place all tissue into plastic bags in the –20 frost free freezer to
reduce drying out during storage.
10) Slice sections at 8 to 10µm on the cryostat. Cold sections will readily transfer to
room temperature super frost plus or subbed slides
11) Let new slides dry (at least 2h) at room temperature in hood
12) Dunk slides in cold acetone (1-3 min) and let dry for at least 1h
13) Label slides as normal
WHOLE TISSUE
1) Whole pieces of tissue that have been cyroprotected (see above) can be frozen by
placing on an aluminum “boat” floating on top a dry ice/methanol solution (see
below).
* note large pieces of tissue (brains) often don’t need to be embedded in OTC
2) Slice sections at 8 to 10µm on the cryostat. Cold sections will readily transfer to
room temperature super frost plus or subbed slides
3) Let new slides dry (at least 2h) at room temperature
4) If necessary, dunk slides in cold acetone (1-3 min) and let dry for at least 1h (helps
the section adhere)
5) Label slides
PARAFFIN SECTIONS (Protocol from Brauer Lab)

1. After fixation, rinse tissue with PBS until fixative is completely removed.
2. Dehydrate tissue using EtOH series.*
50% EtOH for 10 min.
3. Exchange EtOH with Citrisolve
2:1 EtOH:Citrisolve for 10-15 min
100% Citrisolve for 10-15 min
4. Exchange Citrisolve with paraffin. The following steps are done in a vacuum oven set
for 54-58oC. We use Paraplast X-tra or Paraplast Plus (the Plus has DMSO added to
facilitate infiltration-- both from Fisher). Do not let paraffin exceed 60oC for prolong
periods of time because this will degrade the paraffin polymers and make it more
brittle.
2:1 Citrisolve:Paraffin for 30 min
100% Paraffin for 1-2 hr
100% Paraffin for 1-2 hr or overnight
5. Embed in fresh new paraffin and orient tissue as desired before it hardens (vertical for
embryos).
Estimation of reducing sugar by DNS method
Aim: To estimate the amount of reducing sugar by DNS method.
Principle: When an alkaline solution of 3, 5 - dinitro salicylic acid is heated with a reducing
sugar, the acid is reduced to give a orange red colour. This test is of interest chiefly because it
demonstrates the reduction of a non-metallic compound by a reducing sugar. But the reaction has
been used successfully as the basis of quantitative method for the determination of reducing
sugar.
Reagents:
 Stock Glucose: Dissolve 1.0 gm of glucose in 100 ml of distilled water to get 10 mg/ml
 Standard Glucose: Dilute 10 ml of stock solution to 100ml with distilled water to get
1mg/ml.
 3, 5 - dinitro salicylic acid (DNS) Reagent: suspend 1 g of D.N.S in 20 ml of 2N NaOH
Solution Add this suspension carefully to 50 ml of water containing 30 gm of sodium
potassium tartarate and make up to 100ml. Filter the solution and store in brown bottle.
Procedure:
 Pipette out 0.2 - 1.0 ml of standard glucose (1mg ̸ ml) into different test tubes. Make the
volume to 1.0ml in each case with distilled water. Add 1.0 ml of D.N.S. Reagent to each
tube and then place all the tubes in a boiling water bath for 15 min. Cool and add 8.0 ml
of distilled water. Mix the contents of the tube thoroughly and read the absorbance of the
solution in a colorimeter at 540 nm against blank solution.
Observation:
Sl.No Vol. of glucose d.H2O Conc Volume DNS d.H2O O.D. at
Incubate on boiling water

(ml) (ml) (mg/ml) Reagent (ml) (ml) 540 nm
bath for 15 minutes

1 0.0 1.0 0
2 0.2 0.8 0.2
3 0.4 0.6 0.4
4 0.6 0.4 0.6 1.0 8.0
5 0.8 0.2 0.8
6 1.0 0 1.0
UK ?
Results: The concentration of reducing sugar in the given solution is ______________mg/ml

Estimation of Protein by Lowry’s method
Dr. Mahesha H B., Yuvaraja’s College, Mysore.
Aim: To estimate the protein using Lowry’s method.
Principle: The –CO-NH- bond (peptide) in polypeptide chain reacts with copper sulphate in an
alkaline medium to give a blue colored complex. In addition, tyrosine and tryptophan residues of
protein cause reduction of the phosphomolybdate and phosphotungstate components of the
Folin-Ciocalteau reagent to give bluish products which contribute towards enhancing the
sensitivity of this method.
Reagents Required:
1. Reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide.
2. Reagent B: 0.5% copper sulphate (CuSO4.5H2O) in 1% potassium sodium tartarate. Prepare
fresh by mixing stock solutions.
3. Alkaline copper solution (Reagent C): Mix 50mL of reagent A and 1 mL of reagent B prior
to use.
4. Diluted Folin’s reagent (Reagent D): Dilute Folin-Ciocalteau reagent with an equal volume
of 0.1 N NaOH
5. Standard: Dissolve 50mg BSA in 50mL of distilled water in a volumetric flask. Take
10mL of this stock standard and dilute to 50 mL in another flask for working standard
solution. One mL of this solution contains 200 µg protein.
Apparatus and Glass wares required: Test tubes, Pipettes, Colorimeter, etc.,
Procedure:
1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of labeled test
tubes.
2. Pipette out 1 mL of the sample in another test tube.
3. Make up the volume to 1 mL in all the test tubes. A tube with 1 mL of distilled water
serves as the blank.
4. Now add 5 mL of reagent C to all the test tubes including the test tubes labeled 'blank'
and 'unknown'.
5. Mix the contents of the tubes by vortexing / shaking the tubes and allow to stand for 10
min.
6. Then add 0.5 mL of reagent D rapidly with immediate mixing well and incubate at room
temperature in the dark for 30 min.
7. Now record the absorbance at 660 nm against blank.
1
8. Then plot the standard curve by taking concentration of protein along X-axis and
absorbance at 660 nm along Y-axis.
9. Then from this standard curve calculate the concentration of protein in the given sample.
Result: The given unknown sample contains ----µg protein/ml.
Observations and Calculations
Volume Volume of Concentration Volume Volume
of distilled of Protein of of
standard water (ml) (µg) reagent C Incubate reagent D Incubate A660
BSA (ml) (ml) At (ml) At dark
0.0 1.0 00 5 Room 0.5 room 0.00
0.2 0.8 40 5 Temp 0.5 temp.
0.4 0.6 80 5 for 0.5 for
0.6 0.4 120 5 10 0.5 30
0.8 0.2 160 5 min 0.5 min
1.0 0.0 200 5 0.5
1.0 UK 0.0 ? 5 0.5
-----------------
2
ESTIMATION OF SUGAR BY TITRIMETRIC (HAGEDORN - JENSON) METHOD
Dr. Mahesha H B, Yuvaraja’s College, Mysore.
Aim: To estimate reducing sugars by Hagedorn and Jensen method.
Principle: The potassium ferricyanide is reduced to potassium ferrocynide when
heated with alkaline solution by reducing sugars. The ferrocynide formed is
precipitated as potassium zinc salt. The remaining ferricyanide is determined from the amount of
iodine liberated.
The principle reactions are
2K3 [Fe(CN) 6] + 2KI → 2K4[Fe(CN)6] + I2
2K4 [Fe(CN)6] + ZnSO4 → K2Zn [Fe(CN)6] + 3K2SO4
Reagents Required
1. Iodide sulfate chloride solution: Dissolve 25 gms of ZnSO4 and 12.5 gms of NaCl2 in 500
mL of water. To this, add 12.5 gms of KI on the day of the experiment.
2. 3% acetic acid.
3. Potassium ferricyanide solution: Dissolve 0.825 gm of K3[Fe(CN)6] and5.3 gms of
anhydrous sodium carbonate in 500 mL water. Store it in a dark bottle.
4. Preparation of standard sugar solution: Dissolve 100 mg of standard sugar in 100 mLof
water. Take 10 mL of stock and make it up to 100 mL.
5. 0.1N Na2S2O3solution: 12.4 gms of sodium thiosulfate in 500 mL of distilled water. Take 5 mL of stock
and make it up to 100 mL for 0.005NNa2S2O3solution.
6. Starch indicator: 1 gm of starch in 100 mL of H2O and 5 gms of NaCl.
Procedure:
1. Pipette out 0.0, 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of labeled test
tubes.
2. Pipette out 1 ml of the given sample in another test tube.
3. Make up the volume to 2 ml in all the test tubes. A tube with 2 ml of distilled water serves as
the blank.
4. Now add 2 ml of potassium ferricyanide reagent to all the test tubes including the test tubes
labeled 'blank' and 'unknown'.
5. Mix the contents of the tubes by vortexing / shaking the tubes and incubate in a boiling water
bath for 15 min.
6. Then add 3 ml of Iodine sulfate chloride solution, mix well and add 2 ml of 3% acetic acid.
7. Now add few drops of starch solution as indicator and totrate against sodium thiosulphate.
Titrate until the blue color disappears. Note the readings.
8. Then plot the standard curve by taking concentration of glucose along X-axis and difference of
burette readings along Y-axis.
9. From this standard curve calculate the concentration of glucose in the given sample.
1
Volume of Volume Concentration Potassium Incu Iodine 3% Volume of Differe
standard of of creatinine ferricyanide bate sulfate acetic sodium nce in
glucose distilled (µg) reagent (ml) in chloride acid. thiosulphate burette
(ml) water boili solution (ml) used (ml) reading
(ml) ng (ml)
wate
0.0 2.0 00 2 r 3 2 0.00
bath
0.2 1.8 200 2 for 3 2
0.4 1.6 400 2 15 3 2
Min
0.6 1.4 600 2 & 3 2
Coo
0.8 1.2 800 2 3 2
1.0 1.0 1000 2 3 2
1.0 1.0 To be 2 3 2
Sample estimated
----------------
2
EtBr Decontamination Procedure
Decontamination should be performed using the following solution:
4.2 grams of sodium nitrite (NaNO2)

20 milliliters of 50% hypophosphorous acid solution (H3PO2)
300 milliliters of water
To perform decontamination:
− Soak a paper towel in the decontamination solution and thoroughly wash the
contaminated area.
− Once the contaminated area has been thoroughly washed with the decontamination
solution, rinse the area 5 times with tap water using a clean paper towel for each
rinse.
− Soak all spent paper towels in the decontamination solution for one hour. Gently
wring out excess solution and dispose of as hazardous waste with contaminated
gloves, pipette tips or any other solid EtBr debris.
(Taken from http://www.purdue.edu/REM/hmm/ethidbr.htm)

FLUOROPHORE TABLE
Dye Absorbance Wavelength Emission Wavelength Visible color
Hydroxycoumarin 325 386 blue
methoxycoumarin 360 410 blue
Alexa fluor 345 442 blue
aminocoumarin 350 445 blue
Cy2 490 510 green (dark)
FAM 495 516 green (dark)
Alexa fluor 488 494 517 green (light)
Fluorescein FITC 495 518 green (light)
HEX 535 556 green (light)
Cy3 550 570 yellow
TRITC 547 572 yellow
Alexa fluor 546 556 573 yellow
Alexa fluor 555 556 573 yellow
R-phycoerythrin (PE) 480;565 578 yellow
Rhodamine Red-X 560 580 orange
Tamara 565 580 red
Cy3.5 581 581 596 red
Rox 575 602 red
Alexa fluor 568 578 603 red
Red 613 480;565 613 red
Texas Red 615 615 red
Allophycocyanin 650 660 red
Cy5 650 670 red
Cy5.5 675 694 red
TruRed 490;675 695 red
Cy7 743 770 red
Nucleic acid probes:
Dye Absorbance Wavelength Emission Wavelength Visible color

DAPI 345 455 blue
Hoechst 33258 345 478 blue
SYTOX blue 431 480 blue
Hoechst 33342 343 483 blue
YOYO-1 509 509 green
SYTOX green 504 533 green
TOTO 1, TO-PRO-1 509 533 green
SYTOX orange 547 570 yellow
Chromomycin A3 445 575 yellow
Mithramycin 445 575 yellow
Propidium iodide 536 617 red
Ethidium bromide 493 620 red
www.abcam.com/technical 1
SDS-PAGE Gel Preparation Chart
Resolving Gel
Component 7% 7.5 % 8% 8.5 % 9% 9.5 % 10 % 10.5 % 11 % 11.5 % 12 % 12.5 %

Acrylamide 2.33 mL 2.5 mL 2.66 mL 2.83 mL 3 mL 3.16 mL 3.33 mL 3.5 mL 3.66 mL 3.83 mL 4 mL 4.16 mL
Double distilled Water 5.03 mL 4.86 mL 4.7 mL 4.53 mL 4.36 mL 4.2 mL 4.03 mL 3.86 mL 3.7 mL 3.53 mL 3.36 mL 3.2 mL
10%SDS 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl
1.5M Tris Hcl (pH 8.8) 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5mL 2.5 mL
TEMED 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl
10% Ammonium per
33 µl 33 µl 33 µl 33 µl 33 µl 33 µl 33 µl 33 µl 33 µl 33 µl 33 µl 33 µl
sulphate (APS)
Total Volume 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL 10 mL
Stacking Gel Component

4.5 %
Acrylamide 600 µl
Double distilled Water 2.336 mL
10%SDS 40 µl
1.5M Tris Hcl (pH 8.8) 1 mL
TEMED 4 µl
10% Ammonium per sulphate (APS) 20 µl
Total Volume 4 mL
9/18/2015 Gel Solubilization Buffer Composition
GIL .. BIOSCI/Bionet News .. Biosequences .. Software .. FTP
Gel Solubilization Buffer Composition
Suresh Kumar via methods%40net.bio.net (by kumar from lifesensors.com)
Mon Jan 11 16:13:39 EST 2010
Previous message: Methods Digest, Vol 56, Issue 10 (Message 2:Joining two PCR products together)
Next message: Methods Digest, Vol 56, Issue 10 (Message 2:Joining two PCR products together)
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Here is couple of different buffer composition.
Gel solubilization buffer ‐I
60% Guanidine Thiocyanate (5.36M)
140mM MES (2‐[N‐Morpholino] ethanesulfonic acid)
0.006% Phenol Red

Use 3 Volume of buffer to 1 gel volume.
Reference: http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007750
Phenol Red is only a pH indicator. It changes color from yellow to red over the pH range 6.8 to 8.4.
Gel solubilization buffer ‐II
6M Guanidine Thiocyante
50mM Tris‐HCl pH 7.5
20mM EDTA pH 8.0
Use 3 Volume of buffer to 1 gel volume.
Hope this helps.
Suresh
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Message: 2
Date: Sat, 9 Jan 2010 22:10:12 +0530
From: Jagadish Katam <jagadish.kv from hotmail.com>
Subject: Gel Solubilization Buffer Composition
To: <methods from magpie.bio.indiana.edu>
Message‐ID: <SNT124‐W189ABD0BA2F045D7F1CB96E76F0 from phx.gbl>
Content‐Type: text/plain; charset="iso‐8859‐1"
Hi,
I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilizatio
Here i would know the composition of this gel solubilization buffer.
Any suggestions will be greatly appreciated.
Thanks and Best Regards,
Jag

_________________________________________________________________
Windows 7: Find the right PC for you. Learn more.
http://windows.microsoft.com/shop
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Message: 3
Date: Sat, 9 Jan 2010 12:40:27 ‐0800 (PST)
From: Nick Theodorakis <nick.theodorakis from gmail.com>
Subject: Re: Gel Solubilization Buffer Composition
To: methods from net.bio.net
Message‐ID:
<f87be422‐8f3e‐4683‐9cda‐f1d3c01ac53e from j19g2000yqk.googlegroups.com>
Content‐Type: text/plain; charset=ISO‐8859‐1
On Jan 9, 11:40 am, Jagadish Katam <jagadish... from hotmail.com> wrote:
> Hi,
>
> I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilizat
>
> Here i would know the composition of this gel solubilization buffer.
>
In general, agarose gel solubilization can be done with chaotropic
salts such as NaI, sodium perchlorate, or guanidine HCl. These also
work well in conjunction with silica‐based binding resins because DNA
binding to silica is promoted in high salt. I doubt the company will
tell you exactly what is in their buffer, but it is likely similar to
one the salts I mentioned, possibly with additives added as a
stabilizer or other use.
Nick
‐‐
Nick Theodorakis
nick_theodorakis from hotmail.com
http://www.bio.net/mm/methods/2010January/104521.html 1/2
9/18/2015 Gel Solubilization Buffer Composition
contact form:
http://theodorakis.net/contact.html
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Message: 4
Date: Sat, 9 Jan 2010 18:19:29 ‐0700 (MST)
From: "Dr. Hiranya S. Roychowdhury" <hroychow from nmsu.edu>
Subject: Re: hi .......help me plz
To: "Amol Ghodke" <amoltej from gmail.com>
Cc: methods from magpie.bio.indiana.edu
Message‐ID: <1120.71.33.47.214.1263086369.squirrel from webmail.nmsu.edu>
Content‐Type: text/plain;charset=iso‐8859‐1
Do you have access to a library? If you do, perhaps you may check out a
few of the Methods in Enzymology volumes for zymogram protocols. If you
have access to the internet, Google search of the protocols (both zymogram
and protein elution from gels ‐ whether acryl. or starch) will show you
several protocols. Protein/peptide elution from gels is quite simple ‐
works with electrophoresis or simple diffusion/dialysis methods.
> hi all...
> i have to screen proteinase inhibitors!
> for that i have to do zymography.
> can any one plz give me the detail protocol for that
> and plz tell me how to recover screened protein from gel efficiently
> without
> using highly sofasticated instuments
> plz help me
> thanx
>
> ‐‐
> Mr. Amol Bharat Ghodke
> M.Sc. Agril.Biotechnology
> A/P ‐ Kanhur Pathar
> Tal.‐ Parner,
> Dist.‐ Ahmednagar
> Pin ‐ 414303
> Mob. 09970865283/09021257663
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
‐‐
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
Message: 5
Date: Sun, 10 Jan 2010 11:06:34 +0200
From: Yoram Gerchman <gerchman from research.haifa.ac.il>
Subject: yeast estrogen screen?
To: methods from oat.bio.indiana.edu
Message‐ID: <1263114394.4b49989acbeb4 from webmail.haifa.ac.il>
Content‐Type: text/plain; charset=windows‐1255
Greetings all
I am trying to get some version of the Yeast Estrogen Screen Assy for some
enviormental work (beta‐gal, GFP, ...). Does anyone know were does one get the
yeast and plasmid from?
Many thanks
Yoram
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐
This message was sent using IMP, the Webmail Program of Haifa University
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General Guidelines for Decontaminating Pipettes when Working with Different Liquids
Supporting Information
General Guidelines for Decontaminating Pipettes
when Working with Different Liquids
Liquid Handling Special features Decontamination

Aqueous Pipettes are calibrated with Open pipette, rinse contaminated parts
solutions distilled water. Results are well with distilled water, and allow to dry
and buffers extremely accurate. at maximum 60°C in dryer compartment.
Lubricate piston if neccessary using the
grease that comes with the pipette.
Inorganic It is advisable to occasionally rinse The plastics used in Finnpipettes
acids the lower part of the pipette with are acid resistant. However,
distilled water if high-concentration aerosols from the acids can enter the
acids are pipetted frequently. Using lower part of the pipette and affect the
Filtertips is also recommended. performance of the pipette. Clean as
described above in “Aqueous solutions and buffers”.
Alkalis It is advisable to occasionally rinse the The plastics used in Finnpipettes
lower part of the pipette with distilled are alkali resistant. However,
water if high-concentration alkalis are aerosols from the alkalis can enter the
pipetted frequently. Using Filtertips is lower part of the pipette and affect the
also recommended. performance of the pipette. Clean as
described above in “Aqueous solutions
and buffers”.
Potentially To avoid contamination, Filtertips Use 70% ethyl alcohol to disinfect.
infectious should be used. Alternatively, positive The tip cone and tip ejector can be
liquids displacement systems can be used. left in an ethyl alcohol bath overnight.
Autoclave the Finnpipette Digital in one
piece at 121°C for 20 min. Viruses and
spores can be inactivated by wiping the
the pipette with a tissue moistened with
5% sodium hypochlorite. Moisten a clean
tissue with distilled water and wipe the surface well.
Cell cultures To guarantee sterility, Thermo Proceed as described above with
Filtertips should be used. “potentially infectious liquids”.
Organic 1.Density is different than that of water. This evaporation process is normally
solvents Therefore, it is necessary to adjust sufficient for liquids with a high vapour
the pipette. pressure. Alternatively, immerse the
2.Pipetting should be carried out rapidly, contaminated parts in detergent. Rinse
due to the high vapor pressure and well with distilled water and dry as
changes in the wetting pressure. described above.
3.After pipetting is finished, open
the pipette and allow the liquid
to evaporate.
Radioactive To avoid contamination, Filtertips should Open pipette and place contaminated
solutions be used. An alternative would be to use parts in complex solutions or special
positive displacement systems. cleaning solutions. Rinse well with
distilled water and dry as described above.
Proteins To avoid contamination, Filtertips should Open pipette, rinse pipette with
be used. An alternative would be to use detergent. Rinse well with distilled
positive displacement systems. water and dry as described above.
Lightly lubricate piston.
Nucleic acids To avoid contamination, Filtertips or If you have an autoclavable pipette,
positive displacement systems should autoclave it according to the manufac-
be used torer's instructions. Otherwise open
pipette, wipe with 90% ethanol, followed
by 2 M sodium acetate and again 90% ethanol.

3. General Laboratory Safety
3.1 Standard Operating Procedures
Standard operating procedures (SOPs) are detailed work practices, which are developed to
provide guidance for the safe handling of hazardous chemicals. Site-specific standard operating
procedures (SOPs) must be written for each potentially hazardous substance used in the
laboratory. SOPs include but are not limited to procurement, distribution, storage, labeling,
equipment usage, general lab practices, and disposal and emergency procedure practices for the
particular chemical, work or hazard group. Information about each chemical can be obtained
using the MSDS resource link posted on the EHS&RM department’s website.
The SOPs regarding various lab components provided in this manual offer generic safety
guidelines for the laboratories on the campus of Stephen F. Austin State University. This
document contains only a minimum set of guidelines, regulations, and recommendations
required to maintain a safe working environment, and do not provide laboratory workers,
research students, faculty and students with specific standard operating procedures necessary to
work in their respective laboratories. It is the responsibility of the principal investigator or
teaching faculty to develop specific standard operating procedures for their work place or
laboratory.
3.1.1 General Safety Guidelines
The following guidelines have been established to minimize or eliminate hazards in the
laboratory. These guidelines have also been provided to maintain a safe laboratory environment.
It is the responsibility of each person that enters into the laboratory to understand the safety and
health hazards associated with potential hazardous materials and equipment in the laboratory. It
is also the individual’s responsibility to practice the following general safety guidelines at all
times while working in the laboratories of Stephen F. Austin State University.
• Always wear proper eye protection in chemical work, handling and storage areas.
• Always know the hazards associated with the materials that are being utilized in the lab.
• Eyewashes should be flushed weekly and documented on eyewash tags.
• Properly label chemical waste with specific contents. Keep label attached to the
container at all times. Always replace old and deteriorated labels.
• Keep chemical waste containers closed.
• Never remove chemicals, biological agents or any other laboratory related item from the
laboratory without proper authorization.
• Never perform unauthorized work, preparations or experiments.
• Never engage in horseplay, pranks or other acts of mischief in chemical or biological
work areas.
• Chemical fume hood sashes should be kept closed whenever possible. Maintain the
minimum possible opening when working. Do not store chemicals in fume hoods.
• Do not store or consume food and drinks in labs or in any place where hazardous
materials are either present or used.
• Food or drinks and chemicals should not be stored in the same refrigerator.
• Do not wear shorts or open-toed shoes in labs. Wear appropriate personal protective
equipment when working in labs where hazardous materials are present.
• Always wash hands and arms with soap and water before leaving the work area. This
applies even if you are wearing gloves.
• Remove clutter and practice good housekeeping.
• Confine long hair and loose clothing.
• Do not use cell phones, Bluetooth and MP3 players while working in the lab.
• Never leave an open flame unattended. Know the location of the nearest fire extinguisher.
Never leave an experiment unattended while it is being heated or is rapidly reacting.
• Do not keep or work with flammable substances near a flame.
• Secure gas cylinders properly and keep safety caps on cylinders when not in use.
• Have appropriate spill supplies available and follow response procedures.
• Eliminate extension cords and power strips in series. No exposed wiring.
• Keep exits and aisles clear of obstructions.
• Emergency equipment should be clear of obstructions. Be familiar with the location of
emergency equipment – fire alarm, fire extinguisher, emergency eye wash and safety
shower. Know the appropriate emergency response procedures.
• Glass chemical bottles should not be stored on the floor.
• Do not store any lab equipment or chemicals in corridors.
• Keep equipment back from the edge of the lab bench to prevent spillage.
• Report any accident immediately, however minor or irrelevant you might think it might
be.
Primer guidelines, Howard Judelson, 10.06, p. 1
GUIDELINES FOR DESIGNING PRIMERS
Proper primer design is important for applications in PCR, DNA sequencing, and
hybridization. Here are some tips to help you design primers, especially using the
Oligo program. This is based upon Oligo 4.0; there may be some changes
compared to the current version.
A. General recommendations
The ideal primer generally has the following characteristics:
1. Melting temperature (Tm) between 55 and 65°C (usually corresponds to 45-55%

G+C for a 20-mer).
2. Absence of dimerization capability.
3. Absence of significant hairpin formation (usually >3 bp).
4. Lack of secondary priming sites in the template.
5. Low specific binding at the 3' end, to avoid mispriming.
Usually a 20-24 nt primer works well. Oligo (as well as other programs) will pick decent
primers automatically. However, often the “standard” parameters used by such
programs don’t work with a given sequence. In such a case, primers need to be
picked manually. The following hints should help in this process:
B. Melting (and annealing) temperatures
As mentioned above, a Tm of 55-65°C works best in most applications. You may have
noted that there are different ways to calculate Tm. The nearest neighbor
thermodynamic calculation is the most accurate (make sure that you are looking at
reaction conditions appropriate to your experiment (PCR, sequencing, etc.). Oligo
will calculate Tm’s for you (from the pull-down menus, select Analyze, then
Composition and Tm):
For automated sequencing, primers with Tm's above 65-70°C can lead to secondary
priming artifacts and noisy sequences. Remember, the sequence facility doesn’t
tailor the reactions to your specific primer as they use a “generic” annealing
temperature.
Remember that you are dealing with TWO primers in PCR. Their Tm’s should be within
5°C of each other; the closer the better! If Tm’s are mismatched, amplification will
be inefficient: the primer with the higher Tm will misprime at lower temperatures,
while the primer with the lower Tm may not work at higher temperatures. If the Tm
difference is high, add or subtract bases from a primer. An easy way to see if two
primers have similar Tms using Oligo is to use the Analyze>DNA Amplification
command in Oligo, which will show the Tms of both primers at the same time. Note
that Tm is calculated at 50 mM salt, which is standard for PCR. If you were using
labelled oligonucleotides for hybridizations in SSPE, you would need to calculate Tm
differently (the Oligo program can also show a table that adjusts for differences in
salt and formamide).
The annealing temperature (Ta) for a primer pair is generally calculated as 5°C lower
than the estimated melting temperature. The optimal temperature for PCR often
needs to be determined empirically; ideally, the primers should anneal to the
template before the template reanneals to itself. One way you can get into trouble
designing primers is if you use AT-rich primers that flank a GC-rich region of DNA in
PCR. Oligo will warn you of such problems, as well as suggest an optimal
annealing temperature as shown above.
A special case in primer design (for PCR) is when you need to add extra bases to a
primer, for example a restriction site. Typically one might design a primer that
contains 18 nt perfectly matching the template, plus 6-nt representing the restriction
site, and then about 2-nt more to assist in the restriction digestion (some enzymes
need to "sit" upon a sequence larger than the restriction site itself; see the New
England Biolabs catalog for more information). With this type of primer, there are
essentially two different Tm's. Initially, the 18-nt region matching the template will
define the Tm. After several rounds of PCR, most template will have incorporated
the primer site, so the entire primer length will define the Tm. For optimal results,
one might consider doing a two-phase PCR program, shifting the Tm up by a few
degrees after 5-10 cycles, however this may not really be necessary; consider
doing this if the amplification appears to be working poorly.
One way to start off with primers of similar Tms is to use the Tm and ∆G windows in
Oligo as a guide to select candidate primers. The scale at left indicates the ∆G of
hybridization (this can be toggled to Tm if you wish), based on the size of the primer
currently selected (press “L” to see that size) It is easy to see that the Tm of the left
(upper) primer is going to be higher than that of the lower primer. Either the lower
primer should be fitted to another region, or made longer.
C. Specificity
Apart from Tm, a prime consideration in designing primers is ensuring that the likelihood
of annealing to sequences other than the chosen target is very low. This can occur
if the same sequence is present in the template DNA more than once, or when a
primer is poorly designed.
To avoid mispriming, primers should not be very “sticky” on their 3’ ends. A "sticky" 3'
end would be one with a high G/C content (high Tm). Either the middle of the primer
or the 5’ end should be "stickier". The display in Oligo makes it easy to pick primers
with good specificity, by using the “Internal Stability” window at the bottom of the
screen or the Analyze>Internal Stability command (both are shown below). For
example, the following primer (5’-CAGTAACAGA TACGGGCA) would show poor
specificity since its 3’ end is GC rich relative to the rest of the primer. NOTE that
the issue is the 3’ end region, not just the 3’ base!!
BAD!!! BAD!!!
In contrast, the following profiles are “good” for specificity (shown 5’—>3’), since the 3’
end is lower (in this ∆G plot) than the 5’ end or middle of the primers:
Now what about the profile shown below?
This would appear to give good specificity, based on the rules mentioned above.
However, in practice this would be a difficult primer to use since at it’s Tm the 3’ end
would anneal poorly to the template (remember that Tm is the 50% point in a melting
curve). This brings up the “rule of extremes” in primer design:
Avoid primers with long polyG or polyC stretches that can promote non-specific
annealing.
Avoid polyA and polyT as these will “breath” and open the primer-template complex.
Avoid polypyrimidine (T, C) and polypurine (A, G) stretches, which may lead to an
odd shape of the double helix.
Ideally the primer will have a near random mix of nucleotides.
D. What about having a “GC clamp” at the 3’ end?
A "G" or "C" is desirable at the 3' end of primers since this will reduce “breathing” and
thereby increase yield. However, Gs or Cs should not be added if they adversely
influence the overall specificity of the primer!
E. Primer-Primer interactions
When designing primers, it is important to have a minimum of intramolecular or

intermolecular homology. This would result in either hairpins or primer dimerization.
If a primer has a region of self-homology, “snap back” or partially double-stranded
structures can occur which will interfere with annealing to the template. Usually
intraprimer homologies of 3 bp or more should be avoided. The resulting hairpins
are readily detected in Oligo using the Analyze>Duplex (or Hairpin) command, for
example:
Primers should also not contain sequences of nucleotides that would allow one primer
molecule to anneal to another molecule of itself, or to the other primer (if being used
in PCR). Such interactions can also readily detected in Oligo using the
Analyze>Duplex command (remember to check both the individual primer and
upper/lower primer combinations). Also, remember that primer-primer interactions
are stronger at lower temperatures; a small degree of complementarity becomes
less significant as reaction temperatures increase (in other words, something that is
OK with an annealing temperature of 60C may work poorly at an annealing
temperature of 50C).
The worst situation is when the 3’ ends of the primer(s) anneal; this leads to “primer-
dimer” formation:
Internal intermolecular interactions should also be minimized. Oligo provides a ∆G

value for such complexes. Ideally, the smallest ∆G, the better but try to avoid
primers annealing with ∆G values of –7 kcal/mol or higher. Having a
complementary TA sequence at the 3' ends of primers usually doesn't cause
problems since it is not very stable, however a complementary GC region can
cause problems at Tm's below 65C. The primer shown below has a Tm of only
50°C, so the 3’ GC homology (∆G=-3.1 kcal/mole) might be a problem; if the Tm
was more like 60°C, then the primer would probably be OK.
Here are other examples of interacting primers. The following are OK, but not great:
∆G=-5 kcal/mole, OK but not great. ∆G=-9.3 kcal/mole, pretty bad!!
The next is real nice! ∆G=-1.6 kcal/mole, good!!

Homemade Buffer Compositions
• Miniprep Buffers
o Re-suspension Buffer (equivalent of Qiagen Buffer P1)
Tris·HCl – 50 mM
EDTA – 10 mM
RNase A – 100 μg/mL
HCl – final pH 8
(Note: Store RNase A @ -20° C, aliquot buffer and add at time of use, do not
autoclave)
(Note: Do not autoclave RNase A)
o Lysis Buffer (equivalent of Qiagen Buffer P2)
NaOH – 200 mM
SDS – 1% (w/v)
(Note: Do not autoclave SDS, use sterile filter)
o Neutralization Buffer (equivalent of Qiagen Buffer N3)
Gu·HCl – 4.2 M
KOAc – 0.9 M
HOAc – final pH 4.2
o Column Wash/Binding Buffer (equivalent of Qiagen Buffer PB)
Gu·HCl – 5.0 M
Isopropanol – 30 % (v/v)
o Column Wash Buffer (equivalent of Qiagen Buffer PE)
Tris·HCl – 10 mM
Ethanol – 80% (v/v)
HCl – final pH 7.5
• Mini-column Recycling Buffer

o Equilibrium Buffer (equivalent of Qiagen Buffer QBT)
NaCl – 750 mM
MOPS – 50 mM
Isopropanol – 15% (v/v)
Triton X-100 – 0.15% (v/v)
(Note: Do not autoclave MOPS, use sterile filter)
• Protein Purification Buffer

o NID Extraction Buffer
EDTA – 20-50 mM
Tris·HCl – 50 mM
NH₄Cl – 0.75 M
Triton X-100 – 0.5% (v/v)
Sucrose – 5.0% (w/v)
Lysozyme – 100 μg/mL
RNase A – 25 μg/mL
HCl – final pH 8
(Note: Store RNase A & Lysozyme @ -20° C, aliquot buffer and add at time of
use, do not autoclave)
Homemade RNase Zap
5 % H2O2
0.5 % SDS
0.1 M NaOH
1% triton X-100
1mM EDTA
EtBr Decontaminating Solution

0.5M NaNO2 Sodium Nitrite
5.0% H3PO2
Water 300 mL
BD Biosciences Technical Resources Page 1
December, 2010
Immunohistochemistry: Preparation and Staining of Paraffin Sections:

Formalin and Zinc Fixation
This procotol is for use with BD Pharmingen™ reagents. Before you begin, determine the fixation method (formalin or zinc) and
the method of heating slides (microwave or pressure cooker or autoclave) for antigen retrieval, if needed.
I. Tissue Preparation
1. Sacrifice the animal according to prescribed and approved euthanasia techniques.
2. Cut the tissues to be fixed and processed to no thicker than 3 mm.
II. Fixation and Processing of Tissue for Paraffin Sections
A. Fixation of Tissues in 10% Neutral Buffered Formalin
1. Let tissues fix in 10% formalin at room temperature for 8 hours (4 to 8 hours for small-rodent tissue) but no longer than
24 hours.
2. Rinse with running tap water for 1 hour.
3. Follow the processing schedule recommended in section C.
B. Fixation of Tissues in Zinc Fixative
Many antigenic epitopes are masked or even destroyed by 10% formalin fixation. In some cases, fixation in a milder fixative such
as BD Pharmingen™ IHC zinc fixative (Cat. No. 550523) is helpful to preserve the antigenic epitopes.
Use the following recommended fixation times:
Dense tissues such as cardiac muscle, skin, fat 24 to 48 hours

Lung, spleen, thymus, GI tissues 6 to 8 hours
Optimization might be required based on species and the size of the tissue.
1. Place freshly dissected tissues into zinc fixative and fix for the recommended time.
2. Rinse with running tap water for 30 to 45 minutes.
3. Follow the processing schedule recommended in section C.
C. Processing Schedule
The processing, embedding, and sectioning of paraffin blocks requires specialized equipment and expertise and is usually
performed by a histology or pathology laboratory. While hand processing can be performed according to the following protocol, the
results might show marked variation in histology quality and antigenicity.
Station Time (min) Solution

1 Water
2 45 70% alcohol
3 45 80% alcohol
4 45 90% alcohol
5 45 100% alcohol
6 60 100% alcohol
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
bdbiosciences.com
December, 2010

7 60 100% alcohol
Clearing reagent (xylene
8 60 or substitute)
Clearing reagent (xylene
9 60 or substitute)
10 60 Paraffin 1
11 60 Paraffin 2
12 60 Paraffin 3
III. Preparation of Paraffin Sections for Immunohistochemistry
A. Sectioning Protocol
1. Section paraffin blocks at the thickness you want (usually 4 to 5 µm) on a microtome and float on a 40°C water bath
containing distilled water.
2. Transfer the sections onto a Superfrost Plus slide. Allow the slides to dry overnight and store them at room temperature
until ready for use.
B. Deparaffinization and Rehydration of Tissue Slides
1. Place the slides in a 55°C oven for ten minutes to melt the paraffin.
2. Deparaffinize the slides in two changes of xylene or xylene substitute for 5 minutes each.
3. Transfer the slides to 100% alcohol, make two changes for 3 minutes each, and transfer once through 95% alcohol for 3
minutes.
4. Block endogenous peroxidase activity by incubating sections in 3% H2O2 solution in methanol for 10 minutes.
5. Rinse in PBS twice for 5 minutes each time.
6. If the antibody staining requires antigen retrieval to unmask the antigenic epitope, continue with section C. If antigen
retrieval is not required, proceed to section D.
C. Pretreatment of Paraffin Sections with Retrievagen A* (pH 6.0)
Microwave oven method**
1. Make a working solution of BD Pharmingen™ Retrievagen A by mixing 18 mL of solution 1 and 82 mL of solution 2. Bring
the final volume to 1 liter in distilled water.
2. Place slides in a plastic coplin jar filled with the Retrievagen A working solution and heat in a microwave oven to 203°F
(95°C).
3. Mix the Retrievagen A working solution in the coplin jar with a disposable pipet and incubate the slides at 203°F (95°C)
for 10 minutes.
4. Remove the coplin jar with the slides, cover the jar tightly, and allow the solution to slowly cool to room temperature for
20 minutes to enable the protein molecules to fold properly.
5. Rinse the slides in PBS three times for 5 minutes each time.
bdbiosciences.com
December, 2010

*For methods using other antigen retrieval systems, see the instructions in technical data sheets.
**Heating using a microwave oven may require a license under US patent No. 5,244,787.
Pressure Cooker or Autoclave Method

1. Prepare a working solution of Retrievagen A as described in step 1 of the microwave oven method.
2. Place the slides in a glass or metal coplin jar and heat in a pressure cooker or autoclave at 120 to 125°C, 17 to 25 psi, for
5 minutes.
3. When completed (at 0 psi), open the pressure cooker or autoclave and allow the slides to cool to room temperature for
approximately 20 to 30 minutes.
4. Remove the slides from the coplin jar and wash them as described in step 5 of the microwave oven method.
D. Immunohistochemical Staining of Paraffin-embedded Tissues
See Standard Immunohistochemical Staining Procedure (Section III of Immunohistochemical Staining of Frozen Sections)
http://www.bdbiosciences.com/support/resources/protocols/frozen_tissue.jsp.
Begin at step 5 and proceed through coverslipping.
Label No. 23-12512-00
bdbiosciences.com
1019
Immunochemicals
Immunoblotting Troubleshooting Guide

Problem Possible Cause Solution
No signal or weak signal Substrate or conjugate weak Test conjugate and substrate for activity. For example, add enzyme conjugate to
or non-specific bands or no longer active due to age or substrate solution. It should change color.
improper storage
If chemiluminescent detection is being Use fresh film development solutions.
used, the film development solution may
have expired
Incorrect substrate for application Make sure that the substrate selected is appropriate for the enzyme conjugate.
Substrate prepared incorrectly Follow instructions that accompany the substrate. Make sure that fresh H2O2 is
added if necessary.
Incorrect dilution of primary or Check the literature or data sheet for recommended dilutions for the antibodies
secondary antibody being used. Try a range of dilutions.More is not always better, especially with
sensitive detection systems such as chemiluminescence. Most of Sigma's anti
Appendix
bodies are tested with colorimetric substrates that are not as sensitive. For this
reason, the antibody may need to be diluted 5-10 times more if
chemiluminescent detection is being used.
Incorrect primary antibody for the Make sure the antibody has been shown to work in immunoblotting. Not all
application antibodies work in all applications. Primary antibody may not be capable of
reacting with the protein of interest from the species being studied. Check the
literature/data sheet and protein sequence information.
Protein of interest is not present If the positive control worked, check the amount of protein loaded. If
or is present in low amounts necessary, try enriching the amount of protein of interest in the sample loaded
by immunoprecipitation or by purification.
Consult the literature for the best source for the protein of interest.
See "Poor protein transfer" below
Inappropriate secondary antibody The secondary antibody may not be capable of binding to the primary antibody.
for the application Test this by spotting primary antibody on a small piece of membrane. When the
spot dries, block, then probe with diluted secondary, wash and develop with
substrate. A spot should appear if the secondary bound to the primary.
Incubation times inadequate Incubate at least one hour with primary antibody.
Enzyme inhibitor present Sodium azide will inhibit peroxidase reactions.
Over washing Shorten wash times, omit detergents from washing buffers
Poor protein transfer Check transfer of the proteins to the membrane by staining the membrane
with Ponceau S (Product No. P7170) prior to blocking.Make sure membrane
wets uniformly before transfer. Test transfer times. Small proteins (< 10,000)
may have transferred through the membrane (try including a second membrane
behind the first). Larger proteins may require longer transfer times.Check
transfer buffer - high methanol concentrations may prevent transfer of the
protein from the gel. 0.005-0.01% SDS in the transfer buffer may increase the
transfer of protein from the gel, but it can also interfere with protein binding to
the membrane. If the pI of the protein is >9.0 try using CAPS, pH 9 as the
transfer buffer.
High background or Insufficient blocking Try different blocking strategies: Longer blocking times, higher % of blocker, a
Non-specific bands different blocker, inclusion of blocker protein in antibody dilutions or try a
fresh batch of the same blocker.
Primary antibody may not be specific Try a monoclonal or an affinity purified polyclonal antibody.
enough
High concentration of primary Check literature or data sheet for recommended dilution of primary. Try a range
antibody of dilutions in order to optimize your system.
High concentration of secondary Test this by running an extra sample lane and omitting the primary antibody
antibody incubation from the procedure. If this "secondary only"control results in non-
specific staining, try further dilutions of the secondary antibody or try another
secondary.
Secondary antibody is cross-reacting Dilute the secondary antibody in buffer containing 1-5% normal serum from
www.sigma-aldrich.com
with other proteins in the sample the same species as the sample
Multiple bands may be proteolytic Make sure protease inhibitors are present from the first step of sample
fragments of the protein of interest preparation. Store and handle sample preparations to reduce the chance of
proteolysis. Try fresh samples when possible.
Antibody incubation times longer than Shorten the incubation times.
necessary
Overincubation with colorimetric substrate Decrease the staining time. Expose the membrane to substrate until a positive
solution signal is seen. Stop the reaction by washing the membrane before the back
ground develops.
Inadequate washing Increase the number or stringency of the washes
Contaminating enzymes present in sample Test sample with substrate alone to check for contaminating enzyme activity in
protein sample.
A. Tissue Culture Cells
• Grow cultured cells on sterile glass cover slips or slides (Cover Glasses and Micro Slides for Immunohistochemistry)
overnight at 37º C. Wash briefly with PBS (Buffers and General Solutions) and fix cells by one of the following
procedures:
1) 5 minutes in -10º C methanol, air dry (recommended method); or
2) 2 minutes in cold acetone, air dry; or
3) 10 minutes in 1% formalin in PBS (keep wet).
• Wash in three changes of PBS.
B. Frozen Tissue Sections
• Freeze tissue in block in liquid nitrogen according to standard procedures. Block may be stored at -70º C for up to 2
weeks before sectioning.
• Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Or use pre-treated slides.
• Cut 4- to 10-micron thick sections. Adhere sections to room temperature slides. Slides may be stored at -70º C. Thaw
slides at room temperature prior to fixing and staining.
• Fix slides in cold acetone for 10 minutes and keep refrigerated (or choose other fixation procedure). Wash in three
changes of PBS (Buffers and General Solutions).
NOTE: For tissues containing high levels of endogenous biotin (which may result in higher background staining), we
recommend following the Formalin-Fixed, Paraffin-Embedded Tissue Sections protocol, as endogenous biotin is
normally destroyed in paraffin-embedded tissue.
C. Formalin-Fixed, Paraffin-Embedded Tissue Sections
• Fix tissue sections in formalin and embed in paraffin blocks according to standard procedures.
• Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Or use pre-treated slides.
• Cut 4–6 micron thick tissue sections, and apply to slides. Deparaffinize in xylenes using three changes for 5 minutes
each. Hydrate sections gradually through graded alcohols: wash in 100% ethanol twice for 10 minutes each, then 95%
ethanol twice for 10 minutes each. Wash in deionized H O for 1 minute with stirring. Aspirate excess liquid from slides.
2
Optional: Antigen unmasking may be performed at this point. Certain antigenic determinants are masked by formalin
fixation and paraffin embedding and may be exposed by one of several methods:
1) Heat treatment (recommended method): Place slides in a container and cover with 10 mM sodium citrate
buffer, pH 6.0; or with 50 mM glycine-HCl buffer (glycine: sc-29096), pH 3.5, with 0.01% (w/v) EDTA
(EDTA: sc-29092). Heat at 95º C for 5 minutes. Top off with fresh buffer and heat at 95º C for 5 minutes
(optimal incubation time may vary for each tissue type). Allow slides to cool in the buffer for
approximately 20 minutes. Wash in deionized H O three times for 2 minutes each. Aspirate excess liquid
2
from slides.
2) Pepsin: Incubate sections for 10–20 minutes in 0.1% pepsin in 0.01 N HCl at room temperature. Wash
slides several times in deionized H O. Aspirate excess liquid from slides.
2
3) Saponin: Incubate sections for 30 minutes in 0.05% saponin in deionized H O at room temperature.
2
Wash at least three times in PBS. Aspirate excess liquid from slides.
D. Immunofluorescence Staining
• Use suction to remove reagents after each step, but avoid drying of specimens between steps. Use sufficient
reagent to cover the specimen (approximately 100–500 µl per slide is adequate).
• Incubate specimens with 10% normal blocking serum (Normal Sera for Immunohistochemistry) in PBS (Buffers
and General Solutions) for 20 minutes to suppress non-specific binding of IgG. Blocking serum ideally should be
derived from the same species in which the secondary antibody is raised. Wash with PBS.
• Incubate with primary antibody for 60 minutes. Optimal antibody concentration should be determined by titration;
recommended range is 0.5–5.0 µg/ml in PBS with 1.5% normal blocking serum. Wash with three changes of PBS
for 5 minutes each.
• Incubate for 45 minutes with either biotin-conjugated or fluorochrome-conjugated secondary antibody (Secondary
Antibodies for Immunohistochemistry) diluted to 1–5 µg/ml in PBS with 1.5%–3% normal blocking serum. Optimal
antibody concentration should be determined by titration. Wash with three changes of PBS. If fluorochrome-
conjugated secondary antibody is used, incubate in a dark chamber and omit the next step.
• Incubate with streptavidin-fluorescein for 15 minutes in a dark chamber. Optimal streptavidin conjugate
concentration for a given application should be determined by titration; recommended range is 10–20 µg/ml in
PBS. Wash extensively with PBS.
• Mount coverslip with aqueous mounting medium or 90% glycerol in PBS.
NOTE: For a listing of mounting media for Immunohistochemistry including Organo/Limonene and UltraCruz™ Mounting,
see Mounting Media for Immunohistochemistry.
• Examine using a fluorescence microscope with appropriate filters. Store slides in a dark location at room
temperature (UltraCruz™ Mounting Medium: sc-24941) or at 4º C (glycerol/PBS mount).
ISOLATION AND PURIFICATION OF GENOMIC DNA
Gurinder Jit Randhawa

NRC DNA Fingerprinting, NBPGR, New Delhi
For plant cells with a rigid cell wall, the disruption of cells usually requires the
tissue to be ground using a pestle and mortar in liquid nitrogen. The powdered plant
tissue is then transferred to an extraction buffer that contains detergent to disrupt the
membranes. Cetyltrimethyl ammonium bromide (CTAB) is commonly used for this
purpose. The extraction buffer also contains a reducing agent (β- mercaptoethanol) and a
chelating agent (ethylenediamine tetraacetic acid, EDTA). This helps to inactivate
nucleases that are released from the plant cell and can cause serious degradation of the
genomic DNA. Keeping the reactions cold, when possible can minimize their effects.
Phenolic compounds may also be released on disruption of plant tissues and these may
interfere with subsequent uses of the DNA (e.g. if it is to be used in the PCR). Polyvinyl
pyrolidone (PVP) can be added to the extraction buffer to remove phenolic compounds.
Phenol extraction can be used to remove any traces of proteins and the genomic
DNA can be precipitated using either ethanol or isopropanol. Precipitated DNA can be
hooked out of the solution or collected by centrifugation. It is important that DNA is not
sheared, for this reason the DNA should not be vortexed or pipetted repeatedly using a
fine tipped pipette and all manipulations should be as gentle as possible.
DNA extraction has three main steps:
1. Lysis of cell walls and membranes to release DNA into solution.

2. Purification of DNA by precipitating proteins and polysaccharides.
3. Precipitation of DNA and resuspension in a buffer.
Equipments required
1. High speed centrifuge

2. Microfuge
3. Auto-pippets 2-20μl, 20-200μl, 200-1000μl
4. Waterbath
5. -20oC Deep freezer
6. Refrigerator.
A number of published methods are available for the extraction of genomic DNA, some
of these are discussed here:
1. Saghai-Maroof et al. (1984)
Reagents
1. CTAB Buffer:
1.4 M NaCl
100 mM Tris-Cl
20 mM EDTA
2% β-Mercaptoethanol
1.5% CTAB
Adjust pH to 8.0 with HCl and autoclave before use.
2. Isopropanol
3. Saturated phenol
4. Chloroform: isoamyl alcohol (24:1) mixture
5. 10:1 TE:
10mM Tris
1mM EDTA
Adjust pH to 8.0 with HCl and autoclave before use.
6. RNase A (10mg / ml):
Dissolve RNase A in 10mM Tris-Cl, pH 7.5, 15 mM Na Cl. Heat at 100 oC
for 15 min. Cool to room temperature. Store as aliquots at -20 oC.
7. 70% ethanol
Protocol
1. Weigh 5 g of clean young leaf tissue and grind to fine powder with a pestle and
mortar after freezing in liquid nitrogen.
2. Transfer to 50 ml centrifuge tube with 20 ml CTAB buffer maintained at 60oC in
a water bath. Mix vigorously or vortex.
3. Incubate at 60oC for one hour. Mix intermittently.
4. Fill the tube with chloroform: isoamyl alcohol. Mix gently by inverting for 5 min.
5. Spin at 17,000 rpm for 10 min. with ss34 rotor in Sorval RC-5C centrifuge at
25oC.
6. Transfer aqueous phase to a fresh centrifuge tube. Add equal amount of
isopropanol and let the DNA to settle down for 20 min.
7. Spool out the DNA. If necessary we can centrifuge in the microfuge for 2
minutes. Drain out the excess chemicals with a pipette.
8. Add 0.5 ml of 70% ethanol. Mix gently and incubate for 30 min. Decant and
repeat the 70% ethanol treatment. Decant off and dry the pellet under vaccum.
9. Dissolve DNA in minimum volume of 10 : 1 TE.
10. Add RNase (0.2 ml) and incubate at 37o C for one hour.
11. Add equal volume of phenol: chloroform (1:1), mix properly and spin for 5 min.
Take out the DNA supernatant and after this perform two chloroform:isoamyl
alcohol extractions as before. Spin after each extraction.
12. Precipitate DNA by adding 1/10 volume of 3M NaOAc and 2.5 times the total
volume of chilled ethanol. Mix and spool out the DNA. Remove extra salts by
two washings with 70% ethanol. Dry under vacuum.
13. Add minimum volume of TE (10:1). Dissolve at room temperature. Store frozen
at -20o C.
2. Dellaporta et al. (1983)
Reagents
1. Extraction buffer
100 mM Tris-Cl (pH-8.0)
50mM EDTA (pH-8.0)
500 Mm NaCl
10mM β-mercaptoethanol
2. 20% SDS
3. 5M KAc
4. Resuspension buffer I
50mM Tris - Cl (pH-8.0)
10mM EDTA (pH-8.0)
5. Resuspension buffer II
10mM Tris - Cl (pH-8.0)
1mM EDTA (pH-8.0)
6. 3M NaOAc
7. Isopropanol
Protocol
1. Weigh 0.5 to 1 g of tissue. Freeze the tissue rapidly in liquid nitrogen and
grind to a powder with a pestle and mortar as the liquid nitrogen boils off.
Add a little more liquid nitrogen, if necessary, to keep the powder from
thawing while grinding. It is important not to let the tissue thaw once frozen,
until it is added to the buffer.
2. Transfer the frozen powder into a 50 ml centrifuge tube containing 15 ml of
extraction buffer (100 mM Tris- HCI pH 8.0; 50 mM EDTA pH 8.0; 500 mM
Na CI) and 10mM β-mercaptoethanol using a spatula. The tubes must be
placed in an ice bucket.
3. Add 1.0 ml of 20% SDS, mix thoroughly by vigorous shaking and incubate
the tubes at 650C for min (a water bath at 650C must be prepared ready).
4. Add 5.0 ml 5M potassium acetate working solution, shake vigorously and
incubate at 00C for 20 min (minimum time). Most of the proteins and
polysaccharides are removed as a complex with the insoluble potassium
dodecyl sulphate precipitate.
5. Balance the tubes by adding extraction buffer and spin at 15000 rpm
(20,000g) for 20 min. Pour the supernatant through a sterile small funnel
containing two layers of gauze, or mira cloth, into a clean 50ml tube
containing 10ml of cold (-20 0 C) isopropanol kept in an ice bucket. Mix
gently, by inverting at –20 0C for 30 min. A DNA clot should appear.
6. Balance the tubes by adding isopropanol or extraction buffer. Produce a pellet
of DNA by centrifuging at 15,000 rpm for 15 min. Gently pour off the
supernatant and lightly dry the pellets by inverting the tubes on paper towels
for 1-2 min. he pellet must be clear; if it is white, it will contain
polysaccharides, if dark phenolic compounds.
7. Redissolve the DNA pellet in 0.7 ml of TE 50:10 pH 8.0 at room temperature
for 20-30 min or for the time required to be sure that all the pellet is dissolved.
Help to resuspend the pellet by pipetting very gently with a blue tip, but do
not overdo it.
8. Transfer the DNA to an eppendorf tube. All centrifugation from this point in
eppendorf microfuges. Spin the tubes in a microfuge for 12 min at 12 000 rpm
to remove the insoluble debris.
9. Transfer the supernatant to a new eppendorf tube and add 75μ of 3M sodium
acetate and 500μ l of isopropanol. Mix well without vortexing and pellet the
clot of DNA by centrifuging for 1-5 min at 12,000 rpm in microfuge.
10. Discard the supernatant and save the pellet. Add 700μl cold (-200C) 80%
ethanol and dislodge the pellet from the bottom of the tube by tapping the tube
gently with your fingertips. The diluted ethanol removes salts.
11. Centrifuge for 1-5 min at 12,000 rpm. Discard the ethanol and dry the pellet
eliminate to alcohol completely. It can be dried by leaving the tubes open to
the air or by using a vacuum desiccator.
12. Add 500 μl sterile distilled deionized water or TE 10:1 pH 8.0 and maintain at
room temperature for 1h to redissolve the DNA. It may help to pipette up and
down with a blue tip, very gently1 if the DNA cannot be completely
dissolved, centrifuge at 12 000 rpm the supernatant. Discard the insoluble
debris. There is an option to stop at this point and to store the material at -
200C.
13. Add 10μl of RNase solution, incubate for at least 1.5 h at 37 0 C to ensure that
all the remaining RNA is digested.
14. Add 500μl (one volume) PCIA (phenol: chloroform: isoamyl alcohol) to
eliminate RNase and other contaminants. Shake the solution, do not vortex.
Centrifuge at 12, 000 rpm for 10 min to separate the phases.
15. Collect the aqueous phase in a new eppendorf tube, measuring the volume
recovered.
16. Add the same volume of chloroform: isoamyl alcohol. Shake the solution, do
not vortex. Centrifuge at 12,000 rpm for 10 min. to separate the phases. Any
remaining phenol is removed by this procedure.
17. Recover the upper aqueous layer in a new eppendorf tube, measuring the
volume recovered. Add 1/10 volume 3M sodium acetate and 2-3 volume of
cold 100% ethanol. Maintain for 1 h at –200C. There is an option to stop at
this point.
18. Centrifuge for 15 min at 13,000 rpm discard the supernatant. Wash the pellet
with 700μl of cold 70% ethanol to dissolve the remaining salts and dislodge
from the tube wall by tapping the tube gently with your finger tips. If the
pellet is white, salts may still be present. Try to disaggregate the pellet and
wash again with 70% ethanol at room temperature.
19. Centrifuge for min at 12,000 rpm and discard the supernatant.
20. Dry the pellet to eliminate the ethanol.
21. Dissolve the pellet in sterile distilled deionized water or TE 10:1. As the yield
should be 50-100 μg per g of tissue, dissolve in 50-100 μl to obtain a final
concentration of 1μg / μl DNA.
22. Store at -200C.
Miniprep protocol for the isolation of plant genomic DNA
a) CTAB miniprep DNA extraction protocol (Taylor and Powell, 1982)
Reagents
1. Extraction buffer: (50 mM Tris HCl pH 8.0, 10mM EDTA, 0.7M NaCl, 1.0%
CTAB and 0.1% β-mercaptoethanol)
2. Chloroform
3. 10mg/ml RNAase A
4. Isopropanol
5. 70% Ethanol
6. TE buffer: (10mM Tris-HCL pH 8.0, 1mM EDTA)
Protocol
1. Weigh out 0.05 g (dry weight) of freeze-dried tissue powder into a 1.5ml
eppendorf tube.
2. Add 500 μl of CTAB extraction buffer, pre-warmed to 600C and disperse the
lumps by inverting several times.
3. Incubate at 600C for 15 min with occasional mixing by inversion.
4. Add 500 μl of chloroform and mix by inverting until an emulsion is formed.
5. Centrifuge at 13,000 rpm in a bench top centrifuge for 10 min at room
temperature.
6. Place the aqueous phase in a fresh eppendorf tube and mix with 2.5 μl
RNAase A mix gently and incubate at 370 C for 30 min.
7. Transfer the aqueous phase, which should be clear, to a fresh tube and add 0.6
volumes of isopropanol, mix by inversion to precipitate the DNA. Wash the
pellet with 70% ethanol.
8. Resuspend the pellet in 25 μl of 1 x TE.
b) Isolation of genomic DNA for PCR (Edwards et al. 1991)
Reagents
1. Extraction buffer (200mM Tris HCl pH 7.5, 25 mM EDTA, 250 mM NaCl, 0.5%
SDS)
2. Isopropanol
3. 1x TE buffer (10 mM Tris-HCl pH 8.0, 1mM EDTA)
Protocol
1. Remove leaf samples (a few mg of leaf tissue will be sufficient) in a 1.5 ml

eppendorf tube. Leaf samples may be collected using the lid of the tube to punch a
disc of leaf material straight into the tube. This not only ensures uniform sample
size but is also reduces contamination.
2. Grind the samples in the original eppendorf tube at room temperature, without
buffer, for 15 to 20 sec, or until liquid exudes from the issue.
3. Add 400μl of extraction buffer and mix the samples for 5sec. At this stage
samples may be left at room temperature until they have all been processed.
4. Centrifuge the samples at full speed (13,000 rpm) for 5 min at room temperature.
5. Remove 300 μl of the aqueous fraction, place in a fresh eppendorf tube and add
300 μl of isopropanol.
6. Mix the samples and leave at room temperature for 30 to 60 min before
centrifuging at full speed for 10 min.
7. Remove all of the supernatant from the pellet and vacuum dry for about 20 min.
8. Dissolve the pellet in 100μl of 1x TE.
9. Use 1μl of the above for the usual 25μl PCR.
Quantification of DNA
DNA quantification is an important step to know the amount of isolated DNA.
DNA Quantification by UV Spectroscopy
1. Take 5μl of the DNA sample in a quartz cuvette. Make up the volume to 1ml with
distilled water.
2. Measure absorbance of the solution at wavelengths 230, 260, 280 and 300nm.
3. Calculate the ratios A230 / A260 and A280 / A260.
4. A good DNA preparation exhibits the following spectral properties: A300< 0.1
O.D. units A230 / A260 < 0.45 O D units A280 / A260 < 0.55) O D units.
5. Calculate DNA concentration using the relationships for double stranded DNA, 1
O D at 260 nm = 50μg / ml. This estimate is influenced by the contaminating
substances like RNA and very low molecular weight DNA in the solution.
6. Prepare a working stock of samples of about 100μl with concentrations of
10 ng/μl.
DNA Quantification by Fluorimetry
The DNA extraction procedures do not eliminate RNA. Therefore, the estimatied
DNA concentration by UV spectrophotometry may not be very accurate. RNase
treatment may help in reducing the errors. However, fluorimetric estimations are more
reliable as it measures the fluorescence emitted by the double stranded DNA- Hoechst
33258 dye complex, which is directly proportional to the amount of DNA in the sample.
Since Hoechst 33258 dye does not bind to single stranded DNA and very small fragments
of DNA this procedure gives more reliable estimates of the DNA concentrations in the
sample.
Reagents
1. 10x TNE (1000ml, buffer stock solution)

100mM Tris
1M NaCl
10 mM EDTA
(Dissolve in 800 ml distilled water. Adjust pH to 7.4 with HCl. Add distilled
water to make volume 1000ml. Filter and Autoclave before use. Store at 40C for
up to 6 months in an amber bottle.)
2. Hoechst 33258 dye stock
Hoechst 33258: 1 mg /ml in distilled water.
(Add 10 ml distilled water to 10 mg H33258. Do not filter. Store at 40C for up to
6 months in an amber bottle).
Protocol
1. Prepare the assay and DNA standard solutions as described below:

DNA assay solution
Low range (A):
(10-500 ng/ml final DNA concentration)
H33258 stock solution: 10.0 μl
10x TNE buffer: 10.0 ml
Distilled filtered water: 90.0 ml
High range (B):
(100-500 ng/ml final DNA concentration)
H33258 stock solution: 100.0 μl
10x TNE buffer: 10.0 ml
Distilled filtered water: 90.0 ml.
2. Turn on the fluorimeter (Hoefer) at least 15 min before using.
3. Zero the instrument: Prepare an assay blank using 2 ml of appropriate assay
solution (A or B for high DNA concentration). Dry the sides of a cuvette. Insert
the cuvette into the well, close the lid, and press < ZERO>. After “0” displays,
remove the cuvette.
4. Calibrate the instrument: Deliver 2 μl of the appropriate DNA standard solution
(low or high range) to 2 ml of assay solution in the cuvette. Mix by pipetting
several times into a disposable transfer pipette. Place cuvette in well, close the lid
and press <CALIB>. Enter 100 for the low range assay, 1000 for the high range
assay and press < ENTER>. After the entered value displays, remove the cuvette.
5. Zero the instrument: Empty and rinse the cuvette. Dry by draining cuvette and
blot in upside down on a paper towel. Add 2 ml of the same Assay Solution used
in step 2, insert the cuvette into the well, close the lid, and press <ZERO>. After
“0” displays remove the cuvette.
6. Measure the sample and mix well. Place the cuvette in the well, close the lid, and
record the measurement.
7. Measure subsequent samples. Repeat steps 5 and 6 for each sample.
References
Dellaporta, S.L. Wood, J. and Hicks, J.B. 1983. A plant DNA mini preparation:
Version II. Plant Molecular Biology Reporter 1: 19-21.
Edwards, K.J., Johnstone, C and Thompson, C.1991. A rapid and simple method for
the preparation for plant genomic DNA for PCR analysis. Nucleic Acids Research 19,
No.6: 1349.
Saghai-Marrof, M.A, Soliman, K.M., Jorgensen, R.A. and Allard, R.W. 1984.
Ribosomal DNA spacer-length polymorphism in barley: Mendelian inheritance,
chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81: 8014-
8018.
Taylor, B. and Powell, A. 1982. Isolation of Plant DNA and RNA. Focus 4 : 4-6.
1. Isolation of P.falciparum DNA
If material from P. falciparum cultures is used, then there is no problem with

contaminating DNA from human cells. However, if material from patients is used (blood
samples) then DNA contamination with human DNA is unavoidable. For most applications,
for example, PCR and DNA hybridization, this can be ignored, but if, for example, genomic
libraries are to be produced from uncultured material, this poses a serious problem. The DNA
content of a human cell is approximately 200 times that of a ring stage parasite and thus
Plasmodium DNA might be highly underrepresented in a given sample. Separating
lymphocytes from the blood sample by buffy-coat elimination or density-gradient
centrifugation will substantially reduce the amount of human DNA but will never eliminate
human DNA completely.
Materials and Methods

Equipments: Bench-top centrifuge, Microfuge, Water bath or heating block, Pipettes, Gel-
electrophoresis equipment.
Reagents
1. Phosphate-buffered saline (PBS): 11.5 g Na2HPO4, 2.96 g NaH2PO4·2H2O, 5.84 NaCl
per liter.
2. PBS with 0.05% (w/v) saponin (Sigma 8047-15-2).
3. Tris-EDTA (TE) buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 7.6.
4. Tris-borate-EDTA (TBE)-buffer: 1 M Tris-HCl, 0.8 M boric acid, 2 mM EDTA, pH 8.0.
5. Proteinase K: 20 mg/mL in 10 mM Tris-HCl pH 8.0.
6. Sodium dodecyl sulfate (SDS): 20% (w/v) in H2O.
7. TE-saturated phenol (pH 8.0).
8. Chloroform.
9. 3M sodium acetate, pH 4.5.
10. Ethanol, 99 and 75%.
11. DNA-grade agarose.
12. DNA-size-marker (e.g., 1-kb ladder; Gibco-Life Technologies).
13. Ethidium bromide.
14. Blood samples, culture material.
Method
1. Transfer cultured material to a sterile tube and sediment erythrocytes by centrifugation at
800g for 5 min.
2. Discard the supernatant and add 5 pellet-volumes of ice-cold PBS with 0.05% (w/v)
saponin. Mix and leave for 10 min on ice.
3. Pellet parasites for 5 min at 4000g, Discard the supernatant and wash pellet twice with
PBS.
4. Resuspend the pellet after the last wash in 1/100 of original culture volume TE.
5. Add proteinase K to a final concentration of 20 µg/mL followed by 20% SDS to a final
concentration of 0.5 %; carefully mix by inverting the tube slowly & Incubate mixture for 2–
12 h at 56–65°C.
6. Add 1 vol of TE-saturated phenol and 1 vol of chloroform & Invert tubes for approx 10
min until a complete mixture of the organic and aqueous phase occurs. Do not vortex & Spin
at 15,000g for 10 min.
7. Transfer the upper aqueous phase into a new tube and repeat the phenol– chloroform step.
8. Spin at 15,000g for 10 min.
9. Transfer the upper aqueous phase into a new tube, add 1 vol of chloroform, and mix both
phases carefully for 5 min & Spin at 15,000g for 5 min.
10. Transfer aqueous phase to new tube and add 1/10 vol of 3 M Na acetate, pH 4.5 and 2.5
vol of 99% ethanol.
11. Invert tube several times to mix solution completely & freeze at –20°C for at least 1 h.
12. Spin at ~15,000g for 10 min; discard the supernatant & add 500 –1000 µL of 70% ice-
cold ethanol.
13. Quickly vortex the tube & Spin at ~15,000g for 5 min & Discard the supernatant and air-
dry tube.
14. Dissolve dried pellet in TE to an approximate concentration of 0.5–1 mg/mL DNA.
15. Check the quality of DNA by running 0.5–1 µg of the preparation on a 0.5% agarose gel.
The DNA should give a broad but distinct band at approx 20 kb. The smear below the band
should not extend further down than 10–15 kb. Store DNA at –20°C.
2. Isolation of genomic DNA from falciparum

Schizont-infected erythrocytes of P. berghei, P. yoelii and P. falciparum were lysed
with tris pH 8.8 followed by centrifugation at 15,000 rpm (JA-20 rotor Beckman) for 10
minutes to obtain Schizont pellets. The pellets were then homogenized in 600u1 TES buffer
(10mM Tris-HCL p117.6, 50 mM EDTA pH8.0, 0.1 % SDS) containing 1mg/ml proteinase
K. Lysed erythrocytes were then incubated in a 37ºC water bath for 30 minutes. 500µl phenol
chloroform (1:1) mix was used to extract schizont DNA and the DNA precipitated in 70%
ethanol and resuspended in distilled water. Isolated DNA was separated by agarose gel
electrophoresis using a 0.7% agarose gel, visualized using ethidium bromide. Isolated DNA
was used as templates for PCR. DNA concentration was obtained using Beckman DU Series
500 Spectrophotometer.
3. DNA isolation from Plasmodium falciparum
Small-scale genomic DNA isolation from Plasmodium falciparum
Equipment: centrifuge (4 °C), speed-vac, agarose electrophoresis unit, spectrophotometer.

Materials and Reagents:
• Infected erythrocytes (a few mL with about 10% parasitemia)
• Phosphate-buffered saline (PBS) (pH 7.2)
• 5% saponin solution
• Lysis buffer: 40 mM Tris-HCl (pH 8.0), 80 mM EDTA (pH 8.0), 2% SDS, 0.1
mg/mL
• Proteinase K (Add the proteinase K just before using the buffer.)
• Phenol equilibrated with 0.1 M Tris–HCl (pH 7.0)
• Chloroform
• RNase
• 3 M Sodium Acetate (pH 5.0) use (CH3COONa.3H2O)
• Ice cold Absolute ethanol
• 70% ethanol
• TE-buffer: 10 mM TrisHCl (pH 8.0) 1 mM EDTA (pH 8.0)
Procedure:
1. Centrifuge the infected erythrocytes at 3,000 ´ g for 2 min. Wash the cells once in
cold PBS.
2. Resuspend the cells from one microfuge tube (1.7 mL) in 1 mL of PBS.
3. Add and gently mix 10 mL of 5% saponin (for a final concentration of 0.05%).
4. Immediately centrifuge the tube at 6,000 ´ g for 5 min after lysis is observed &
remove supernatant.
5. Add 25 mL of lysis buffer and 75 mL of distilled water to the pellet & incubate the
tube at 37 °C for ~3 h with intermittent stirring by hand.
6. Add 100 mL of distilled water, then 200 mL of phenol. Mix well and centrifuge at
2,000 g for 8 min.
7. Extract likewise with 200 mL of chloroform & add 2 mL of RNase-It cocktail for 30
min at 37°C.
8. Extract with phenol and chloroform as above.
9. Precipitate the genomic DNA by adding one-tenth volume of sodium acetate and 2.5
volumes of ice cold absolute ethanol; allow the tube to sit for 2 hours or overnight at -
20 °C. The DNA can be stored this way as well.
10. Centrifuge the precipitate at 2,000 ´ g for 30 min at 4 °C, wash it gently once with
70% ethanol, dry it in a speed-vac, and gently resuspend the pellet in 25 to 100 mL of
distilled water, depending on its size.
11. Determine the DNA concentration at OD260; 2 mL of culture may yield ~2 mg of
genomic DNA. Visualize ~0.5 mg on a 0.8% agarose gel to see that the genomic
DNA runs as a high-molecular weight, somewhat broad band and is thus unsheared
and free of RNA.
4. Preparation of P. falciparum genomic DNA (A-Phenol/Chloroform precipitation)
Material and Reagents:

• 3M Na Acetate pH5.0
• 95% and 70% ethanol
• TE-buffer
• 18% SDS
• phenol/chloroform
• chloroform
Procedure:
1. Spin culture at 1200 rpm for 5 minutes to pellet parasitised red blood cells and remove
supernatant.
2. Gently resuspend pellet in 4 volumes of buffer A (1.6 mL).
3. Add 1 volume of 18% SDS (0.4 mL) and mix thoroughly then let sit for 2-3 minutes.
4. Add 8 volumes of phenol/chloroform (2.4 mL) and mix thoroughly
5. Spin at 3500 rpm for 10 minutes.
6. Remove aqueous phase into a clean corex tube and ethanol precipitate by adding 1/10
volume of 3M Na Acetate pH5.0 (250 µL) and 2.5 volumes of ethanol (6.5 mL).
7. Leave at -20 °C for at least 1 hour, but the preparation could be stored overnight at -20 °C
at this stage.
8. Pellet DNA by spinning at 10,000 rpm for 10 minutes & drain off the ethanol.
9. Dissolve DNA in 600 µl of TE and transfer to an Eppendorf tube.
10. Extract twice with phenol/chloroform by adding 600 µL of phenol/chloroform,
mixing and spinning at 4,000 rpm for 3 minutes then removing aqueous phase to a clean
tube.
11. Extract once with chloroform by adding 600 µL of chloroform, mixing and spinning
at 4,000 rpm for 3 minutes then removing aqueous phase to a clean tube.
12. Ethanol precipitate DNA by adding 50 µL of 3M Na Acetate pH5.0 (250 µl) and 1 mL
of ethanol.
13. Leave at -20 °C for at least 1 hr, but the preparation could be stored overnight at -20
°C at this stage.
14. Pellet DNA by spinning at 10,000 rpm for 10 mins in a microcentrifuge.
15. Remove supernatant and wash DNA pellet with 1 mL of 70% ethanol
16. Dissolve DNA in 50 µL TE and store at 4 °C.
17. Run 2 µL on a 1% agarose gel to check.
Isolation of IgY from the yolks of eggs by a chloroform polyethylene
glycol procedure
Introduction:
By shaking a dilute suspension of egg yolk with chloroform followed by low
speed centrifugation (1500 g for 30 min) the water soluble proteins which include
chicken IgG (IgY) separate from the emulsion of chloroform and lipophilic
substances. The IgY may then be separated from the associated water soluble
proteins by precipitation with 12% polyethylene glycol Mr 6000. The method called
the chloroform - polyethylene glycol procedure was compared with the polyethylene
glycol procedure which is currently being used. It was found that the chloroform -
polyethylene glycol method yielded 2.57 times more IgY than the conventional
polyethylene glycol method.
Requirements:
• Phosphate Buffered Saline (50 mM, pH 7.5)
• Chloroform
• Poly Ethylene Glycol 6000 Carbowax.
• Egg Yolk
Procedure:
• Yolk was separated from egg white and washed with sterile water once.
• To the yolk 50 mM PBS (40 ml/yolk) was added and stirred in a magnetic
stirrer for 40 min at Room temperature.
• Now add chloroform (10 ml/yolk) was added and stirring was continued for
another 30 min.
• Centrifuge the solution at 10000 rpm for 30 min; Repeat the centrifugation to
get clear supernatant.
• To the supernatant add PEG 6000 at 14% (w/v) one stir on magnetic stirrer for
30 min at RT.
• Centrifuge at 10000 rpm for 30 min at 40C.
• Discard supernatant and dissolve the pellet in minimum 50 mM PBS (5
ml/yolk)
• Freeze them in aliquots for further use.
Isolation of DNA
Aim: To isolate DNA from chiccken liver.
Principle: Extraction of DNA is accomplished by the rupturing of cell wall,

wall cell and nuclear
membrane followed by deproteinization and precipitation of the nucleic acid using ethanol.
Materials Required:
1. Extraction buffer: 100mM Tris-Cl

Tris Cl buffer containing 0.15 M NaCl, 0.1MEDTA, 2.5% 2
SDS pH 7.8.
2. Chloroform and isoamyl alcohol mixture 24:1.
3. 3M sodium acetate
4. Tris-EDTA
EDTA buffer: 10 mM Tris-Cl
Tri pH 7.5, 1mM EDTA.
5. Absolute alcohol, 70% ethanol, Sample, Glass wares,, centrifuge, water bath etc.,
Procedure:
1. Grind 0.5gm of the chhicken liver in 0.5 – 0.75 ml of extraction buffer in a mortar and
pestle.
2. Transfer the homogenized sample in to a micro centrifuge tube and incubate at 65 °C
for 30 min.
3. After incubation centrifuge
centri at 3000 rpm for 10 min at room temperature.
4. Collect the supernatant in to another micro centrifuge tube. To this,this add an equal
amount of chloroform – isoamyl alcohol mixture and shake the contents.
con
5. Centrifuge the contents at 8000 rpm for 10 min at room temperature.
temp ture.
6. After centrifugation, carefully pipette out the upper clear aqueous phase from the
coagulated protein emulsion at the interface in to a fresh tube.
7. To this, add 1/20th volume of 3 M sodium acetate and double volume chilled
absolute ethanol and swirl gently to precipitate DNA. White fibrous DNA appears in
the tube. For further precipitation incubate the tube at -20°C
20°C for 30 – 60 min.
8. Centrifuge the contents for 10 min at 8000 rpm, discard the supernatant and air dry
the DNA pellet. Wash the DNA with 70 % ethanol twice.
9. Suspend the pellet in 100 µl TE buffer and store at -20°
20° for future use.
Result: DNA is isolated from plant source, which is observed as white fibrous material.
Flow chart:
0.5 g chicken liver
0.5 – 0.75 ml of extraction buffer
1
Homogenize and incubate at 65 °C for 30 min
Centrifuge the homogenate at 3000

300 rpm for 10 min
Transfer the supernatant in to a fresh tube
Add equal volume of Chloroform-isoamyl

Chloroform alcohol mixture, mix and centrifuge at 8000
800 rpm
for 10 min
Collect the upper clear supernatant in to fresh tube and add 1/20th volume of sodium
acetate and double volume of chilled absolute ethanol and swirl gently
Fibrous White DNA appears
Keep the tube at -20°C for 30-60 min for further precipitation
Centrifuge 8000 rpm for 5 min; discard the supernatant and air dry the pellet
Wash in 70% ethanol (1-2

2 times) and suspend in 100 µl TE buffer and preserve for future use
2
Isolation of DNA
Aim: To isolate DNA from plant source.
Principle: Extraction of DNA is accomplished by the rupturing of cell wall,

wall cell and nuclear
membrane followed by deproteinization and precipitation of the nucleic acid using ethanol.
Materials Required:

Tris Cl buffer containing 0.15 M NaCl, 0.1MEDTA, 2.5%
2
SDS pH 7.8.
4. Tris-EDTA
EDTA buffer: 10 mM Tris-Cl
Tri pH 7.5, 1mM EDTA.
5. 70% ethanol, Sample, Glass wares, centrifuge, water bath etc.,
Procedure:
1. Grind 0.5gm
gm of the sample material in 0.5 – 0.75 ml of extraction buffer in a mortar
and pestle.
2. Transfer the homogenized sample in to a micro centrifuge tube and incubate at 65 °C
for 30 min.
4. Collect the supernatant in to an another micro centrifuge tube. To this,
this add an equal
amount of chloroform – isoamyl alcohol mixture and shake the contents.
con
temp ture.
6. After centrifugation, carefully pipette out the upper clear aqueous phase from the
coagulated protein emulsion at the interface in to a fresh tube.
7. To this, add 1/20th volume of 3 M sodium acetate and double volume chilled ethanol
and swirl gently to precipitate DNA. White fibrous DNA appears in the tube. For
further precipitation incubate the tube at -20°C for 30 – 60 min.
8. Centrifuge the contents for 10 min at 8000 rpm, discard the supernatant and air dry
the DNA pellet. Wash the DNA with 70 % ethanol twice.
Result: DNA is isolated from plant source, which is observed as white fibrous material.
Flow chart:
0.5 g coconut endosperm
1

300 rpm for 10 min

Chloroform alcohol mixture, mix and centrifuge at 8000
800 rpm
for 10 min
Collect the upper clear supernatant in to fresh tube and add 1/20th volume of sodium
acetate and double volume of chilled ethanol and swirl gently
Keep the tube at -20°C for 30-60 min for further precipitation

2 times) and suspend in 100 µl TE buffer and preserve for future use
2
Isolation of DNA
Aim: To isolate DNA from plant source.
Principle: Extraction of DNA is accomplished by the rupturing of cell wall, cell

and nuclear membrane followed by deproteinization and precipitation of the
nucleic acid using ethanol.
Materials Required:

Tris Cl buffer containing 0.15 M NaCl,
0.1MEDTA, 2.5%
5% SDS pH 7.8.
4. Tris-EDTA
EDTA buffer: 10 mM Tris-Cl pH 7.5, 1mM EDTA.
5. 70% ethanol, Sample, Glass wares,, centrifuge, water bath etc.,
Procedure:
1. Grind 0.5gm
gm of the sample (mulberry leaf) material in 0.5 – 0.75 ml of
extraction buffer in a mortar and pestle.
2. Transfer the homogenized sample in to a micro centrifuge tube and
incubate at 65 °C for 30 min.
4. Collect the supernatant in to an another micro centrifuge tube. To this,
this add
an equal amount of chloroform – isoamyl alcohol mixture
mixtur and shake the
contents.
temp
6. After centrifugation, carefully pipette out the upper clear aqueous phase
from the coagulated protein emulsion at the interface in to a fresh tube.
7. To this, add 1/20th volume of 3 M sodium acetate and double volume
chilled ethanol and swirl gently to precipitate DNA. White fibrous DNA
appears in the tube. For further precipitation incubate the tube
tu at -20°C for
30 – 60 min.
8. Centrifuge the contents for 10 min at 8000 rpm, discard the supernatant
and air dry the DNA pellet. Wash the DNA with 70 % ethanol twice.
Result: DNA is isolated from plant source, which is observed as white fibrous
material.
Flow chart:
0.5 g Mulberry Leaf /any plant tissue
1

300 rpm for 10 min

Chloroform isoamyl alcohol mixture, mix and centrifuge at
8000 rpm for 10 min
Collect the upper clear supernatant in to fresh tube and add 1/20th volume of
sodium acetate and double volume of chilled ethanol and swirl gently
Keep the tube at -20°C for 30-60

60 min for further precipitation

2 times) and suspend in 100 µl TE buffer and preserve for
future use
2
Isolation of DNA
Aim: To isolate DNA from chicken liver.
Principle: Extraction of DNA is accomplished by the rupturing of cell wall, cell

and nuclear membrane followed by deproteinization and precipitation of the
nucleic acid using ethanol.
Materials Required:

Tris Cl buffer containing 0.15 M NaCl,
0.1MEDTA, 2.5%
5% SDS pH 7.8.
4. Tris-EDTA
EDTA buffer: 10 mM Tris-Cl pH 7.5, 1mM EDTA.
5. Absolute alcohol, 70% ethanol, Sample, Glass wares,, centrifuge, water
bath etc.,
Procedure:
1. Grind 0.5gm
gm of the silkworm midgut (or any tissue) in 0.5 – 0.75 ml of
extraction buffer in a mortar and pestle.
2. Transfer the homogenized sample in to a micro centrifuge tube and
incubate at 65 °C for 30 min.
4. Collect the supernatant in to another micro centrifuge tube. To this,
this add an
equal amount of chloroform – isoamyl alcohol mixture and shake the
contents.
temp
6. After centrifugation, carefully pipette out the upper clear aqueous phase
from the coagulated protein emulsion at the interface in to a fresh tube.
7. To this, add 1/20th volume of 3 M sodium acetate and double volume
chilled absolute ethanol and swirl gently to precipitate DNA. White fibrous
DNA appears in the tube. For further precipitation incubate the tube at -
20°C for 30 – 60 min.
8. Centrifuge the contents
ntents for 10 min at 8000 rpm, discard the supernatant
and air dry the DNA pellet. Wash the DNA with 70 % ethanol twice.
Result: DNA is isolated from plant source, which is observed as

a white fibrous
material.
Flow chart:
0.5 g Midgut tissue
1

300 rpm for 10 min

Chloroform isoamyl alcohol mixture, mix and centrifuge at
8000 rpm for 10 min
Collect the upper clear supernatant in to fresh tube and add 1/20th volume of
sodium acetate and double volume of chilled absolute ethanol and swirl gently
Keep the tube at -20°C for 30-60

60 min for further precipitation

2 times) and suspend in 100 µl TE buffer and preserve for
future use
2
Isolation of RNA
Aim: To isolate RNA from yeast.

yeast
Principle: Total
otal yeast RNA is obtained by extracting a whole cell homogenate with phenol.
The concentrated solution of phenol disrupts hydrogen bonding in the macromolecules,
causing denaturation of protein. The turbid suspension is centrifuged and two phases appear;
the lower phenol phase contains DNA, and the upper aqueous phase contains carbohydrates
and RNA. Denaturated protein, which is present in both the phases, is removed by
centrifugation. The RNA is then precipitated with alcohol.
Materials Required:
1. Dried Yeast.
2. Phenol solution: 900g/litre.
3. Potassium acetate: 200g/litre, pH 5.
4. Absolute ethanol.
5. Diethyl ether.
6. Glass wares,, centrifuge, water bath etc.,
Procedure:
1. Suspend 0.15 g of dried yeast in 0.60 ml of warm (37 ºC) water and incubate in an
water bath for 15 min at 37 ºC.
2. Then add 0.8 ml of concentrated phenol solution and stir the suspension mechanically
for 30 min at room temperature, then centrifuge at 3000g for 15 min in cold (5 ºC) to
break the emulsion.
3. Carefully collect the upper aqueous layer withwith a Pasteur pipette and centrifuge at
10000 g for 5 min in cooling centrifuge to sediment denatured protein.
ation collect supernatant into a fresh tube and add 1/10th volume of
4. After centrifugation
potassium acetate and two volumes of ethanol. Cool the solution
solution in ice and leave to
stand for one hr.
5. Collect the precipitate by centrifuging at 2000 g for 5 min at 5 C.
6. Wash the RNA with ethanol: water (3:1), ethanol thanol and finally ether; air dry and
preserve for future use.
Result: RNA is isolated from yeast, which is observed as white fibrous material.
Flow chart
0.15 g yeast granules
0.6 ml of warm water and incubate at 37 ºC for 15 min
1
Add 0.8 ml of concentrated phenol solution and stir the suspension mechanically for
30 min at R T
Centrifuge at 3000g for 15 min in cold (5 ºC) to break the emulsion.
Carefully collect the upper aqueous layer with a Pasteur pipette and centrifuge at
10000 g for 5 min at 5 ºC
Collect supernatant into a fresh tube and add 1/10th volume of potassium acetate and
two volumes of ethanol.
Collect the precipitate by centrifuging at 2000 g for 5 min at 5 ºC.
Wash the RNA with ethanol: water (3:1), ethanol

thanol and finally ether; air dry and
preserve for future use.
2
APPENDIX
Kits, Buffers and Reagents:

All the chemicals, media and reagents used were of Molecular Biology grade.
1M Tris: 12.11 g of Tris-base was dissolved in 80 ml water. Desired pH was adjusted
with the help of concentrated HCL. The final volume was adjusted to 100 ml with the
help of MQ water; pH was adjusted as per requirement and was sterilized by autoclaving.
0.5 M EDTA (pH 8.0): 18.61 g of Disodium salt of Ethylene diamine tetra acetic acid 2
H2O was added to 80 ml water. Stirred vigorously on a magnetic stirrer. NaOH was used
to adjust the pH to 8.0. It was sterilized by autoclaving after making the volume to 100
ml.
5M NaCl: 29.22 g of sodium chloride was dissolved in 80 ml of water, volume raised to

100 ml with water and sterilized by autoclaving.
Protienase K: 0.15 g of powdered proteinase K was dissolved in 10 ml of autoclaved MQ

water. It was filter sterilized.
10X Sera Lysis Buffer (50 ml):

1M Tris pH 8.0 = 0.5 ml
0.5 M EDTA = 1.0 ml
5.0 NacL = 1.5 ml
MQ H2O = 47.0 ml
TE buffer:
10mM Tris (pH 8.0)
1mM EDTA (pH 8.0)
10X TBE (per litre):

Tris = 121g
Boric acid = 61.8 g
EDTA = 9.3 g
Gel Loading buffer:
0.25% bromophenol blue
40% (w/v) Sucrose in water
Proteinase K buffer
1M Tris HCl (pH 7.4) 10 uL
1M CaCl2 2 uL
MQ H2O 988 uL
Equilibrated Phenol: Phenol was melted at 68oC. 8-Hydroxyquinoline was added to make
the final concentration at 0.1%. Then several times extraction was carried out with equal
volumes of 0.1 M Tris (pH 8.0) till pH 7.5-7.8 was reached. Finally two changes in
10mM Tris and 1mM EDTA (pH 8.0) was given and stored at 4oC after the last change.
Ethidium Bromide: 5 mg/ml stock solution was made in water.
20% Sodium Dodecyl sµlphate (20% SDS): 20 gm of SDS was dissolved in 90 ml of

water. Heated at 68oC to assist dissolution. pH was adjusted to 7.2 by adding 1-2 drops of
concentrated HCl. Final volume was adjusted to 100 ml.
Kits:
enzyme immuno assay kits for HAV IgM , Anti-HCV, anti HEV IgM was procured
from the Immuno vision US, General Biologicals Corp.Taiwan,Wantai,Chaina
respectively
Gel extraction, and PCR purification kit, DNA extraction kit was procured from Qiagen.
ELIZA Kit for 8-hydroxy-2-deoxy Guanosine estimation procured from Cayman,US..
Molecular biology reagents:

Taq DNA polymerase-PCR amplification and Sequencing kits were used from Perkin
Elmer, Promega and MBI Fermentas , Germany.
PCR purification and Gel extraction kits were obtained from Qiagen.,Germany
DNA ladders and PCR markers were bought from Promega and MBI Fermentas,
Germany
Restriction enymes were procured from MBI Fermentas ,Germany
dNTP,10X PCR buffer, 25mM Mgcl2 were procured from MBI Fermantus
Ligation Cloning Calculations
Prepared by Ms Alex Aitken
Theory and Example
For ligation of 5’ or 3’ over-hang restriction fragments or A-tail reactions, molar ratios of

INSERT:VECTOR need to be (2:1).
For plasmid ligations a maximum concentration of DNA per ligation reaction should be 500ng.
To calculate ng of insert to include in the ligation reaction:
{ [ng of vector] x [kb size of insert] } / { kb size of vector } x ( insert:vector ratio)

= ng of insert required
E.g., for 166bp insert with 3.956kb plasmid and 10ng of vector in the reaction
{ [10ng x 0.166] / 3.956} x (2/1) = ng of insert
= 0.839ng of insert
To check the calculation above:
INSERT = 166bp
VECTOR = 3956bp
3956/166 = 24
For an equimolar ratio i.e., 1:1 INSERT: VECTOR, then you will need 24 times more weight of
plasmid.
1:24
For a molar ratio of 2:1 INSERT:VECTOR (most common for a ligation reaction), then you
need a weight ratio of 2:24 or 1:12
If the vector concentration is 10ng/μl

If you need x 12 weight of plasmid to insert, then:
12.5/12 = 0.83333ng/μl of insert.

8/19/2015 Making miniprep solutions for spin columns Molecular Biology
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Making miniprep solutions for spin columns ► Spin Column
► Plasmid DNA Extraction
Now that we can reuse the columns... (May/04/2007 ) ► Solution
Pages: Previous 1 2 3 Next
The plot thickens... This paper from 1999 by the same people who did the regeneration of DNA columns paper
describes another way of doing plasmid extraction using silica. Similar principle to the alkaline lysis process but a bit
more optimised for silica binding. Anyway:
QUOTE
Binding of DNA to silica requires the presence of high salt concentration. We used NaCl rather than NaI
(Geneclean
II, BIO 101), guanidium thiocyanate (4), or sodium perchlorate (3) to facilitate DNA binding to silica.
We tested varying concentrations of NaCl ranging from 1 to 4 M and found that 2 M and above allowed maximal
binding. Importantly, use of NaCl instead of the chaotropic salts to promote DNA binding to silica offers the
advantage that NaCl is inexpensive and the solution is stable at room temperature. Moreover, when compared
in parallel, NaCl coeluted the least quantity of bacterial RNA than NaI or guanidium thiocyanate.
Reference number 4 is the Boom paper (source of the silicabinding invention). I just read it and they didn't even try
NaCl! They assumed that a chaotropic agent was required and only tried GuSCN. If only I had time to experiment
with these things...
Zouden
Mini Spin Columns
epochlifescience.com
$95/250 pcs, With Buffer Recipe
for PCR/Plasmid/Gel
Extraction/DNA/RNA
sometimes, we forget that the basis for this site is PROTOCOLS
if you go to the 'protocol' link at the top of the page, dig till you find the ones with plasmid prep solutions. that's where
I got my recipes for my own P1, P2, and P3.
incidentally, back in the old days they put the recipes at the back of the manual. P3 was always KoAc/HoAc; the G
Cl is a newer addition
aimikins
Buffer P3 is for regular miniprep, not for spin columns. For spin columns you use buffer N3 which is a 'secret'
although not a very wellkept one.
Have you used buffer P3 (KoAc/HoAc) in a spin column? Did it work, or did your DNA wash through the column and
not bind?
Actually, it seems like it probably would work (someone should tell Qiagen):
Biotechniques. 2000 Jan;28(1):1079. Highthroughput method for isolating plasmid DNA with reduced
lipopolysaccharide content.
Abstract:
QUOTE
Isolating plasmid DNA from bacteria is a fundamental step in molecular biology. It is often accomplished by an
alkaline lysis of bacteria and the subsequent adsorption of nucleic acids to silica oxide in the presence of
chaotropic substances. Here we show that the addition of such chaotropic reagents is not required for the efficient
DNA isolation with silica oxide. This surprising finding allowed us to purify plasmid DNA with significantly less
lipopolysaccharides (LPS), which is otherwise a common bacterial contaminant of silica oxideisolated DNA and
inhibits subsequent applications. In addition, we have implemented a precipitation step that altogether leads to a
reduction of the LPS content by a factor of 900 relative to published methods. Our novel protocol facilitates an
inexpensive highthroughput analysis of pure plasmids in a 96well format without the addition of hazardous
http://www.protocolonline.org/biologyforums/posts/26940more1.html 1/3
reagents.
Pretty interesting, don't you think?
QUOTE
We stepwise reduced the concentration of guanidine hydrochloride, but even when we excluded this chemical
altogether, we did not observe a significant drop in the yield of plasmid DNA (Table 1). We also found that
chaotropic substances are not required for DNA binding to other silicabased DNA adsorption matrixes such as
commercially available spin columns (data not shown).
The DNA binding is dependent on the presence of silica oxide and potassium acetate in the reaction (data not
shown). Possibly, the high potassium acetate concentration used to neutralize the alkaline lysis of bacteria is
sufficient to facilitate the DNA binding to silica.
Zouden
very interesting indeed.
perneseblue
no, trust me...P3 works fine with columns. there used to be only P3, when I started this. it was what you used when
doing a maxiprep or midiprep with the Qiagen columns in the mid90's. I suspect the alterations in the formula may
improve yield? but they're not necessary. I used to get pretty fantastic yield with homemade P3, and like I said, that
was when Qiagen gave you ALL the recipes in the back of the manual. at that time, they were really the only going
concern for DNA prep, so they didn't have to worry so much about people stealing all their technology
gee, I'm old
aimikins
Ahh, but the midi and maxi columns are completely different to mini columns. You're right that P3 is used for midi
and maxi columns (anion exchange resin), but according to qiagen you have to use proprietry buffer N3 for mini
columns (silica membrane). But I believe you that buffer P3 works on mini columns, it's just that when the silica
technology was invented they didn't think it would work (for some reason they never tried have a look at the Boom
paper from 1990). So the 'official' stance from Qiagen is that you have to buy buffer N3 from them if you want to use
mini silica spin columns they provide the recipes for all the other buffers *except* for N3. They also conveniently
sell bottles of N3...
So based on all this it seems the 'official stance' is wrong, and that buffer P3 will also work on mini columns, or at
least, buffer P3 + 2M NaCl will work. We could write a book called 'Molecular Biology Secrets "They" Don't Want
You to Know About'
Zouden
Could you tell me which protocol you used to regenerate the column? Is this boiling for 10 min?
Thanks,
TC
QUOTE (Zouden @ May 4 2007, 05:55 AM)
Well, I've been using the protocol mentioned in this forum to reuse miniprep spin columns. The whole process is
very easy and I get excellent results (no different to fresh columns) saving lots of money! But I'm running out of
the solutions so I've started making them. I can't do all of them so I'd like to hear your ideas.
Qiagen are kind enough to describe the composition of some of their buffers, which are presumably the same for
all brands as it's a wellestablished protocol.
Buffer P1 resuspension buffer
50mM TrisCl, pH 8.0, 10mM EDTA, 100ug/mL RNase A
Buffer P2 Lysis Buffer
200mM NaOH, 1% SDS
Buffer N3 Neutralization
Q: What is the composition of Buffer N3?
A: The composition of buffer N3 is confidential. It is a proprietary component of the QIAprep Spin Miniprep Kit.
And that's as far as I got. I know that N3 contains a chaotropic salt, probably guanidine hydrochloride, to enable
the DNA to bind to the silica, and probably acetic acid to bring the pH down. But what concentrations?
Any ideas? I've spent quite a bit of time looking around the net and couldn't find it.
thc
Buffer N3 contains 25 50% Guanidinium Chloride and 10 25% Acetic Acid according to the MSDS. Note that
MSDS contents do not necessarily list all components. Density is 1.14 g/cm3 and the pH is 4.3 @ 20Â°C.
25 â€“50% guanidinium chloride equals 2.6 â€“ 5.2 M, but letâ€™s round that off to 2.5 â€“ 5 M. .
Glacial acetic acid is 17.4 M, so 10 25% would equal 1.7 â€“ 4.4 M.
Maniatis utilizes 6 M guanidinium chloride (MW 95.53) and 0.2 M acetic acid (MW 60.05). However, 6 M
guanidinium chloride = 57.32%.
tfitzwater
I just want to write to confirm that buffer P3 standard miniprep (KoAc pH 5.5) neutralization solution does NOT
work in spin columns. The DNA will not bind.
Next I'll try P3 + 2M NaCl.
Zouden
Store the used columns in 1N HCl. Wash with H2O before next use. I regenerate maxi prep column by this method,
too.
[quote name='thc' date='Jul 16 2007, 12:06 PM' post='104788']
Could you tell me which protocol you used to regenerate the column? Is this boiling for 10 min?
Thanks,
TC
WHR
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Molarity Problems Worksheet
M = _n_ - n= # moles
V - V must be in liters (change if necessary)
- Use M or mol/L as unit for molarity
1. What is the molarity of a 0.30 liter solution containing 0.50 moles of NaCl?
2. Calculate the molarity of 0.289 moles of FeCl3 dissolved in 120 ml of solution?
3. If a 0.075 liter solution contains 0.0877 moles of CuCO 4, what is the molarity?
4. How many moles of NaCl are present in 600. ml a 1.55 M NaCl solution?
5. How many moles of H2SO4 are present in 1.63 liters of a 0.954 M solution?
6. How many liters of solution are needed to make a 1.66 M solution containing 2.11 moles of
KMnO4?
7. What volume of a 0.25 M solution can be made using 0.55 moles of Ca(OH) 2?
For all of the problems below you will need to do a mole-mass conversion. Each problem
will involve two steps.
8. What is the molarity in 650. ml of solution containing 63 grams of NaCl?
9. How many grams of Ca(OH)2 are needed to produce 500. ml of 1.66 M Ca(OH)2 solution?
10. What volume of a 0.88 M solution can be made using 130. grams of FeCl2?
Answers: (done quickly; there may be errors)
1. 1.7 M
2. 2.41 M
3. 1.2 M
4. 0.930 moles
5. 1.56 moles
6. 1.27 L
7. 2.2 L
8. 1.7 M
9. 61.5 grams
10. 1.2 L
Dilution Problems Worksheet
1. How do you prepare a 250.-ml of a 2.35 M HF dilution from a 15.0 M stock solution?
2. If 455-ml of 6.0 M HNO3 is used to make a 2.5 L dilution, what is the molarity of the
dilution?
3. If 65.5 ml of HCl stock solution is used to make 450.-ml of a 0.675 M HCl dilution, what is
the molarity of the stock solution?
4. How do you prepare 500.-ml of a 1.77 M H2SO4 dilution from an 18.0 M H2SO4 stock
solution?
Answers:
1. 39.2-ml (Put in paragraph form)
2. 1.1 M
3. 4.64 M
4. 49.2-ml
Take 49.2-ml of 18.0 M H2SO4 stock solution and pour it into a 500-ml volumetric flask.
Fill to the 500-ml line with distilled water to make 1.77M H2SO4 solution.
Extra Molarity Problems for Practice

1. How many moles of LiF would be required to produce a 2.5 M solution with a
volume of 1.5 L?
2. How many moles of Sr(NO3)2 would be used in the preparation of 2.50 L of a 3.5 M solution?
3. What is the molarity of a 500-ml solution containing 249 g of KI?
4. How many grams of CaCl2 would be required to produce a 3.5 M solution with a
volume of 2.0 L?
Answers:
1. 3.75 M
2. 8.75 M
3. 3.00 M
4. 7.8 x 102g
Buffers and Stock solutions Page 1 of 15
agarose 1%
-weigh 2 g agarose in a 500 ml bottle
-ad 200 ml 1* TAE buffer
-in the magnetron (H2) 600 W, 3 min. or autoclave 20 min 120°C
-ad 2 µl ethidiumbromide (10 mg/ml)
-keep it at 60 °C
agarose 1.5%
-weigh 3 g agarose in a 500 ml bottle
-ad 200 ml 1* TAE buffer
-in the magnetron (H2) 600 W, 3 min. or autoclave 20 min 120°C
-ad 2 µl ethidiumbromide (10 mg/ml)
-keep it at 60 °C
APS 10%
make a 10% (w/v) solution of AmmoniumPeroxodiSulfat (mw 228.2) in
milliQ (1 gram in 10 ml) and store 0.5 ml alliquots at -20°C.
CTAB 5%
CTAB (hexadecyltrimethylammoniumbromide) 5 g in 100 ml 120 mM
K2HPO4 pH 8.0
CI
chloroform + isoamylalcohol 24 : 1
EDTA 0.5 M (pH 8.0)

solve 186.1 g Na2EDTA-2H2O (mw = 372.24) in 700 ml aqua by
adjusting the pH to 8.0 with 10 M NaOH (appr. 45 ml) , add bidest to
make the volume 1 liter
Ethidium bromide
10 mg ethidium bromide in 1 ml H20
EDTA 0.5 M pH=8.0

dissolve 186.1 g Na2EDTA.2H2O in 700 ml water ,
adjust pH to 8 with 10 M NaOH (± 50 ml)
add H2O to make 1 liter
Autoclave 20 min 120°C store at room temperature
Gel-dye TTGE/DGGE
0.05 g bromophenol blue in 10 ml 1* TAE
Most_Used_Solutions.doc November 5, 2013

K2HPO4 buffer solution pH 8.0

make 120 mM K2HPO4 (MW = 174.18) 20.9 g/l
make 120 mM KH2PO4 (MW = 136.09) 16.3 g/l
ad 947 ml K2HPO4 and 53 ml KH2PO4 together , adjust the pH with
one of the solutions
Loading buffer (6*) DNA TTGE DGGE

0.05% (w/v) bromophenol blue (0.05 g)
40% (w/v) sucrose (40 g)
0.1 M EDTA pH=8 (20 ml 0.5M EDTA)
0.5% (w/v) SDS (0.5 g)
adjust volume to 100 ml
NaAc 3M
40.82 g SodiumAcetat - trihydrat (mw 136.08) in 100 ml aqua
NaOH 10 M
dissolve 400 g Sodium hydroxide (mw 40.0) in 450 ml H2O. Add H2O to
1 liter.
PCI
phenol + chloroform + isoamylalcohol 25 : 24 : 1 (put some buffer on top)
Tris-Cl 1 M (pH 8.0)

solve 121 g Tris base (mw = 121.14) in 800 ml aqua adjust the pH to 8.0
with conc.HCl (appr. 45 ml) , add bidest to make the volume 1 liter
TE (ph 8.0)
10 mM Tris-Cl 10ml/l 1M Tris-Cl pH 8.0
1 mM EDTA 2ml/l 0.5M EDTA pH 8.0
TAE buffer 50*

242.0 g Tris-base
57.1 ml Acetic Acid glacial
100 ml 0.5 M EDTA or 37.2 g Na2EDTA.2H2O
Check pH (=8) and adjust volume to 1000 ml with dH2O
Autoclave 20 min 120°C store at room temperature.
TAE running buffer 1*

Mix 20 ml 50* TAE with 980 ml H2O

Molarities and Specific Gravities of Concentrated Acids and Basesa
Molecular % by Molarity Specific 1M solution

Acid/base weight weight (approx.) gravity (ml/liter)
Acetic acid (glacial) 60.05 99.6 17.4 1.05 57.5
Ammonium hydroxide 35.0 28 14.8 0.90 67.6
Formic acid 46.03 90 23.6 1.205 42.4
98 25.9 1.22 38.5
Hydrochloric acid 36.46 36 11.6 1.18 85.9
Nitric acid 63.01 70 15.7 1.42 63.7
Perchloric acid 100.46 60 9.2 1.54 108.8
72 12.2 1.70 82.1
Phosphoric acid 98.00 85 14.7 1.70 67.8
Sulfuric acid 98.07 98 18.3 1.835 54.5
a
CAUTION: Handle strong acids and bases carefully.

1 M HCl
(TCA); however, this recipe is cheaper, easier to prepare, and just as efficient.
Ammonium hydroxide, concentrated stock solution
See Table A.2A.1.

Add H2O to 500 ml
Ammonium sulfate, saturated
76 g ammonium sulfate
100 ml H2O
Heat with stirring to just below boiling point
Let stand overnight at room temperature
ATP, 100 mM
1 g ATP (adenosine triphosphate)

12 ml H2O
Adjust volume to 16.7 ml with H2O
Store in aliquots indefinitely at -20°C
BBS (BES-buffered solution), 2x
50 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES; Calbiochem)

280 mM NaCl
1.5 mM Na2HPO4, pH 6.95
800 ml H2O
Adjust pH to 6.95 with room temperature 1 N NaOH
H2O to 1 liter
Filter sterilize through a 0.45-um nitrocellulose filter (Nalgene)
Store in aliquots at -20°C (can be frozen and thawed repeatedly)
The pH of this solution is critical (pH 6.95 to 6.98). When a new batch of 2x BES buffer is
prepared, its pH should be checked against a reference stock prepared (and tested) earlier

BSA (bovine serum albumin), 10% (w/v)
Dissolve 10 g BSA (e.g., Sigma) in 100 ml H2O. Filter sterilize using a low-protein-binding
0.22-um filter. Store indefinitely at 4°C.
Lower-concentration stock solutions (e.g., 1%), which are useful for various applications,
can be made by diluting 10% stock appropriately with sterile water.
BSA is available in various forms that differ in fraction of origin, preparation, purity, pH,
and cost; the most commonly used is fraction V. Use the form that is appropriate for the
application; this may need to be optimized empirically.
CaCl2, 1 M
147 g CaCl2·2H2O
H2O to 1 liter
Carbonate buffer
1.6 g Na2CO3 (15 mM final)

2.9 g NaHCO3 (35 mM final)
0.2 g NaN3 (3.1 mM final)
H2O to 1 liter
Adjust to pH 9.5
CAUTION: Sodium azide is poisonous; follow appropriate precautions for handling,

storage, and disposal.
CMF-DPBS (calcium- and magnesium-free Dulbecco's phosphate-buffered saline)
8.00 g NaCl (0.137 M)

0.20 g KCl (2.7 mM)
2.16 g Na2HPO4·7H2O (8.1 mM)
0.20 g KH2PO4 (1.1 mM)
H2O to 1 liter
Store at room temperature
Denhardt solution, 100x
10 g Ficoll 400
10 g polyvinylpyrrolidone
10 g bovine serum albumin (Pentax Fraction V; Miles Laboratories)
H2O to 500 ml
Filter sterilize and store at -20°C in 25-ml aliquots

DEPC (diethylpyrocarbonate)-treated solutions
Add 0.2 ml DEPC to 100 ml of the solution to be treated. Shake vigorously to dissolve the
DEPC. Autoclave the solution to inactivate the remaining DEPC.
CAUTION: Wear gloves and use a fume hood when using DEPC, as it is a suspected
carcinogen.
Many investigators keep the solutions they use for RNA work separate to ensure that "dirty"
pipets do not go into them.
Do not treat solutions containing Tris with DEPC, as Tris inactivates the DEPC.
DMEM (Dulbecco's modified Eagle medium), supplemented
Dulbecco's modified Eagle medium, high-glucose formulation (see APPENDIX 2B; e.g., Life
Technologies), containing:
5%, 10%, or 20% (v/v) FBS, heat inactivated (optional; see recipe below)
1% (v/v) nonessential amino acids
2 mM L-glutamine
100 U/ml penicillin
100 ug/ml streptomycin sulfate
Filter sterilize if anything nonsterile has been added
DMEM containing this set of additives is sometimes called "complete DMEM." The
percentage of serum used is indicated after the medium name--e.g., "DMEM/5% FBS."
Absence of a number indicates no serum is used. DMEM is also known as Dulbecco's
minimum essential medium.
Ham's F-12 nutrient mixture (APPENDIX 2B; available commercially, e.g., from Life
Technologies), is sometimes added to DMEM; the resulting medium is known as DMEM/F-
12.
Because of the higher bicarbonate content, DMEM requires ~10% CO2 to maintain pH 7.4.
Culture media containing glutamine and penicillin should be warmed to 37°C as few times
as possible since components, especially glutamine, degrade rapidly at 37°C.
DPBS (Dulbecco's phosphate-buffered saline)
8.00 g NaCl (0.137 M)

0.20 g KCl (2.7 mM)
0.20 g KH2PO4 (1.1 mM)
0.10 g MgCl2·6H2O (0.5 mM)
2.16 g Na2HPO4·7H2O (8.1 mM)
0.10 g anhydrous CaCl2 (0.9 mM)

H2O to 1 liter
DTT (dithiothreitol), 1 M
Dissolve 1.55 g DTT in 10 ml water and

filter sterilize.
Store in aliquots at -20°C.
EDTA (ethylenediaminetetraacetic acid), 0.5 M (pH 8.0)
Dissolve 186.1 g disodium EDTA dihydrate in 700 ml water.

Adjust pH to 8.0 with 10 M NaOH (~50 ml; add slowly).
Add water to 1 liter and filter sterilize.
Begin titrating before the sample is completely dissolved. EDTA, even in the disodium salt
form, is difficult to dissolve at this concentration unless the pH is increased to between 7 and
8.
Ethidium bromide, 10 mg/ml
Dissolve 0.2 g ethidium bromide in 20 ml H2O

Mix well and store at 4°C in dark
CAUTION: Ethidium bromide is a mutagen and must be handled carefully.
FBS (fetal bovine serum)
Thaw purchased fetal bovine serum (shipped on dry ice and kept frozen until needed). Store
3 to 4 weeks at 4°C. If FBS is not to be used within this time, aseptically divide into smaller
aliquots and refreeze until used. Store £1 year at -20°C. To heat inactivate FBS, heat serum
30 min to 1 hr in a 56°C water bath with periodic gentle swirling during the first 10 to 15
min to ensure uniform heating.
Repeated thawing and refreezing should be avoided, as it may cause denaturation of the
serum.
Heat-inactivated FBS (FBS that has been treated with heat to inactivate complement protein
and thus prevent an immunological reaction against cultured cells) is useful for a variety of
purposes. It can be purchased commercially or made in the lab as described above.

HBSS (Hanks' balanced salt solution)
0.40 g KCl (5.4 mM final)

0.09 g Na2HPO4·7H2O (0.3 mM final)
0.06 g KH2PO4 (0.4 mM final)
0.35 g NaHCO3 (4.2 mM final)
0.14 g CaCl2 (1.3 mM final)
0.10 g MgCl2·6H2O (0.5 mM final)
0.10 g MgSO4·7H2O (0.6 mM final)
8.0 g NaCl (137 mM final)
1.0 g D-glucose (5.6 mM final)
0.2 g phenol red (0.02%; optional)
Add H2O to l liter and adjust pH to 7.4 with 1 M HCl or 1 M NaOH
Filter sterilize and store up to 1 month at 4°C
HBSS may be made or purchased without Ca2+ and Mg2+ (CMF-HBSS). These components
are optional and usually have no effect on an experiment; in a few cases, however, their
presence may be detrimental. Consult individual protocols to see if the presence or absence
of these components is recommended.
Bottles should be kept tightly closed to prevent CO2 loss and subsequent alkalinization.
HCl, 1 M

913.8 ml H2O
HeBS (HEPES-buffered saline) solution, 2x
16.4 g NaCl
11.9 g HEPES acid
0.21 g Na2HPO4
800 ml H2O
Add H2O to 1 liter
Filter sterilize through a 0.45-um nitrocellulose filter
Store in 50-ml aliquots at -20°C
If the solution is to be used for transfection, the pH should be between 7.05 and 7.12, and
should be tested for transfection efficiency.
KCl, 1 M
74.6 g KCl
H2O to 1 liter

LB medium
Per liter:
10 g tryptone
5 g yeast extract
5 g NaCl
1 ml 1 M NaOH
Autoclave 25 min
Although the pH is adjusted to near 7 with NaOH, the medium is not very highly buffered,
and the pH of a culture growing in the medium drops as the culture nears saturation.
The medium may also contain antibiotics (e.g., 50 ug/ml ampicillin, 12 ug/ml tetracycline),
galactosides (e.g., 20 ug/ml Xgal, 0.1 mM IPTG), or other nutritional supplements added
after the medium has been autoclaved.
To make LB agar for LB plates, add 15 g/liter agare.
MgCl2, 1 M
20.3 g MgCl2·6H2O
H2O to 100 ml
MgSO4, 1 M
24.6 g MgSO4·7H2O
H2O to 100 ml
MOPS buffer
0.2 M MOPS [3-(N-morpholino)-propanesulfonic acid], pH 7.0

0.5 M sodium acetate
0.01 M EDTA
Store in the dark and discard if it turns yellow
NaCl, 5 M
292 g NaCl
H2O to 1 liter
NaOH, 10 M

Add H2O to 1 liter

10x stock solution, 1 liter:

80 g NaCl
2 g KCl
11.5 g Na2HPO4·7H2O
2 g KH2PO4
Working solution, pH ~7.3:

137 mM NaCl
2.7 mM KCl
4.3 mM Na2HPO4·7H2O
1.4 mM KH2PO4
8.00 g NaCl (0.137 M)

0.20 g KCl (2.7 mM)
0.24 g KH2PO4 (1.4 mM)
1.44 g Na2HPO4 (0.01 M)
H2O to 1 liter
PCR amplification buffer, 10x
500 mM KCl
100 mM Tris·Cl, pH 8.3 (see recipe below)
x mM MgCl2
0.1% (w/v) gelatin
Store in aliquots at -20°C
This solution can be sterilized by autoclaving. Alternatively, it can be made from sterile
water and stock solutions, and the sterilization omitted.
15 mM MgCl2 is the concentration (x) used for most PCR reactions. However, the optimal
concentration depends on the sequence and primer of interest and may have to be
determined experimentally (see APPENDIX 3).
PMSF (phenylmethylsulfonyl fluoride), 100 mM
Dissolve 0.174 g PMSF in 10 ml of 100% ethanol, isopropanol, or methanol. Store in

aliquots up to 2 years at -20°C.
CAUTION: Phenylmethylsulfonyl fluoride is toxic.
Make fresh dilutions from the alcohol stock for each use, because the half-life of PMSF in
aqueous solution is <30 min at room temperature and a few hours on ice.
If PMSF is being added to a solution without detergent, the solution should be stirred
vigorously during PMSF addition because PMSF has a tendency to form an insoluble
precipitate in aqueous solution.

Polylysine-coated tissue culture surfaces
Prepare a stock solution by dissolving 100 mg polylysine in 100 ml water (poly-L-lysine or

poly-D-lysine can be used; check specific protocol for choice of isomer) and filter sterilize
through a 0.22-um filter. Store in 5-ml aliquots at -20°C. When ready to use, dilute 1 part
stock solution with 19 parts water to prepare a 50 ug/ml working solution.
To coat culture dishes, multiwell plates, or chamber slides: Fill tissue culture dishes,
multiwell plates, or slide wells with the working solution and incubate 1 hr in a 37°C
incubator, then remove solution by vacuum aspiration and allow surface to dry.
To coat coverslips: Sterilize coverslips by autoclaving or by incubating them in 95% ethanol

and drying before coating. Place coverslips in a single layer in a petri dish containing
working solution and incubate 1 hr at 37°C. Remove coverslips using sterile forceps and
allow surface to dry.
Store coated tissue culture ware up to 3 months at 4°C. Use diluted solutions only once.
Solution B: 19.6 g potassium acetate (KC2H3O2) per liter (0.2 M) in water.
Referring to Table A.2A.2 for desired pH, mix the indicated volumes of solutions A and B,
then dilute with water to 100 ml. Filter sterilize if necessary. Store up to 3 months at room
temperature.
Solution A: 27.2 g KH2PO4 per liter (0.2 M final) in water.
Solution B: 34.8 g K2HPO4 per liter (0.2 M final) in water.
temperature.
This buffer may be made as a 5- or 10-fold concentrate simply by scaling up the amount of

potassium phosphate in the same final volume. Phosphate buffers show concentration-
dependent changes in pH, so check the pH of the concentrate by diluting an aliquot to the
final concentration.
Saponin, 10% (w/v)
Dissolve 1 g saponin in 10 ml PBS (see recipe above)

Store in 500-ul aliquots at -20°C
Once thawed, the 10% solution is stable for several months when stored at 4°C.
SDS, 20% (w/v)
Dissolve 20 g SDS (sodium dodecyl sulfate or sodium lauryl sulfate) in H2O to 100 ml total
volume with stirring. Filter sterilize using a 0.45-um filter.
It may be necessary to heat the solution slightly to fully dissolve the powder.
SDS electrophoresis buffer, 5x
15.1 g Tris base

72.0 g glycine
5.0 g SDS
Distilled, deionized H2O to 1 liter
Dilute to 1x before use
Do not adjust the pH of the stock solution; the pH is 8.3 when diluted to 1x.
Use purified SDS if appropriate.
SED (standard enzyme diluent)
20 mM Tris·Cl, pH 7.5
500 ug/ml bovine serum albumin (Pentax Fraction V)
10 mM a-mercaptoethanol
Store at 4°C for up to 1 month
Sodium acetate, 3 M
Dissolve 408 g sodium acetate trihydrate (NaC2H3O2·3H2O) in 800 ml H2O

Adjust pH to 4.8, 5.0, or 5.2 (as desired) with 3 M acetic acid (see Table A.2A.1)
Add H2O to 1 liter
Filter sterilize

Solution B: 27.2 g sodium acetate (NaC2H3O2·3H2O) per liter (0.2 M) in water.
temperature.
Solution A: 27.6 g NaH2PO4·H2O per liter (0.2 M final) in water.
Solution B: 53.65 g Na2HPO4·7H2O per liter (0.2 M) in water.
temperature.
This buffer may be made as a 5- or 10-fold concentrate by scaling up the amount of sodium
phosphate in the same final volume. Phosphate buffers show concentration-dependent
changes in pH, so check the pH by diluting an aliquot of the concentrate to the final
concentration.
SSC (sodium chloride/sodium citrate), 20x
3 M NaCl (175 g/liter)

0.3 M Na3citrate·2H2O (88 g/liter)
Adjust pH to 7.0 with 1 M HCl
STE buffer
10 mM NaCl
1 mM EDTA, pH 8.0

TAE (Tris/acetate/EDTA) electrophoresis buffer
50x stock solution:

242 g Tris base
37.2 g Na2EDTA·2H2O
H2O to 1 liter
Working solution, pH ~8.5:
40 mM Tris·acetate
2 mM Na2EDTA·2H2O
TBE (Tris/borate/EDTA) electrophoresis buffer, 10x

960 ml H2O
40 ml 0.5 M EDTA, pH 8.0 (20 mM final; see recipe above)
TBS (Tris-buffered saline)

0.9% (w/v) NaCl
10 mM Tris·Cl, pH 7.4, 7.5, or 8.0 (or other pH; see recipe below)
50 mM triethanolamine, pH ~11.5
0.1% (v/v) Triton X-100
0.15 M NaCl
Add Triton X-100 as a 10% stock sterilized by Millipore filtration and stored in the dark to
prevent photooxidation (stock is stable 5 years at room temperature). (see recipe below).

150 mM NaCl
TM buffer, 10x

100 mM MgCl2

Tris-buffered saline (TBS)
100 mM Tris·Cl, pH 7.5 (APPENDIX 2)

0.9% (w/v) NaCl (150 mM)
Tris·Cl, [tris(hydroxymethyl)aminomethane], 1 M

Filter sterilize if necessary
Store up to 6 months at 4°C or room temperature
Approximately 70 ml HCl is needed to achieve a pH 7.4 solution, and ~42 ml for a solution
that is pH 8.0.

decreasing approximately 0.028 pH units per 1°C. Tris-buffered solutions should be
adjusted to the desired pH at the temperature at which they will be used. Because the pKa of
Tris is 8.08, Tris should not be used as a buffer below pH ~7.2 or above pH ~9.0.
Triton X-100, 10% (w/v)
1 g Triton X-100
H2O to 10 ml
Stir to dissolve
Filter sterilize through a 0.45-um filter
Store protected from light up to 6 months at room temperature
TTBS (Tween 20/TBS)
Dissolve 0.1% (w/v) polyoxyethylenesorbitan monolaurate (Tween 20) in TBS (see recipe
above). Store up to several months at 4°C.

RNA Isolation for Frozen Mouse Livers and Reverse Transcription
I. Introduction
RNA is typically isolated from tissue or cells based on the procedure originally
described by Chomczynski and Sacchi in 1987. In this method tissue or cells are
disrupted in a solution containing guanidinium thiocyanate, a strong chaotropic
denaturant, which lyses cells and inactivates cellular RNases. The sample lysate was then
mixed with a sodium acetate, pH 4, water saturated phenol and chloroform:isoamyl
alcohol (49:11 v/v) solution. After subsequent cooling and centrifugation, the RNA was
removed from the aqueous phase whereas DNA and protein were present in the
interphase and phenol phase. The RNA was then precipitated with isopropanol,
reprecipitated, washed and finally solubilized in 0.5% SDS.
Recent technologies have allowed for RNA isolations based on the silica-based
filters, thus avoiding the use of organic solvents required for the extraction step. We will
be using Ambion’s RNAqueous®-4PCR Kit. After mixing the lysate with ethanol the
resulting solution is added to the silica filter that selectively binds mRNA and larger 18S
and 28S ribosomal RNAs; very small RNAs such as tRNA and 5S ribosomal RNA are
not quantitatively bound. The filter is then washed to remove DNA, protein, and other
contaminants. The RNA is then eluted in nuclease-free water containing a trace amount
of EDTA to chlelate heavy metals. To ensure that there is no trace DNA contamination
that may produce a false positive for a reverse transcriptase PCR (RT-PCR), the eluted
RNA may be treated with DNase 1. If the RNA has been treated with DNase 1, it, and
divalent cations, must be removed for further downstream applications. The
concentration of RNA is determined, after diluting the recovered RNA with T.E (10mM
Tris, 0.1 mM EDTA, pH 8), using the following equation:
[RNA], µg/mL = A260 x dilution factor x 40 µg/mL
Though yields will vary according to type and amount of sample, 1-10 µg per mg tissue
are expected. Purity of the RNA is indicated by A260/A280 ratio values in the range of 1.8 -
2.1.
Though freshly dissected tissues give the best yields, tissues treated with a tissue
storage/RNA stabilization solution, such as Ambion’s RNAlaterTM can be used with very
good results. Additionally, as RNases are much more resilient than DNases, special care
must be made to ensure an RNase free work environment. Thus, RNase decontamination
solutions such as Ambion’s RNaseZap® will be used to inactivate RNases on work
surfaces and on equipment.
As PCR requires a DNA template to amplify DNA with DNA polymerases, RNA
cannot be used directly as a template. With the discovery of the enzyme reverse
transcriptase in the late 1980s, it is now possible to copy RNA into its complementary
DNA sequence (cDNA). The process of subjecting RNA to reverse transcription followed
by PCR is commonly known as RT-PCR and can be used to generate inserts for cloning
into plasmid vectors, to make templates for in vitro transcription, or to assess the
mutational status of expressed sequences. There are a couple of ways in which to
generate cDNA with the reverse transcriptase; one with oligo (dT)18 primers that
correspond to the 3’ poly(A) tails of mRNA and the other with random decamers which
is useful if the mRNA template is partially degraded and lacking the poly(A) tails.
Appropriate controls are required to detect the presence of genomic DNA in the RNA
isolation.
II. Materials
1. Frozen mouse liver, ~ 75 mg
2. Ambion Ambion’s RNaseZap®
3. Ambion RNAqueous®-4PCR Kit containing various components
4. Kinematica AG Polytron PT1200CL tissue homogenizer with a 7 mm

generator
5. Digital Pipets and tips
6. 100 % EtOH
7. T.E Buffer: (10mM Tris, 0.1 mM EDTA, pH 8)
8. 37oC, 44oC, 80oC and 92oC water baths

9. Tabletop Centrifuge: Eppendorf Model 5415C
10. Microfuge tubes: 2.0 mL and 1.5 mL capped polypropylene tubes
11. Beckman DU-640 spectrophotometer and UV microcuvettes
12. Ambion RETROscriptTM First Strand Synthesis Kit for RT-PCR
13. Ambion SuperTaqTM recombinant thermostable DNA polymerase, 5 U/µL
14. Thermal Cycler: Eppendorf Mastercycler gradient
15. Agarose: Cambrex Bio Sciences SeaKem LE agarose
16. 1X TBE Buffer: 90 mM Tris, 90 mM boric acid, 0.2 mM Na2EDTA, pH ~8.0
17. Gel Star nucleic acid gel stain: BioWhittaker Molecular Applications
18. Nanofuge: National Labnet Co. Model C-1200 mini centrifuge
19. DNA MW standard: New England Biolabs 100 – 1000 bp, 100 bp step ladder
20. Horizontal gel electrophoresis apparatus: Fotodyne
21. 10X Gel loading buffer: 80% (v/v) glycerol, 100 mM Na2EDTA, 1% (w/v)
SDS, 0.1% (w.v) bromophenol blue, 0.1% (w/v) xylene cyanol FF
III. Methods
1. Wear gloves for the entire procedure.
2. Obtain a frozen mouse liver sample, previously treated with Ambion’s

RNAlaterTM, from your instructor; the sample has been pre-weighed and the
mass written on the 2.0 mL microfuge tube. Record the mass.
3. Estimate the tissue volume and add 10-12 volumes of the Lysis/Binding
Solution; consult with your instructor
4. Carefully, homogenize the tissue with the Polytron homogenizer.
5. Centrifuge the lysate at 11,000 rpm for 1 min to clarify the lysate. Carefully
transfer the supernatant to a clean sterile 2.0 mL microfuge tube. Estimate the
volume and add an equal volume of 64% ethanol. Mix thoroughly by vortex
mixing.
6. Pipet no more than 700 µL of the lysate/ethanol mixture onto the RNAqueous
Filter Cartridge in a collection tube. Centrifuge at 13,000 rpm (do not exceed
this limit as filter can become damaged!) until the lysate/ethanol solution
has passed through the filter (1 min, possibly more); discard the eluate and
reuse the collection tube. Repeat this process with the remainder of the
lysate/ethanol mixture with the same filter (do not exceed 700 µL); discard
the eluate. If needed repeat with the remainder of the lysate/ethanol solution;
discard the eluate and reuse the collection tube.
7. Apply 700 µL of Wash Solution #1 to the filter cartridge and centrifuge at

13,000 rpm until all the wash solution is through the filter (1 min, possibly
more); discard the eluate and reuse the collection tube.
8. Add 500 µL of Wash Solution #2/3 to the filter cartridge and centrifuge at
13,000 rpm until all the wash solution is through the filter (1 min, possibly
more); discard the eluate and reuse the collection tube. Repeat with an
additional aliquot of Wash Solution #2/3. After discarding the second wash
solution centrifuge for an additional 1 min to remove the last traces of the
wash solution. Put the filter cartridge into a new collection tube.
9. Add 50 µL of 80oC Elution Solution to the filter and quickly centrifuge at

13,000 rpm. Using the same collection tube add a second 10 µL aliquot of
80oC Elution Solution to the filter and quickly centrifuge at 13,000 rpm. Save
the eluate and discard the filter.
10. To ensure that there is no DNA contamination the isolated RNA will be
treated with DNase. Add 6 µL of 10X DNase 1 buffer and 1 µL of 2 U/µL
DNase I. Mix gently and place in a 37oC water bath for 20 min.
11. Resuspend the DNase Inactivation Reagent by vortex mixing. Remove the
sample from the water bath and add 7 µL of the re-suspended DNase
Inactivation Reagent to it. Flick the tube gently to disperse the DNase
Inactivation Reagent. Incubate at room temperature for 2 min, flicking the
tube one more time at the 1 min mark.
12. Centrifuge at 11,000 rpm (10,000 x g) for 1 min and carefully transfer the
supernatant to a clean sterile 1.5 mL microfuge tube. Label your RNA sample
appropriately and keep on ice.
13. Take the A260, A280 and A320 absorbance readings of a 1:40 diluted (2 µL RNA
sample plus 78 µL T.E buffer) RNA sample using a Elution Solution diluted
in the same manner as the blank. The A260 reading should be between 0.1 and
1.0. Consult with your instructor if it is not. Subtract the A320 value from the
A260 and A280 values. Determine the RNA concentration (µg/µL) for your
undiluted sample using the equation in the introduction. Determine the total
µg of RNA recovered from you tissue sample. Estimate the volume of your
undiluted sample with a pipet, try an initial setting of 60 µL. Calculate the µg
RNA/mg tissue.
14. Using your calculated RNA concentration determine the volume required for
1 - 2 µg of RNA. Show this to your instructor. Depending on its value (it
should not exceed 10 µL) you may have to concentrate the RNA following a
protocol provided by your instructor. Note- a minimum concentration would
be [RNA] = 0.1 µg/µL.
15. If your RNA is of suitable concentration perform a Two-Step RT-PCR with

heat denaturation RETROScript protocol for reverse transcription reactions
using two samples; one with the Oligo (dT) and the other with Random
Decamers.
16. To three separate, sterile 1.5 mL microfuge tubes, one labeled dT, one labeled
RD, and the third labeled –RT, add the appropriate volume of your RNA
sample to give 1 – 2 µg of RNA. To the sample labeled dT add 2 µL of the 50
µM Oligo (dT) primers solution; to the sample labeled RD add 2 µL of the
50 µM Random Decamers solution. To each of these samples add an
appropriate volume of nuclease-free water so that the final volume is 12 µL.
To the sample labeled –RT add an appropriate volume of nuclease-free water
so that the final volume is 20 µL; this will serve as a negative control for the
subsequent PCR to show if there were any genomic DNA contamination in
your RNA sample. Mix and nanofuge these samples briefly and place in the
80oC water bath for 3 min to denature the RNA.
17. Remove the three samples and place on ice for 1 min. Nanofuge briefly and
place back on ice. To only the samples labeled dT and RD add 8 µL of the
Reverse Transcriptase Master Mix prepared by your instructor in the
following manner:
2 µL 10X RT Buffer (500 mM Tris-HCl, pH 8.3, 750 mM KCl, 30 mM

MgCl2, 50 mM DTT)
4 µL dNTP mixture (2.5 mM each dNTP)
1 µL 10 U/µL Placenta RNase Inhibitor
1 µL 100 U/µL MMLV-Reverse Transcriptase
Your instructor will prepare two positive RT controls with 1 µg of control

mouse liver template RNA; one with the oligo (dT) primers and one with the
random decamer (RD) primers.
18. Mix gently, nanofuge briefly, and incubate these samples as well as the –RT
sample at 44oC for 1 hour. The reverse transcriptase is inactivated by
incubating the samples at 92oC for 10 min. The samples are placed on ice and
can now be stored at -20oC or subjected to PCR.
19. The PCR of any cDNA produced by the RT reaction will use the
RETROscript kit’s Positive Control PCR Primer mixture and should yield a
361 bp segment of a conserved “housekeeping” gene rig/S15 which encodes a
small ribosomal subunit protein. The sequence of the primers are:
Forward primer: 5’ - TTCCGCAAGTTCACCTACC
Reverse primer: 5’ - CGGGCCGGCCATGCTTTACG
20. To three separate PCR tubes add 2.5 µL of your dT, RD and –RT samples,
respectively. To each sample add 22.5 µL of a PCR master mix that has been
prepared in the following manner:
2.5 µL 10X PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM

MgCl2)
1.25 µL dNTP mixture (2.5 mM each dNTP)
1.25 µL Positive Control PCR primer mixture (5 µM each primer)
0.4 µL 5U/µL Ambion SuperTaq
17,1 µL sterile molecular biology grade water
21. The PCR is performed as follows: (Program RIGS15)
Denature at 95oC for 1 min, followed by 30 cycles of
Denature at 94oC for: 30 sec

Anneal at 55oC for : 30 sec
Extend at 72oC for: 30 sec
After 30 cycles an additional Extension at 72oC for 5 min

Storage at 4oC followed by storage at –80oC.
Freeze your various, labeled samples
22. The following week prepare a 2.0% (0.60g agarose/30 mL TBE) agarose gel
in the usual way with a 8-tooth comb.
23. Recover your PCR samples, thaw and keep on ice. Remove 10 µL of each to
separate 1.5 mL microfuge tubes and add 2 µL of the Ambion High
Resolution Gel Loading Solution to each. Vortex briefly and spin down for 10
sec.
24. Load 12 µL of your three samples, 12 µL of a positive control sample

(provided by the instructor), and 3 µL of the 100 bp DNA ladder into separate
wells. Run at 100 V until the blue tracking dye is ~ 1.5 cm from the end of the
gel.
25. Photo-document your gel. Measure the distance, in mm, from the well to each
band of the standards as well as the distance for the samples.
26. Construct a log bp versus distance standard curve and determine the number
of bp for each amplified product. Evaluate your results in context of the
expected results and use of the various controls.
III. Points for Discussion:
What was the mass of the mouse liver tissue? What was the concentration
(µg/µL) of your isolated RNA solution? What was the A260/A280 ratio and its
meaning? What was the µg RNA/mg tissue and how does it compare with what
was expected? What volume was required to give 1 -2 µg RNA for the RT
reaction? Was the RT successful? What was the size of the PCR products for the
positive control, your dT and random decamer samples? Compare these to the
expected value. Also compare the intensities of the these bands with one another
and discuss the significance. Was there any DNA contamination in your RNA
sample?
IV. References:
Chelly, J., Concordet, J., Kaplan, J., and Hahn, A. (1989) Illegitimate
transcription: transcription of any gene in any cell type. Proc. Natl. Acad. Sci.
USA, 8, 2617 – 2621.
Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation

by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytica
Biochemistry, 162, 156-159.
Inoue, C., Shiga, K., Takasawa, S., Kitagawa, M., Yamamoto, H., and
Okamoto, H. (1987) Evolutionary conservation of the insulinoma gene rig
and its possible function. Proc. Natl. Acad. Sci, 84, 6659- 6662.
Kitagawa, M., Takasawa, S. Kikuchi, N., Itoh, T., Teraoka, H., Yamamoto,
H., and Okamoto, H. (1991) Rig encodes ribosomal protein S15; The primary
structure of mammalian ribosomal protein S15. FEBS, 283, 210- 214.
RETROscriptTM First Strand Synthesis Kit for RT-PCR instruction manual,

Version 0011, Ambion, Austin, TX
RNAqueous®-4PCR Kit instruction manual, Version 0304, Ambion, Austin,

TX
OPTIMIZATION OF PCR
[Modified after Optimization of PCRs from Michael A. Innis and David H. Gelfand in PCR Protocols: A
Guide to Methods and Applications(1990) (ed. M. A. Innis et al.) p. 3-12. Academic Press, San Diego, CA]
Polymerase Chain Reaction (PCR) is a very sensitive assay in which a single DNA molecule can be
amplified, and single-copy genes can be extracted out of complex mixtures of genomic sequences. PCR can
also be utilized for rapid screening and/or sequencing of inserts directly from aliquots of individual phage
plaques or bacterial colonies. Enhancements (such as the use of thermostable DNA polymerases and
automation) of the method have fostered the development of numerous and diverse PCR applications (K. B.
Mullis, U.S. patent 4,683,195, July 1987; U.S. patent 4,683,202, July 1987; Saiki et al. 1985; Mullis et al.
1986; Mullis and Faloona 1987). Since no single protocol will be appropriate to all situations, each new PCR
application requires optimization. Some often encountered problems include: no detectable product or a low
yield of the desired product, the presence of nonspecific background bands due to mispriming or
misextension of the primers, the formation of "primer-dimers" that compete for amplification with the
desired product, and mutations or heterogeneity due to misincorporation. The objective here is to expedite
the optimization process by discussing parameters that influence the specificity, fidelity, and yield of the
desired product.
Standard PCR Protocol

Although standard conditions will amplify most target sequences, they are presented only as starting
conditions for designing new PCR applications. All analytical PCR applications should be optimized.
1. Set up a 100 µl reaction in a 0.5-ml microfuge tube, mix, and overlay with 75 ul of mineral oil:
Template DNA (105 to 106 target molecules*)
20 pmol each primer (Tm >55°C preferred)
20 mM Tris-HCl (pH 8.3) (20°C)
1.5 mM MgC12
25 mM KCl
0.05% Tween 20
100 µg/ml of autoclaved gelatin or nuclease-free bovine serum albumin
50 µM each dNTP
2 units of Taq DNA polymerase
* 1 µg of human single-copy genomic DNA equals 3 x 105 targets; 10 ng of yeast DNA equals 3 x 105
targets; 1 ng of Escherichia coli DNA equals 3 x 105 targets; 1% of an M13 plaque equals 106 targets.
2. Perform 25 to 35 cycles of PCR using the following temperature profile:
Denaturation 96°C, 15 seconds (a longer initial time is usually desirable)
Primer Annealing 55°C, 30 seconds
Primer Extension 72°C, 1.5 minutes
3. Cycling should conclude with a final extension at 72°C for 5 minutes. Reactions are stopped by chilling to
4°C and/or by addition of EDTA to 10 m M.
Enzyme Concentration
A recommended concentration range for Taq DNA polymerase is between 1 and 2.5 units (SA = 20
units/pmol) (Lawyer et al. 1989) per 100 ul reaction when other parameters are optimum. However, enzyme
requirements may vary with respect to individual target templates or primers. When optimizing a PCR,
enzyme concentrations ranging from 0.5 to 5 units/100 ul should be tested. If the enzyme concentration is too
Page 1 of 5
high, nonspecific background products may accumulate, and if too low, an insufficient amount of desired
product is made. Note: Taq DNA polymerase from different suppliers may behave differently because of
different formulations, assay conditions, and/or unit definitions.
Deoxynucleotide Triphosphates
Stock dNTP solutions should be neutralized to pH 7.0, and their concentrations should be determined
spectrophotometrically. Primary stocks are diluted to 10 mM, aliquoted, and stored at -20°C. A working
stock containing 1 mM each dNTP is recommended. The stability of the dNTPs during repeated cycles of
PCR is such that approximately 50% remains as dNTP after 50 cycles. Deoxynucleotide concentrations
between 20 and 200 µM each result in the optimal balance among yield, specificity, and fidelity. The four
dNTPs should be used at equivalent concentrations to minimize misincorporation errors. Both the specificity
and the fidelity of PCR are increased by using lower dNTP concentrations. Low dNTP concentrations
minimize mispriming at nontarget sites and reduce the likelihood of extending misincorporated nucleotides
(Innis et al. 1988). The lowest dNTP concentration appropriate for the length and composition of the target
sequence should be determined empirically, e.g., the use of low, uniform dNTP concentrations (12 µM each)
enabled highly sensitive.
Magnesium Concentration
The magnesium concentration may affect: primer annealing, strand dissociation temperatures of both
template and PCR product, product specificity, formation of primer-dimer artifacts, and enzyme activity and
fidelity. Taq DNA polymerase requires free magnesium on top of that bound by template DNA, primers, and
dNTPs. Accordingly, PCRs should contain 0.5 to 2.5 mM magnesium over the total dNTP concentration.
Note: The presence of EDTA or other chelators in the primer stocks or template DNA may disturb the
apparent magnesium optimum.
Other Reaction Components

A recommended buffer for PCR is 10 to 50 mM Tris-HCl (between pH 8.3 and 8.8) when measured at 20°C;
however, an extensive survey of other buffers has not been performed. Tris is a dipolar ionic buffer having a
pKa of 8.3 at 20°C, and a pKa of -0.021/°C. Thus, the true pH of 20 mM Tris (pH 8.3) at 20°C varies
between 7.8 and 6.8 during typical thermal cycling conditions. Up to 50 mM KCl can be included in the
reaction mixture to facilitate primer annealing. NaCl at 50 mM, or KCl above 50 mM, inhibits Taq DNA
polymerase activity (Innis et al. 1988). Gelatin or bovine serum albumin (100 ug/ml) and nonionic
detergents, such as Tween 20 or Laureth 12 (0.05 to 0.1%; Mazer Chemicals, Gurnee, Illinois), are included
to help stabilize the enzyme, although many protocols work well without added protein.
Primer Annealing
The temperature and length of time required for primer annealing depend upon the base composition, length,
and concentration of the amplification primers. An applicable annealing temperature is 5°C below the true
Tm of the primers. Because Taq DNA polymerase is active over a broad range of temperatures, primer
extension will occur at low temperatures, including the annealing step (Innis et al. 1988). The range of
enzyme activity varies by two orders of magnitude between 20 and 85°C. Annealing temperatures in the
range of 55 to 72°C generally yield the best results. At typical primer concentrations (0.2 µM), annealing
will require only a few seconds.
Increasing the annealing temperature enhances discrimination against incorrectly annealed primers and
reduces misextension of incorrect nucleotides at the 3' end of primers. Therefore, stringent annealing
temperatures, especially during the first several cycles, will help to increase specificity. For maximum
Page 2 of 5
specificity in the initial cycle, Taq DNA polymerase can be added after the first denaturation step during
primer annealing. Low extension temperature together with high dNTP concentrations favors misextension
of primers and extension of misincorporated nucleotides. For these reasons, PCRs are better performed using
longer primers and high annealing and extension temperatures, e.g., from 55 to 75°C.
Primer Extension
Extension time depends upon the length and concentration of the target sequence and upon temperature.
Primer extensions are traditionally performed at 72°C because this temperature was near optimal for
extending primers on an M13-based model template. Estimates for the rate of nucleotide incorporation at
72°C vary from 35 to 100 nucleotides second-1 depending upon the buffer, pH, salt concentration, and the
nature of the DNA template (Innis et al. 1988; Saiki and Gelfand 1989). An extension time of one minute at
72°C is considered sufficient for products up to 2 kb in length. However, longer extension times may be
helpful in early cycles if the substrate concentration is very low, and at late cycles when product
concentration exceeds enzyme concentration (approximately 1 nM).
Denaturation Time and Temperature

The most likely cause for failure of a PCR is incomplete denaturation of the target template and/or the PCR
product. Typical denaturation conditions are 95°C for 30 seconds, or 97°C for 15 seconds; however, higher
temperatures and longer times may be appropriate, especially for G+C-rich targets. It only takes a few
seconds to denature DNA at its strand-separation temperature (Tss); however, there may be lag time involved
in reaching Tss inside the reaction tube. Incomplete denaturation allows the DNA strands to "snap back" and,
thus, reduces product yield. In contrast, denaturation steps that are too high and/or too long lead to
unnecessary loss of enzyme activity. (The half-life of Taq DNA polymerase activity is >2 hours, 40 minutes,
and 5 minutes at 92.5, 95, and 97.5°C, respectively.) Due to the possibility of partial loss in enzymatic
activity, Taq is sometimes added last.
Cycle Number
The optimum number of cycles will depend mainly upon the starting concentration of target DNA when
other parameters are optimized. A common mistake is to execute too many cycles. To quote Kary Mullis, "If
you have to go more than 40 cycles to amplify a single-copy gene, there is something seriously wrong with
your PCR." Too many cycles can increase the amount and complexity of nonspecific background products
(see Plateau Effect). Of course, too few cycles give low product yield. Some guidelines for number of cycles
versus starting target concentration are provided:
Number of target molecules Number of cycles
3 x 105 25 to 30
1.5 x 104 30 to 35
1 x 103 35 to 40
50 40 to 45
Primers
Primer concentrations between 0.1 and 0.5 µM are generally optimal. Higher primer concentrations may
promote mispriming and accumulation of nonspecific product and may increase the probability of generating
a template-independent artifact termed a primer-dimer. Nonspecific products and primer-dimer artifacts are
themselves substrates for PCR and compete with the desired product for enzyme, dNTPs, and primers,
resulting in a lower yield of the desired product.
Page 3 of 5
Some simple rules aid in the design of efficient primers. Typical primers are 18 to 28 nucleotides in length
having 50 to 60% G+C composition. To calculate the Tm for a given primer, one can use the rule-of-thumb of
2°C for A or T and 4°C for G or C (Thein and Wallace 1986). Depending on the application, Tm between
55°C and 80°C are desired. Complementarity at the 3' ends of primer pairs should be avoided as this
promotes the formation of primer-dimer artifacts and reduces the yield of the desired product. Also, runs
(three or more) of C's or G's at the 3' ends of primers may promote mispriming at G+C-rich sequences and
should be avoided, as should palindromic sequences within primers. A less obvious reason for some primers
failing to work is the presence of secondary structure in the template DNA. In this case, substitution of 7-
deaza-2'-deoxyGTP for dGTP has been very useful.
Special-purpose primers: Primers may contain 5' extensions or mismatches for incorporating restriction
enzyme sites, an ATG start codon, or promoter sequences into the target sequence. Mismatched bases can be
placed internally for mutagenesis. Degenerate primers can be used to isolate novel genes on the basis of
similarity and/or amino acid sequence. Some authors have suggested using inosine in primers instead of
using degenerate primers (Knoth et al. 1988). When using degenerate primers, it helps to avoid degeneracy at
the 3' ends, because mismatched bases are inefficiently extended.
Plateau Effect
The term "plateau effect" is used to describe the attenuation in the exponential rate of product accumulation
that occurs during late PCR cycles (the accumulation of 0.3 to 1 pmol of the intended product). Depending
on reaction conditions and thermal cycling, one or more of the following may influence plateau: (1)
utilization of substrates (dNPTs or primers); (2) stability of reactants (dNTPs or enzyme); (3) end-product
inhibition (pyrophosphate, duplex DNA); (4) competition for reactants by nonspecific products or primer-
dimer; (5) reannealing of specific product at concentrations above 10-8 M (may decrease the extension rate or
processivity of Taq DNA polymerase or cause branch-migration of product strands and displacement of
primers); and (6) incomplete denaturation/strand separation of product at high product concentration.
An important consequence of reaching plateau is that an initially low concentration of nonspecific products
resulting from mispriming events may continue to amplify preferentially. Optimizing the number of PCR
cycles is the best way to avoid amplifying background products.
Fidelity Considerations
Conditions that promote misincorporation include when deoxynucleotide concentrations are well below the
Km (i.e., <1 µM) or when the concentration of one dNTP is low relative to the other three. A respectable
"chain-termination" sequencing ladder could be generated by limiting one dNTP in each of four separate
sequencing reactions (Innis et al. 1988). Because misincorporated bases cannot be proofread (Taq lacks a 3'
to 5' exonuclease activity), misincorporation errors do occur during PCR promote chain termination.
However, mismatched bases are inefficiently extended; chain termination thus restricts the amplification of
defective molecules and helps to maintain fidelity. In addition, high-fidelity thermophilic polymerase such as
Vent can also be used in place of Taq if serious problems with misincorporation arise.
What determines whether a mismatch is extended? Petruska et al. (1988) showed (for Drosophila DNA
polymerase ) that enzymatic discrimination against elongating mismatched termini is based mainly on Km
differences: a matched A-T terminus was found to be extended 200 times faster than a G-T mismatch, and
1400 and 2500 times faster than C-T and T-T mismatches were, respectively. The same is likely to be true
for Taq DNA polymerase. Therefore, the concentration of dNTPs in the reaction is predicted to have a
substantial effect on the fidelity of PCR at high dNTP concentrations, i.e., >1 mM, mismatches will be
extended more efficiently. In general, high-temperature annealing/extension (>55°C) and low dNTP
concentrations (10 to 50 µM each) give the highest fidelity in the final PCR product.
Page 4 of 5
Literature Cited
Chamberlain, J. S., R. A. Gibbs, J. E. Ranier, P. N. Nguyen, and C. T. Caskey.1988. Deletion screening of
the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16:
11141 - 11156.
Ehlen, T., and L. Dubeau. 1989. Detection of ras point mutations by polymerase chain reaction using
mutation-specific, inosine-containing oligonucleotide primers. Biochem. Biophys. Res. Commun.
160:441-447.
Innis, M. A., K. B. Myambo, D. H. Gelfand, and M. A. D. Brow.1988. DNA sequencing with Thermus
aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.
Proc. Natl. Acad. Sci. USA 85: 9436-9440.
Kim, H.-S., and O. Smithies. 1988. Recombinant fragment assay for gene targeting based on the polymerase
chain reaction. Nucleic Acids Res. 16: 8887-8903.
Knoth, K., S. Roberds, C. Poteet, and M. Tamkun. 1988. Highly degenerate, inosinecontaining primers
specifically amplify rare cDNA using the polymerase chain reaction. Nucleic Acids Res. 16: 10932.
Lawyer, F. C., S. Stoffel, R. K. Saiki, K. Myambo, R. Drummond, and D. H. Gelfand. 1989. Isolation,
characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus
aquaticus. J. Biol. Chem. 264: 6427-6437.
Mullis, K. B., and F. A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain
reaction. Methods Enzymol. 155 :335-350.
Mullis, K., E Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich. 1986. Specific enzymatic amplification of
DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51: 263-273.
Petruska, J., M. F. Goodman, M. S. Boosalis, L. C. Sowers, C. Chaejoon, and 1. Tinoco, Jr. 1988.
Comparison between DNA melting thermodynamics and DNA polymerase fidelity. Proc. Natl. Acad.
Sci. USA 85: 6252-6256.
Saiki, R. K., and D. H. Gelfand. 1989. Introducing AmpliTaq DNA polymerase. Amplifications 1:4-6.
Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim. 1985. Enzymatic
amplification of globin genomic sequences and restriction site analysis for diagnosis of sickle cell
anemia. Science 230: 1350- 1354.
Thein, S. L., and R. B. Wallace. 1986. The use of synthetic oligonucleotides as specific hybridization probes
in the diagnosis of genetic disorders. In Human genetic diseases: a practical approach (ed. K. E.
Davis), p.33-50. IRL Press, Herndon, Virginia.
Page 5 of 5
PREPARATION OF 2/3 N SULFURIC ACID SOLUTION
IN LABORATORY
paramedicsworld.com/biochemistry-practicals/preparation-of-2-3n-sulfuric-acid-h2so4/medical-paramedical-studynotes
A solution is a homogeneous mixture composed of only one phase. In such a mixture, a

solute is a substance dissolved in another substance called as the solvent. The
concentration of a solute in a solution is a measure of how much of that solute is dissolved
in a solvent.
Standard solutions are those in which the exact amount of a substance presents in a
definite volume of solution. To prepare the standard solution, a known weight of solute is
measured on a well-calibrated weighing scale and then dissolved in a solvent (usually
distilled water) to make up a specific volume.
Normality (N) is one of the commonly used units that determine the concentration of the
solution that expresses the gram equivalent weight of solute per liter of the solution. In
other words, Normality is simply the number of active grams of a solute per liter of the
solution.
The 2/3 N Sulfuric Acid solution is prepared as follows…..
APPARATUS REQUIRED…..
Measuring cylinder
Volumetric flask / Beaker
Stirrer
CHEMICALS REQUIRED…..
Distilled water
Concentrated H2SO4 (97%)
CALCULATIONS FOR 2/3 N SULFURIc ACID

SOLUTION…..
Molecular weight of H2SO4 = (2×1) + 32 + (4×16) = 2 + 32 + 64 = 98 gm
N = [Weight / Equivalent weight] × [1000 / Volume (in ml)]
2/3 = [X/98/2] × [1000/500]
X = 1/3 × 49 = 16.3 gm
X = 16.3 gms
1/5
Conversion of grams into volume (ml) –
Volume of acid needed = Grams of acid / (Concentration of acid + Specific gravity)
Specific gravity of H2SO4 = 1.94
Concentration of the acid = 97%
Volume of the acid needed to make 500 ml of 2/3 N H2SO4
(16.3 / 0.97) + 1.94 = 8.7 ml
Thus, 8.7 ml of 97% H2SO4 should be added to the 491.3 ml of Distilled water to make the
500 ml of 2/3 N H 2SO4 Solution.
PROCEDURE FOR 2/3 N SULFURIC ACID SOLUTION…..

⇒ Take measuring cylinder and measure 8.7 ml of H2SO4 [sulfuric acid (97%)].
⇒ Take 250 ml of Distilled water in the Beaker or Volumetric flask.
⇒ Now add 8.7 ml of the H2SO4 slowly in the flask having distilled water.
⇒ Mix well using a stirrer (if you are making the solution in the beaker) or by swirling (in
case you are making the solution in the volumetric flask).
⇒ Make the final volume 500 ml by adding the distilled water.
⇒ Mix well by stirring (in case of the beaker) or swirling (in case of the volumetric flask).
PRECAUTIONS TO BE TAKEN WHILE PREPARING

SULFURIC ACID SOLUTION…..
⇒ Measure accurately the 8.7 ml of 97% concentrated sulfuric acid.
⇒ Always add acid to water and never water to the acid as dilution of acid is a highly
exothermic reaction.
⇒ Work near a running supply of water. If the acid contacts the skin, it must be washed off
rapidly with the copious amount of tap water.
⇒ Concentrated H2SO4 is highly corrosive so handle with care.
2/5
Plant Genomic DNA Extraction using CTAB
Introduction
The search for a more efficient means of extracting DNA of both higher quality and yield
has lead to the development of a variety of protocols, however the fundamentals of DNA
extraction remains the same. DNA must be purified from cellular material in a manner
that prevents degradation. Because of this, even crude extraction procedures can still be
adopted to prepare a sufficient amount of DNA to allow for multiple end uses.
DNA extraction from plant tissue can vary depending on the material used. Essentially
any mechanical means of breaking down the cell wall and membranes to allow access to
nuclear material, without its degradation is required. For this, usually an initial grinding
stage with liquid nitrogen is employed to break down cell wall material and allow access
to DNA while harmful cellular enzymes and chemicals remain inactivated. Once the
tissue has been sufficiently ground, it can then be resuspended in a suitable buffer, such
as CTAB. In order to purify DNA, insoluble particulates are removed through
centrifugation while soluble proteins and other material are separated through mixing
with chloroform and centrifugation. DNA must then be precipitated from the aqueous
phase and washed thoroughly to remove contaminating salts. The purified DNA is then
resuspended and stored in TE buffer or sterile distilled water. This method has been
shown to give intact genomic DNA from plant tissue. To check the quality of the
extracted DNA, a sample is run on an agarose gel, stained with ethidium bromide, and
visualised under UV light.
Materials
CTAB buffer
Microfuge tubes
Mortar and Pestle
Liquid Nitrogen
Microfuge
Absolute Ethanol (ice cold)
70 % Ethanol (ice cold)
7.5 M Ammonium Acetate
55o C water bath
Chloroform : Iso Amyl Alcohol (24:1)
Water (sterile)
Agarose
6x Loading Buffer
1x TBE solution
Agarose gel electrophoresis system
Ethidium Bromide solution
CTAB buffer 100ml
2.0 g CTAB (Hexadecyl trimethyl-ammonium bromide)

10.0 ml 1 M Tris pH 8.0
4.0 ml 0.5 M EDTA pH 8.0 (EthylenediaminetetraAcetic acid Di-sodium salt)
28.0 ml 5 M NaCl
40.0 ml H2O
1g PVP 40 (polyvinyl pyrrolidone (vinylpyrrolidine homopolymer) Mw 40,000)
Adjust all to pH 5.0 with HCL and make up to 100 ml with H2O.
1 M Tris pH 8.0
Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust pH to 8.0 by adding 42 ml of

concentrated HCL. Allow the solution to cool to room temperature before making the
final adjustments to the pH. Adjust the volume to 1 L with H2O. Sterilize using an
autoclave.
5x TBE buffer
54 g Tris base
27.5 g boric acid
20 ml of 0.5M EDTA (pH 8.0)
Make up to 1L with water.
To make a 0.5x working solution, do a 1:10 dilution of the concentrated stock.
1% Agarose gel
1 g Agarose dissolved in 100 ml TBE
Procedure
- Grind 200 mg of plant tissue to a fine paste in approximately 500 μl of CTAB buffer.
- Transfer CTAB/plant extract mixture to a microfuge tube.
- Incubate the CTAB/plant extract mixture for about 15 min at 55o C in a recirculating
water bath.
- After incubation, spin the CTAB/plant extract mixture at 12000 g for 5 min to spin
down cell debris. Transfer the supernatant to clean microfuge tubes.
- To each tube add 250 μl of Chloroform : Iso Amyl Alcohol (24:1) and mix the
solution by inversion. After mixing, spin the tubes at 13000 rpm for 1 min.
- Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge
tube.
- To each tube add 50 μl of 7.5 M Ammonium Acetate followed by 500 μl of ice cold
absolute ethanol.
- Invert the tubes slowly several times to precipitate the DNA. Generally the DNA can
be seen to precipitate out of solution. Alternatively the tubes can be placed for 1 hr at
-20 o C after the addition of ethanol to precipitate the DNA.
- Following precipitation, the DNA can be pipetted off by slowly rotating/spinning a
tip in the cold solution. The precipitated DNA sticks to the pipette and is visible as a
clear thick precipitate. To wash the DNA, transfer the precipitate into a microfuge
tube containing 500 μl of ice cold 70 % ethanol and slowly invert the tube. Repeat.
((alternatively the precipitate can be isolated by spinning the tube at 13000 rpm for a
minute to form a pellet. Remove the supernatant and wash the DNA pellet by adding
two changes of ice cold 70 % ethanol )).
- After the wash, spin the DNA into a pellet by centrifuging at 13000 rpm for 1 min.
Remove all the supernatant and allow the DNA pellet to dry (approximately 15 min).
Do not allow the DNA to over dry or it will be hard to re-dissolve.
- Resuspend the DNA in sterile DNase free water (approximately 50-400 μl H2O; the
amount of water needed to dissolve the DNA can vary, depending on how much is
isolated). RNaseA (10 μg/ml) can be added to the water prior to dissolving the DNA
to remove any RNA in the preparation (10 μl RNaseA in 10ml H2O).
- After resuspension, the DNA is incubated at 65o C for 20 min to destroy any DNases
that may be present and store at 4o C.
- Agarose gel electrophoresis of the DNA will show the integrity of the DNA, while
spectrophotometry will give an indication of the concentration and cleanliness.
DNA quality confirmation
– Prepare a 1 % solution of agarose by melting 1 g of agarose in 100 mL of 0.5x TBE

buffer in a microwave for approximately 2 min. Allow to cool for a couple of
minutes then add 2.5 μl of ethidium bromide, stir to mix.
– Cast a gel using a supplied tray and comb. Allow the gel to set for a minimum of 20
min at room temperature on a flat surface.
– Load the following into separate wells
o 10 μL 1kb ladder
o 5 μL sample + 5 μL water + 2 μL 6x Loading Buffer
– Run the gel for 30 min at 100 V
– Expose the gel to UV light and photograph (demonstration)
– Confirm DNA quality, presence of a highly resolved high molecular weight band
indicates good quality DNA, presence of a smeared band indicates DNA
degredation.
Plant Genomic DNA Extraction using Qiagen
plant mini kit
The advantages of using DNA isolation kits over crude methods (described above), is
they are fast, simple, do not contain harmful chemicals such as phenol or chloroform and
involves minimal handling. The technology makes use of spin columns, which contain a
silica-gel-based membrane that binds the DNA. The DNA while bound to the membrane
can be washed and cleaned from contaminants and then eluted from the column
(membrane) using water. The DNA obtained is usually more pure and clean than DNA
isolated from the crude method described above. One disadvantage of the kits is the cost,
with kits ranging in price from $250 to $300+ for 50 reactions.
Below is an exert from the QIAGEN DNeasy Plant Mini Kit Handbook, which can be
viewed on the QIAGEN web site at:-
http://www1.qiagen.com/literature/handbooks/PDF/GenomicDNAStabilizationAndPurifi
cation/FromAnimalAndPlantIssues/DNY_MinMax/1026510HB_DNY_012004WW_LR.
pdf
Protocol: Isolation of Total DNA from Plant Tissue Using

the DNeasy Plant Mini Kit
Important points before starting
■ If using the DNeasy Plant Mini Kit for the first time please read “Important Notes”
(page 12).
■ Buffer AP1 may develop a yellow color upon storage. This does not affect the
procedure.
■ All centrifugation steps are carried out at room temperature (15–25°C) in a microcentrifuge.
Things to do before starting
■ Buffers AP1 and AP3/E concentrate may form precipitates upon storage. If
necessary, warm to 65°C to redissolve (before adding ethanol to Buffer AP3/E).
Do not heat Buffer AP3/E after ethanol has been added.
■ Buffers AW and AP3/E are supplied as concentrates. Before using for the first time,
add the appropriate amount of ethanol (96–100%) as indicated on the bottle to
obtain a working solution.
■ Preheat a water bath or heating block to 65°C.
Manual disruption
Grind plant or fungal tissue under liquid nitrogen to a fine powder using a mortar and
pestle. Transfer the tissue powder and liquid nitrogen to an appropriately sized tube
and allow the liquid nitrogen to evaporate. Do not allow the sample to thaw. Proceed
immediately to the DNA preparation protocol.
DNA Preparation
1. Add 5 ml of Buffer AP1 (preheated to 65°C) and 10 µl of RNase A stock solution
(100 mg/ml) to a maximum of 1 g of ground tissue and vortex vigorously.
No tissue clumps should be visible. Vortex or pipet further to remove any clumps.
Clumped tissue will not lyse properly and will therefore result in lower DNA yields.
Note: Do not premix Buffer AP1 and RNase A prior to use.
2. Incubate the mixture for 10 min at 65°C. Mix 2–3 times during incubation by
inverting the tube.
This step lyses the cells.
3. Add 1.8 ml of Buffer AP2 to the lysate, mix, and incubate for 10 min on ice.
This step precipitates detergent, proteins, and polysaccharides.
4. Centrifuge lysate at 3000–5000 x g for 5 min at room temperature.
A pellet will form, but some particles will also float.
5. Decant supernatant into the QIAshredder Maxi Spin Column (lilac) placed in a
50 ml collection tube and spin at 3000–5000 x g for 5 min at room temperature
(15–25°C) in a swing-out rotor. Transfer flow-through, without disturbing the pellet
in the collection tube, to a new 50 ml tube (not supplied), and record the volume.
Typically, 5–6 ml of lysate is recovered. After centrifugation of the sample, most of
the debris and precipitates will be retained in the filter but there will also be a pellet
in the collection tube. Avoid disturbing the pellet when transferring the supernatant.
6. Add 1.5 volumes of Buffer AP3/E (see “Things to do before starting”) directly to
the cleared lysate and mix immediately by vortexing.
For example, to 5 ml of cleared lysate add 7.5 ml of Buffer AP3/E. Reduce the
amount of Buffer AP3/E accordingly if the volume of lysate is smaller. A precipitate
may form after the addition of Buffer AP3/E but this does not affect the DNeasy
procedure.
Note: Ensure ethanol has been added to Buffer AP3/E (see “Things to do before
starting”).
Note: It is important to pipet the Buffer AP3/E mixture directly into the cleared
lysate and to mix immediately.
7. Apply sample to the DNeasy Maxi Spin Column (colorless spin column) including
any precipitate which may have formed (maximum loading volume 15 ml). Centrifuge
at 3000–5000 x g for 5 min. Discard flow-through and reuse collection tube.
8. Add 12 ml Buffer AW to the DNeasy Maxi Spin Ccolumn and centrifuge for 10 min
at 3000–5000 x g to dry the membrane. Discard flow-through and collection tube.
It is important to dry the membrane of the DNeasy Maxi Spin Column since residual
ethanol may interfere with subsequent reactions. This spin ensures that no residual
ethanol will be carried over during elution.
After washing with Buffer AW, the DNeasy Maxi Spin Column membrane is usually
only slightly colored. In the rare case that the membrane remains significantly
colored after washing with Buffer AW, refer to “Darkly colored membrane” in the
Troubleshooting Guide on page 25.
9. Transfer the DNeasy Maxi Spin Column to a new 50 ml tube (supplied). Pipet
0.75–1 ml of Buffer AE directly onto the DNeasy Maxi Spin Column membrane
and leave for 5 min at room temperature (15–25°C). Centrifuge for 5 min at
3000–5000 x g to elute.
Note: Elution may also be performed with 0.5 ml of Buffer AE (instead of 0.75–1 ml).
This increases the final DNA concentration in the eluate, but also reduces overall
DNA yield. See “Elution”, page 13.
10. Add another 0.75–1 ml of Buffer AE and repeat the elution step as described in
step 9.
The first and second eluates may be combined or collected separately. For separate
collection of the eluates, see “Elution” on page 13.
Experiment 3 for CSS451
Plasmid Isolation Using Alkaline Lysis
Plant Biotechnology Resource & Outreach Center, Michigan State University
Plasmid Isolation Protocol
1. 5 ml LB medium containing proper antibiotics were inoculated with a single

bacterial colony. The tube was incubated at 37 ˚C overnight with vigorous
shaking at 360 rpm.
2. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
3. Discard the supernatant.
4. Resuspend bacterial pellet in a total of 1 ml ice-cooled solution I (50 mM). Pipet
up and down or vortex as necessary to fully resuspend the bacteria.
5. Add 2 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix
thoroughly by repeated gentle inversion. Do not vortex.
6. Add 1.5 ml ice-cold Solution III to the lysate. Mix thoroughly by repeated gentle
inversion. Do not vortex.
7. Centrifuge at 15,500 x g for 30 minutes at 4C.
8. Recover resulting supernatant.
9. Add 2.5 volume isopropanol to precipitate the plasmid DNA. Mix thoroughly by
repeated gentle inversion. Do not vortex.
10. Centrifuge at 15,500 x g for 30 minutes at 4C.
11. Removal of resulting supernatant. The pellet is plasmid DNA.
12. Rinse the pellet in ice-cold 70% EtOH and air-dry for about 10 minutes to allow
the EtOH to evaporate.
13. Add ddH2O or TE to dissolve the pellet. After addition of 2ul RNase A
(10mg/ml), the mixture was incubated for 20 minutes at room temperature to
remove RNA.
Note:
1. Spin down your cells. Your DNA is still in the cells, so it is in the pellet at this
stage.
2. Discard the supernatant and to even invert the tube and wipe the lip with a
Kim-wipe or Q-tip.
3. Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent
metals (primarily magnesium and calcium). Removal of these cations destabilizes
the cell membrane. It also inhibits DNases. Glucose should also be added to
maintain osmolarity and prevent the buffer from bursting the cells.
1
4. Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline
solution gave rise to the name of this technique. Mix this by gentle inversion and
incubate on ice for five minutes (but no longer, or your DNA will be irreversibly
denatured). Three things happen during this stage:
a. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate)
is a detergent found in many common items such as soap, shampoo and toothpaste.
b. NaOH loosens the cell walls and releases the plasmid DNA and sheared
cellular DNA.
c. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands
are separated. Plasmid DNA is circular and remains topologically constrained.
5. Renature the plasmid DNA and get rid of the garbage. Add potassium
acetate (KAc), which does three things:
a. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured

as single stranded DNA (ssDNA).
b. The ssDNA is precipitated, since large ssDNA molecules are insoluble in high
salt.
c. Adding sodium acetate to the SDS forms KDS, which is insoluble. This will
allow for the easy removal of the SDS from your plasmid DNA.
Now that you've made it easy to separate many of the contaminants, centrifuge to remove
cell debris, KDS and cellular ssDNA. Your plasmid DNA is in the supernatant, while all
of the garbage is in the pellet.
6. Precipitate the plasmid DNA by alcohol precipitation (ethanol or

isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium
chloride or sodium acetate) and spin this down. DNA is negatively charged, so
adding a salt masks the charges and allows DNA to precipitate. This will place
your DNA in the pellet.
7. Rinse the pellet—your plasmid DNA—in ice-cold 70% EtOH and air-dry for
about 10 minutes to allow the EtOH to evaporate.
8. Resuspend your now clean DNA pellet in buffer (often Tris) and EDTA plus
RNases to cleave any remaining RNA. Your DNA is now back in solution.
DNA of this purity is good for a number of uses, such as in vitro transcription or
translation or cutting with some enzymes. If you are sequencing or transforming this
DNA into mammalian cells, you'll want to use additional purification techniques such as
phenol extraction, Qiagen column purification, or silica-based purification.
2
Isolation of the Plasmid after Alkaline Lysis
The plasmid "miniprep " method is useful for preparing partially purified plasmid DNA
in small quantities from a number of transformants. It relies on an alkaline SDS lysis to
free the plasmid DNA from the cell, leaving behind the E. coli chromosomal DNA with
cell wall debris. The protocol described involves three basic steps: growth of bacteria and
amplification of the plasmid; harvesting and lysis of the bacteria; and purification of the
plasmid DNA.
These purification procedures exploit in one way or another the two major differences
between Escherichia coli DNA and plasmid DNA:
1. The E. coli chromosome is much larger than the DNA of plasmids used as vectors.
2. The bulk of E. coli DNA extracted from cells is obtained as broken, linear
molecules. By contrast, most plasmid DNA is extracted in a covalently closed,
circular form.
The purification protocol therefore involves a differential precipitation step, in which the
long strands of E. coli DNA, entangled in the remnants of lysed cells, are preferentially
removed. Because each of the complementary strands of plasmid DNA is a covalently
closed circle, the strands cannot be separated (without breaking one of them) by
conditions such as exposure to mild alkali (up to pH 12.5), which break most of the
hydrogen bonds of DNA. Closed circular molecules regain their native configuration
when returned to neutral pH.E. coli remains in the denatured state. This method
provides enough purified plasmid DNA for sequencing.
5 ml LB medium were inoculated with a single bacterial colony. The tube was
incubated at 37psy176 C overnight with vigorous shaking. 4.5 ml of the culture were
centrifuged for 20 minutes at 3500 rpm at 4psy176 C. The remainder of the overnight
culture was stored at 4psy176 C. The medium was removed, leaving the bacteria pellet as
dry as possible. The pellet was resuspended in 150 l ice-cold Lysis buffer I (50 mM
glucose, 10 mM EDTA, 25 mM Tris, pH 8.0) and stored for 5 minutes at room
temperature. After adding 300 l freshly prepared and mixing by inversion, the mixture
was incubated for 5 minutes on ice. Now 225 l ice-cold 3M KAc/5M HAc (pH 6.0) were
added and mixed gently. The tube was stored on ice for 5 minutes, precipitating the
chromosomal bacteria DNA. After centrifugation for 15 minutes at 13000 rpm at
4psy176 C, the supernatant was transferred to a fresh tube. Proteins were removed by
vortexing with 400 l phenol (see section phenol), adding 300 l Sevac and centrifuging
for 2 minutes at 13000 rpm.
500-600 l of the aqueous layer were removed and mixed with 1 ml ethanol to precipitate
the DNA (see section EtOH). After incubating for 5 minutes at room temperature, the
Eppendorf tube was centrifuged for 10 minutes at 13000 rpm at 4psy176 C. The
3
supernatant was removed, the pellet washed with 70% ethanol and recentrifuged. After
vacuum drying,
Solution I (Lysis buffer I): 50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0. Store

at 0˚C
a. 10ml 500mM Glucose

b. 2ml 500mM EDTA pH 8.0
c. 2.5ml 1M Tris pH 8.0
d. 85.5ml H2O
e. Autoclave and store at 4°C
Solution II (Lysis buffer II): Freshly prepared 0.2 N NaOH, 1% SDS. Store at room
temperature (RT)
Isopropanol: Stored at -20 0˚C
Solution III (Lysis buffer III): 3M KOAc, pH 6.0
a. 60ml 5M potassium acetate (49.07g potassium acetate in 100ml H2O)

b. 11.5ml glacial acetate
c. 28.5ml H2O
4
Precipitation techniques
Purpose: to concentrate the sample and remove small interfering species, such as salts & detergents, for
downstream applications e.g. 2D gels.
For small amounts of protein (< low microgram) it may be necessary to add a carrier protein such as
insulin.
Acetone Precipitation Protocol

1. Cool the required volume of acetone to -20°C.
2. Place protein sample in acetone-compatible tube.
3. Add four times the sample volume of cold (-20°C) acetone to the tube.
4. Vortex tube and incubate for 60 minutes at -20°C.
5. Centrifuge 10 minutes at 13,000-15,000 x g.
6. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet.
Optional: If additional cycles of precipitation are necessary to completely remove the interfering
substance, then repeat steps 2-5 before proceeding to step 7.
7. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do
not over-dry pellet, or it may not dissolve properly.
8. Resuspend in appropriate buffer.
TCA Precipitation Protocol
1. Add an equal volume of 20% TCA (trichloroacetic acid) to protein sample.

2. Incubate 30 min on ice.
3. Spin in microfuge at 4 deg. For 15 min.
4. Carefully remove all supernatant.
5. Add ~300 ul cold acetone and spin 5 min at 4 degrees.
6. Remove supernatant and dry pellet.
7. Resuspend samples in desired buffer.
Warning: TCA is a strong acid and should be handled with care
Chloroform/Methanol Precipitation
1. To sample of starting volume 100 ul

2. Add 400 ul methanol
3. Vortex well
4. Add 100 ul chloroform
5. Vortex
6. Add 300 ul H2O
7. Vortex
8. Spin 1 minute @ 14,0000 g
9. Remove top aqueous layer (protein is between layers)
10. Add 400 ul methanol
11. Vortex
12. Spin 2 minutes @ 14,000 g
13. Remove as much MeOH as possible without disturbing pellet
14. Speed-Vac to dryness
15. Bring up in 2X sample buffer for PAGE
Reference: Wessel, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143

Preparation of primary human monocytes/macrophages
Cell numbers and expected yield
With a typical yield of 1 – 2 x 106 mononuclear cells per ml of blood, we usually obtain 5 x 108 to 1 x
109 mononuclear cells/ leukopack (50 ml leukocytes enriched from 450 ml whole blood). Assuming
10% monocytes, expected yield of monocytes is ~ 5 x 107.
Supplies required
Leukopack (Buffy coat) - We obtain pathogen-negative leukopacks from the Gulf Coast Blood
Regional Blood Center.
Isolymph (Gallard & Schlesinger #759-001) or Ficoll-Paque (Amersham #17-1440-02)
Phosphate-buffed saline (PBS)
RPMI + 1% human serum, SIGMA # H1388
RPMI + endotoxin-free 10% fetal bovine serum (FBS) + pen/strep (= Complete RPMI)
50 ml conical tubes (polypropylene)
Fenwal sampling site coupler (punctures blood bag), Baxter Transfusion Therapy: 4C2405
Vacutainer needle (transfer from blood bag to draw tube), Fisher #02-665-20, 21 gauge, 1 inch
Serum tube, Fisher #02-683-81, 15 ml, 16 x 125 mm
RBC lysis buffer (155mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) – filter sterilized.
Trypan blue (0.4%)
GM-CSF, R & D Systems # 215-GM
Note: All supplies must be sterile. Media should be warmed to 37°C prior to use. Isolymph, PBS and
RBC lysis buffer are used at room temperature (R.T.).
Note: Take appropriate measures to sterilize waste and dispose of materials properly.
Procedure - Adherence Method

This procedure should be performed under sterile BL-2 conditions.
1. Carefully remove content of leukopack and transfer to conical tubes.

Peel back opening and puncture tube with site coupler (beveled end).
Carefully insert needle and collect blood into serum tubes.
Transfer to 5 50 ml conical tubes (~10 ml/tube).
2. Dilute 1:2 with PBS.

Add PBS to bring volume up 30 ml/tube.
3. In a separate set of five 50 ml conical tubes, add 20 ml Isolymph/Ficoll-Paque per tube.
4. Very gently, layer the diluted blood on top of the Isolymph/Ficoll-Paque. Do not allow layers to mix.
Using a sterile pipet, transfer the blood to the tube containing the Isolymph/Ficoll-Paque.
Position the tip of the pipet against the wall of tube just above the Isolymph/Ficoll-Paque.
Pipet slowly as to not disturb the interface.
5. Centrifuge at 500g for 30 min. at 20°C - No brake.
Rice & Herrmann Laboratory/ Protocols/Monocyte Isolation Page 1 of 3

Written by Christine H. Herrmann
6. Recover lymphocyte layer (buffy coat).
Using pipet, remove plasma down to
plasma & platelets
about 1 cm above buffy coat (lymphocyte)
layer. lymphocytes
With clean pipet, collect the buffy coat and Isolymph/Ficoll-Paque
transfer to a 50 ml conical tube (collect ~5
ml/ tube). erythrocytes &
granulocytes
Split layers between 2 clean conical tubes.
7. Wash 1X with PBS.

Add PBS to 45 ml.
Centrifuge at 400g for 10 min. at R.T. (Resume use of brake).
8. Lyse red blood cells with RBC lysis buffer.

Gently resuspend pellet by adding 10 ml RBC lysis buffer (rake bottom of tube across rack
before adding buffer to loosen cell pellet).
Incubate 5 min. at R.T with occasional gentle mixing. Ensure pellet is completely
resuspended.
After incubation, add 30 ml PBS.
9. Centrifuge at 200g for 5 min. at R.T. (If RBCs still visible, repeat lysis steps #8 & 9).
10. Wash 1X with PBS.

Gently resuspend pellet with 40 ml of PBS (remove any aggregates with Pasteur pipet).
Centrifuge at 200g for 5 min. at R.T.
11. Gently resuspend pellets from both tubes in a total of 40 ml PBS (combine pellets).
12. Count aliquot of cells to determine cell number and cell viability.
To Eppendorf tube containing 160 µl PBS + 200 µl trypan blue, add 40 µl cells (1:10 dilution).
Place on hemacytometer and immediately count cells in 2 quadrants (longer incubation with
dye can lead to cell death).
Calculate total cell #: cell count /2 X 104 X 10 (D.F.) X 40 (vol.)
Calculate % viable cells: (total # of viable cells per ml of aliquot x 100)
total # of cells per ml of aliquot
13. Centrifuge at 200g for 5 min. at R.T.
14. Resuspend cells at a concentration of 2.5 x 106 cells/ml in RPMI + 1% heat-inactivated human
serum AB.
15. Plate 10 ml of cell suspension/ 10 cm dish (reduce volume accordingly when plating in smaller
dishes).
16. Allow cells to incubate for 1 hr. at 37°C, 5% CO2.
17. Remove nonadherent cells and wash adherent cells 2 - 3X with warm PBS.
18. Add 5 ml complete RPMI (with 10% FBS)/ dish. Incubate 2 hrs. at 37°C, 5% CO2.
19. Wash 2 - 3X with warm PBS.

20. Add 10 ml Complete RPMI + 10 U/ml GM-CSF and incubate until appropriate time to harvest.
Cells should be fed every 4 – 5 days with Complete RPMI.
Procedure - Negative Selection

This alternate procedure should be performed if highly purified monocytes are required at time points
shortly after isolation (Days 0 –2).
1. Follow procedure above for adherence method through Step 13.
2. Use the Miltenyi Biotec Monocyte Isolation Kit II (#130-091-153), following the manufacturer’s
instructions.
3. Count cells.
4. Harvest cells for D0 time point or resuspend cells at a concentration of 1 x 106 cells/ml in RPMI +
1% heat-inactivated human serum.
5. Plate 10 ml of cell suspension/ 10 cm dish.
6. Allow cells to incubate for 1 hr. at 37°C, 5% CO2.
7. Remove nonadherent cells and add 10 ml Complete RPMI + 10 U/ml GM-CSF and incubate until
appropriate time to harvest. Cells should be fed every 4 – 5 days with Complete RPMI.
To harvest monocytes/macrophages
1. Wash cells 2 –3 times with cold PBS.
2. Incubate in 2 - 3 ml of ice-cold 3 mM EDTA/PBS for 10 – 15 min on ice.
3. Firmly tap dish to loosen cell attachment.
4. Scrape gently. Collect cells by centrifugation.
5. Cell pellet may be stored at –80oC or prepared appropriately for analysis.

Protein Precipitation Protocols
Notes: All reagents need to high purity/HPLC quality. All tubes used should be new or hand cleaned thoroughly
with Micro90 detergent. High quality water needs to be used for all the solutions and steps. For this protocol, I
used terminology MilliQ water to mean that equivalent quality of water needs to be used. Therefore, never use
dH2O or ddH2O water. At all steps of the sample preparation, use powder-free nitrile gloves (no aloe). “When
using precipitation as a cleanup step, it is recommended to have a starting protein concentration in the range
of 1–10 mg/mL. When protein samples are too diluted, it will be difficult to quantitatively recover proteins
following precipitation cleanup (Friedman, D.B., Hoving, S., and Westermeier, R. (2009) Isoelectric focusing
and two-dimensional gel electrophoresis. Methods in Enzymology 463: 515-540).” I find this may not be
possible for many samples.
Acetone Precipitation
[Modified from the protocol posted by the Nevada Proteomics Center]

http://www.unr.edu/inbre/Proteomics/Acetone_Precipitation_Protocol.html
1) Prepare 80% acetone solution and store at -20 ºC in a brown bottle.

2) Add 4 volumes chilled acetone/milliQ water stock to the sample extract.
3) Vortex well.
4) Incubate tube overnight at -20 ºC. (The tubes can be left up to 1 week with minimal protein degradation
or modification (GE 2-D Clean-Up Kit)).
5) Microfuge at top speed (at least >12,000xg), 4 ºC for 10 min or for larger tubes, centrifuge at >8,000xg,
4 ºC for 10 min. Remove tubes from the centrifuge as soon as it stopped.
6) Gently dump off the supernatant.
7) Positioning the tube same as before (pellet should towards the outer wall of the rotor), microfuge briefly
to bring any remaining liquid to the bottom of the tube.
8) Pipet off the remaining sup, making sure no visible liquid is left behind.
9) Wash the pellet with 1 ml (microfuge tubes) or 10 ml (larger tubes) chilled acetone/milliQ. The pellet
needs to be thoroughly broken up with a glass rod.
10) Repeat steps #5-8.
11) Repeat wash 1 more time (steps #9-10).
12) Air dry the pellet for 5 min. Do not over dry the pellet or it will be difficult to resuspend.
13) Resuspend pellet as described below. (You can freeze the pellets at -80 ºC until resuspension step.)
TCA Precipitation with Acetone Wash
1) Make a 100% w/v TCA stock and store the solution at 4°C in a brown bottle. Place a brown bottle of
100% acetone in the -20 ºC.
2) To two samples, add chilled 100% TCA at1/10 of the sample volume.
3) Mix sample by inverting several times.
4) Incubate one of the samples on ice for 15 min and incubate the other overnight at -20 ºC. Overnight
increases yields but could damage sialic acid on glycoproteins.
5) Thaw -20 ºC sample.
7) Gently dump off the supernatant.
10) Suspend the pellet in 100 μl (microfuge tube) and 1 ml (large centrifuge tube) milliQ water using a glass
rod. The pellet should be dispersed, but will not be dissolved by the water.
11) Add 1 ml (microfuge tube) and 10 ml (large centrifuge tube) chilled 100% acetone.
1
12) Vortex each tube for 30 sec.
13) Over a course of 1 hr, vortex the tube three times for 30 sec. Continue incubating the tubes at -20 ºC
between each vortexing.
14) Incubate tubes at -20 ºC overnight. (You can skip this step but your yields are lower. The tubes can be
left up to 1 week with minimal protein degradation or modification (GE 2-D Clean-Up Kit)).
15) Repeat steps #6-9.
TCA/Acetone Precipitation
[Modified from the protocol described by Gorg, A.; Obermaier, C.; Boguth, G.; Harder, A.; Scheibe, B.;
Wildgruber, R.; Weiss, W. (2000) The current state of two-dimensional electrophoresis with immobilized pH
gradients. Electrophoresis 21, 1037-53.]
1) Make a 13.3% w/v TCA in acetone stock and 100% acetone stock. Store the solutions in a brown bottle
at -20 ºC. Add 0.2% w/v DTT to both solutions just prior to use.
2) Add three volumes of the chilled 13.3% w/v TCA in acetone containing 0.2% w/v DTT stock to the
sample (final concentration is 10% TCA). Mix well by inverting tube.
3) Incubate for 1.5 hrs or overnight at -20 ºC. Overnight increases yields but could damage sialic acid on
glycoproteins.
5) Dump off the supernatant.
8) Add 1 ml (microfuge tube) and 10 ml (large centrifuge tube) -20 ºC chilled, acetone containing 0.2%
DTT stock. Break up the pellet using a glass rod. The pellet should be dispersed, but will not be
dissolved by the water.
9) Vortex each tube for 30 sec.
10) Over a course of 1 hr, vortex the tube three times for 30 sec.
11) Repeat centrifugation steps #4-7.
12) Repeat the acetone Wash 1X (steps #8-11).
Peterson TCA-DOC Precipitation with Acetone Wash
[This method was recommended by Hediye Erdujument-Bromage from the Proteomics Core of the Memorial
Sloan-Kettering Cancer Center for precipitating samples with low concentrations of proteins. TCA-DOC part of
this protocol was slightly modified by us from the manuscript from Peterson, G. (1977) A Simplification of the
Protein Assay Method of Lowry et al. Which is More Generally Applicable. Analytical Biochemistry 83: 346-
356.]
1) Make a 72% TCA stock and store the solution at 4 ºC in a brown bottle. Place a brown bottle of 100%
acetone in the -20°C. Make a 0.15% solution of sodium deoxycholate and store at room temperature.
2) To the sample, add 1/10 of the volume of 0.15% sodium deoxycholate. Mix by inverting the tube
several times.
3) Incubate on ice for 15 min.
4) Add 1/10 of the original sample volume of the chilled 72% TCA. Mix by inverting the tube several times.
2
10) Add 1 ml (microfuge tube) and 10 ml (large centrifuge tube) -20 ºC chilled, acetone. (The tubes can be
left up to 1 week with minimal protein degradation or modification (GE 2-D Clean-Up Kit)).
11) Vortex each tube for 30 sec.`
Arnold TCA-DOC Precipitation with Acetone Wash
[This is method was modified from the original method published by Arnold and Ulbrich-Hofmann (Arnold, U.
and Ulbrich-Hofmann, A. (1999) Quantitative Protein Precipitation from Guanidine Hydrochloride-Containing
Solutions by Sodium Deoxycholate/Trichloroacetic Acid Analytical Biochemistry 271: 197-199). The original
method was referenced by Friedman et al. ]
1) Make a 50% TCA stock and store the solution at 4 ºC in a brown bottle. Place a brown bottle of 100%
acetone in the -20°C. Make a 1% solution of sodium deoxycholate and store at room temperature.
2) To the sample, add 1/10 of the volume of 1% sodium deoxycholate. Mix by inverting the tube several
times.
4) Add 1/5 of the original sample volume of the chilled 50% TCA. Mix by inverting the tube several times.
10) Add 1 ml (microfuge tube) and 10 ml (large centrifuge tube) -20 ºC chilled, acetone. (The tubes can be
left up to 1 week with minimal protein degradation or modification).
11) Vortex each tube for 30 sec.`
Methanol and Chloroform Precipitation
[This method was adapted by David Friedman (Friedman, D.B., Hoving, S., and Westermeier, R. (2009)
Isoelectric focusing and two-dimensional gel electrophoresis. Methods in Enzymology 463: 515-540) from the
method described by Wessel and Flugge (Wessel, D., and Flugge, U. I. (1984). A method for the Quantitative
Recovery of Protein in Dilute Solution in the Presence of Detergents and Lipids. Analytical Biochemistry 138:
141–143).
1) Bring up predetermined amount of protein extract to 100 mL with water.

2) Add 300 mL (3-volumes) water.
3) Add 400 mL (4-volumes) methanol.
3
4) Add 100 mL (1-volume) chloroform.
5) Vortex vigorously and centrifuge; the protein precipitate should appear at the interface.
6) Remove the water/MeOH mix on top of the interface, being careful not to disturb the interface. Often
the precipitated proteins do not make a visibly white interface, and care should be taken not to disturb
the interface.
7) Add another 400 mL methanol to wash the precipitate.
8) Vortex vigorously and centrifuge; the protein precipitate should now pellet to the bottom of the tube.
9) Remove the supernatant and briefly dry the pellets in a vacuum centrifuge.
10) Resuspend the pellets in a suitable amount of 2D gel-compatible buffer
Acidified Acetone and Methanol Precipitation
[This method was posted by Kevin Hakala on the ABRF forum (3/6/13) in response on how to remove SDS-
PAGE Sample Buffer from a protein sample. He noted the following: “It recovers dilute protein from SDS-
sample buffer. I have not tried to digest the precipitated material, I only re-suspended the pellet in a smaller
volume of SDS-sample buffer to concentrate the original sample. I don't have any data regarding recovery.]
1) Prepare acidified acetone: 120ml acetone + 10µl HCl (1mM final concentration).
2) Prepare precipitation reagent: Mix equal volumes of acidified acetone and methanol and keep at
-20ºC.
3) To one volume of protein solution add 4 volumes of cold precipitation reagent. Mix and keep ON at
-20ºC.
4) Spin 15min 4ºC in microfuge at maximum speed (15000g). Carefully discharge supernatant and
retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).
5) Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone or methanol residue
(small tubes).
Resuspending the Pellets for Traditional SDS-PAGE Gel Electrophoresis
1) Store pellets at -80 ºC until ready to run the gel.

2) Resuspend the pellet by pipetting in 1X SDS sample buffer mixed with fresh reducing agent.
3) Briefly vortex the sample. (Skip this step if you are interested in studying protein modifications due to
oxidization.)
4) Incubate sample at 70°C for 10 min or 95°C for 5 min.
5) Load sample onto the gel after cooling.
Resuspending the Pellets for 2-D Gel Electrophoresis
1) Thaw the standard Rehydration Solution (7 M Urea + 2 M Thiourea + 4% CHAPS + 0.5% Ampholytes
3-10 + 0.0002% Bromophenol Blue). Add 0.004g DTT.
2) Add 300 μl of standard Rehydration Solution + DTT to each pellet.
3) Resuspend the pellets by pipetting. Don’t vortex since it will oxidize the proteins.
4) Leave the sample resting on top of ice for 2 hrs, pipetting every 30 min to get the sample into solution.
If your sample goes into solution sooner then proceed onto step #10.
5) If the sample has still not gone into solution, freeze the samples at -80 ºC to help the sample go into
solution.
6) Thaw the samples at RT.
7) Resuspend the pellets by pipetting. If your sample goes into solution sooner then proceed onto step
#10.
8) If the sample has still not go into solution repeat freeze thaw and resuspension steps (#5-7). If your
sample goes into solution sooner then proceed onto step #10.
4
9) If the pellet still has not gone into solution, leave sample overnight at 4ºC. Resuspend the pellet the
next morning by pipetting.
10) Transfer the samples to 1.5 ml microfuge tube.
11) Microfuge at top speed for 5 min at 4ºC.
12) Transfer the sup to another 1.5 ml microfuge tube. Make sure you pipet away from the pellet.
13) Take 30 μl of each sample to measure the protein conc.
14) Store the remaining samples and the 30 μl aliquots at -80 ºC.
Note: If your pellet has not been solubilized after step #9, sonication maybe used. However, this should be
used as a last resort since extreme care must be taken in not heating sample since urea breaks down during
heating. The sample should be sonicated with bursts lasting no longer than several seconds, and should be
chilled between bursts by placing on ice.
5
Measuring Protein Concentration of Cleaned-Up Samples with Modified Bradford Assay
[Scaled down from the protocol described by Ramagli, L. S., Quantifying protein in 2-D PAGE solubilization
buffers. Methods Mol Biol 1999, 112, 99-103.]
1) Thaw standard rehydration solution and add 0.004 g of DTT (0.4%) for every 1 ml rehydration solution.
2) Prepare fresh ovalbumin standards in Rehydration buffer (Standards can be stored at -20ºC and
reused):
For 10 mg/ml: Add 0.005g per 500 μl Rehydration buffer + DTT
For 4 mg/ml: Add 200 μl 10mg/ml stock to 300 μl Rehydration buffer + DTT
For 1 mg/ml: Add 50 μl 10mg/ml stock to 450 μl Rehydration buffer + DTT
3) Prepare Bio-Rad Protein Reagent by diluting 1:4 in milliQ water. Diluted reagent does not need to be
freshly made.
4) Add 20 μl 0.12 N HCl to all the standard and sample tubes. (Assuming concentration HCl is 12.1 N, add
10 ml conc HCl to 990 ml nanopure water to make a 0.12 N HCl)
5) Prepare protein standard curve (Do in duplicate):
Final Conc. of Amt. of

Std. Stock Used Amt. of Std. Stock
Ovalbumin (Std.) Rehyd. Sol’n
1) Blank 5 μl 0 μl 0 μl
2) 1 μg 4 μl 1 mg/ml 1 μl
6) 16 μg 1 μl 4 mg/ml 4 μl
7) 32 μg 1.8 μl 10 mg/ml 3.2 μl
6) Prepare the following samples (Do in quadruplicate):
Amt. of Amt. of Sample

Sample
Rehyd. Sol’n
Sample 0 μl 5 μl
Sample (if the blue color is higher
4 μl 1 μl
than the standard curve)
7) Add 875 μl of Bio-Rad diluted dye to each tube. Immediately after addition, close the lid on the tube
and mix by vortexing tube.
8) Incubate the tubes for 5 min at room temp.
9) Transfer 200 μl from each tube to a 96-well plate. Prior to transfer, mix the tube by vortexing.
10) Measue A595 before 1 hr.
11) Use a linear curve to fit the standards.
Melissa Sondej 7/6/11
6
Protocol: Micro-preparation of DNA from Plant Tissue
David M. Francis
Horticulture and Crop Science
The Ohio State University/ OARDC
1680 Madison Ave
Wooster, OH 44691
Reagents:
Extraction Buffer: per liter:

0.35 M Sorbitol 63.75 g
0.1 M Tris 12.10 g Tris-base
0.005 M EDTA 1.86 g
-Adjust pH to 7.5 with HCl

-Add NaBisulfite (3.8 g/l (0.02 M)) just before use
-Do not autoclave, store in refrigerator
Nuclei lysis buffer: per liter:

0.2 M Tris 24.2 g Tris-base
0.05 M EDTA 18.6 g
2.0 M NaCl 116.9 g
2% CTAB 20.0 g Hexadecyltrimethylammonium bromide
-Adjust pH to 7.5 with HCl

-Autoclave
-Store at room temperature
5% Sarcosyl:
5 g N-lauryl sarcosine per 100 ml dd water
-Sterilize by filtration
Chloroform/isoamyl alcohol:
In the hood, mix isoamyl alcohol and chloroform in a ratio of 4 ml isoamyl to 100 ml
chloroform
T1/10E Stock: per 100 ml:

10 mM Tris (7.5-8.0) 1 ml 1 M Tris-base pH 7.5
0.1 mM EDTA 0.2 ml 0.5 M EDTA
Procedure:
Collect a single leaflet (size 1-2 X your thumb nail) from seedlings into polyethylene
bags (0.004 gauge) and label the bags. Ziplock bags will also work if grinding is gentle.
Add 1 ml of “extraction buffer” to the bag.
Grind the leaflet in the extraction buffer by rubbing with thumb.
Transfer 300 ul to a labeled 1.5 ml tube. Take care not to transfer large fragments of
tissue.
Add 300 ul of CTAB “lysis buffer”.
Add 150 ul of 5% Sarcosine.
Incubate at 65 C for 15 min (range 10-20). Gently invert the tubes 1-2 times during the
65 C incubation to mix.
Add 700 ul of Chloroform:isoamyl alcohol. Gently invert the tubes 10-20 times to mix.
Spin in the microfuge for 15 min.
Remove the aqueous phase (top phase) to a fresh, labeled, 1.5 ml tube.
Repeat the Chloroform extraction (Add 700 ul of Chloroform:isoamyl alcohol. Gently

invert the tubes 10-20 times to mix. Spin in the microfuge for 15 min.)
Remove the aqueous phase (top phase) to a fresh, labeled, 1.5 ml tube.
Add 1-0.8 volume of isopropanol (room temp).
Spin in the microfuge for 15 to 20 min to ppt. the DNA.
Dry the DNA (spin vac for 5 min, or air dry with tubes inverted for 30 min)
Resuspend the DNA in 200 ul of T1/10E

Purification of IgG by Precipitation with Sodium Sulfate or Ammonium
Sulfate
Mark Page and Robin Thorpe
1. Introduction
Addition of appropriate amounts of salts, such as ammonium or sodium sulfate, causes
precipitation of IgG (1) from all mammals, and can be used for serum, plasma, ascites fluid,
and hybridoma culture supernatant. Although such IgG is usually contaminated with other
proteins, the ease of these precipitation procedures coupled with the high yield of IgG has led
to their wide use in producing enriched IgG preparations. They are suitable for many
immunochemical procedures, e.g., production of immunoaffinity columns, and as a starting
point for further purification. It is not suitable however for conjugating with radiolabels,
enzymes, or biotin since the contaminating proteins will also be conjugated, thereby reducing
the efficiency of the labelling and the quality of the reagent. The precipitated IgG is usually
very stable, and such preparations are ideally suited for long-term storage or distribution and
exchange between laboratories. Ammonium sulfate precipitation is the most widely used and
adaptable procedure, yielding a 40% pure preparation; sodium sulfate can give a purer
preparation for some species, e.g., human and monkey.
2. Materials
2.1. Ammonium Sulfate Precipitation

1. Saturated ammonium sulfate solution: Add excess (NH4)2SO4 to distilled water (about
950 g to 1 L), and stir overnight at room temperature. Chill at 4°C, and store at this
temperature. This solution (in contact with solid salt) is stored at 4°C.
2. PBS: 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4. Store at 4°C.
2.2. Sodium Sulfate Precipitation

This requires solid sodium sulfate.
3. Methods
3.1. Ammonium Sulfate Precipitation

1. Prepare saturated ammonium sulfate at least 24 h before the solution is required for
fractionation. Store at 4°C.
2. Centrifuge serum or plasma for 20–30 min at 10,000gav at 4°C. Discard the pellet (see
Note 1).
3. Cool the serum or plasma to 4°C, and stir slowly. Add saturated ammonium sulfate
solution drop wise to produce 35–45% final saturation (see Note 2). Alternatively, add solid
ammonium sulfate to give the desired saturation (2.7 g of ammonium sulfate/10 mL of fluid
= 45% saturation). Stir at 4°C for 1–4 h or overnight.
4. Centrifuge at 2000–4000gav for 15–20 min at 4°C (alternatively for small volumes of 1–5
mL, microfuge for 1–2 min). Discard the supernatant, and drain the pellet (carefully invert
the tube over a paper tissue).
5. Dissolve the precipitate in 10–20% of the original volume in PBS or other buffer by
careful mixing with a spatula or drawing repeatedly into a wide-gage Pasteur pipette. When
fully dispersed, add more buffer to give 25 50% of the original volume and dialyze against
the required buffer (e.g., PBS) at 4°C overnight with two to three buffer changes.
Alternatively, the precipitate can be stored at 4 or –20°C if not required immediately.
3.2. Sodium Sulfate Precipitation (see Note 3)

1. Centrifuge the serum or plasma at 10,000gav for 20–30 min. Discard the pellet, warm the
serum to 25°C, and stir.
2. Add solid Na2SO4 to produce an 18% w/v solution (i.e., add 1.8 g/10 mL), and stir at 25°C
for 30 min to 1 h.
3. Centrifuge at 2000–4000gav for 30 min at 25°C.
4. Discard the supernatant, and drain the pellet. Redissolve in the appropriate buffer as
described for ammonium sulfate precipitation (Subheading 3.1., step 5).
4. Notes
1. If lipid contamination is excessive in ascites fluids, thereby compromising the salt
precipitation, add silicone dioxide powder (15 mg/ mL) and centrifuge for 20 min at 2000gav
(2) before adding the ammonium or sodium salt.
2. The use of 35% ammonium sulfate will produce a pure IgG preparation, but will not
precipitate all the IgG present in serum or plasma. Increasing saturation to 45% causes
precipitation of nearly all IgG, but this will be contaminated with other proteins, including
some albumin. Purification using (NH4)2SO4 can be improved by repeating the precipitation,
but this may cause some denaturation. Precipitation with 45% (NH4)2SO4 is an ideal starting
point for further purification steps, e.g., ion-exchange or affinity chromatography and FPLC
purification (see Chapters 139 and 140).
3. Sodium sulfate may be used for precipitation of IgG instead of ammonium sulfate. The
advantage of the sodium salt is that a purer preparation of IgG can be obtained, but this must
be determined experimentally. The disadvantages are that yield may be reduced depending on
the IgG characteristics of the starting material, IgG concentration, and composition.
Fractionation must be carried out at a precise temperature (usually 25°C), since the solubility
of Na2SO4 is very temperature dependent. Sodium sulfate is usually employed only for the
purification of rabbit or human IgG.
References
1. Heide, K. and Schwick, H. G. (1978) Salt fractionation of immunoglobulins, in Handbook
of Experimental Immunology, 3rd ed. (Weir, D. M., ed.), chap. 7. Blackwell Scientific,
Oxford, UK.
2. Neoh, S. H., Gordon, C., Potter, A., and Zola, H. (1986) The purification of mouse MAb
from ascitic fluid. J. Immunol. Meth. 91, 231.
PCR optimization for Pyrosequencing
Ver. 010206
PSQ™ 96 System
PCR optimization for Pyrosequencing
1) Select PCR primers to, if possible, form a fragment < 300 bp. The PCR primers should typically
be about 18-25 bp in length, of approximately the same GC-content as the fragment as a whole,
and with approximately the same melting temperatures. The primers should preferably be more
GC rich in the 5’-end and less in the 3’-end for good specificity. They should not form heavy
hairpin loops or dimers with themselves or the other primer. Check the biotinylated PCR primer
extra carefully for hairpin loops and duplexes, as excess biotinylated primer might cause
background in the Pyrosequencing assay.
If Oligo 6.0 is used for primer design, you can let Oligo find a primer set automatically. The
program will also present calculated optimal and highest annealing temperatures for the selected
primer set, and a good starting point is to choose an annealing temperature between these
values.
2) Biotinylated PCR primer concentrations should be kept low to avoid interference with the
Pyrosequencing assay. Keep the primer concentrations at 0.2 uM (i.e. 10 pmol in a 50 ul PCR
reaction).
3) Biotinylated PCR primers are particularly sensitive to storage. Keep stock primer, and aliquoted,
diluted primers (10uM), in the freezer, not in the fridge!
4) The effective, free Magnesium concentration will depend on nucleotide and DNA concentrations
(because Magnesium binds to nucleotides and DNA). Therefore, keep DNA and nucleotide
concentrations constant during PCR optimization. As a standard, we use 10 ng DNA in a 50 ul
PCR reaction and 125 uM of each nucleotide.
5) In general, increased Magnesium concentrations will lead to higher incorporation rate and
efficiency for TaqGold, but may also make the enzyme slightly more “sloppy” and increase the
risk for incorporation errors. Furthermore, it will stabilize dimer formation increasing the risk for
mis-priming in the template and stabilizing secondary structures in the DNA template, which
particularly may decrease the amplification efficiency for GC-rich templates. Lowered Magnesium
concentrations will in general make the amplification reaction more stringent, but also less
efficient, leading to lower yields.
6) Very GC-rich fragments (>70%) often benefit from adding 5% DMSO and exchanging part of the
deoxyguanosine with deoxyinosine (dI:dG 3:1 is a good starting point).
7) TaqGold is gradually activated during the amplification reaction and therefore requires more cycles
than a protocol with ordinary Taq. For best yield, and consumption of all biotinylated PCR primers
which is important for Pyrosequencing, run 45-50 PCR cycles.
8) The parameters that may need optimization is the annealing temperature and Magnesium
concentration. The annealing temperature typically falls in the range 54°C – 64°C. Select two
DNA samples that can be used for all PCR optimizations. With TaqGold, a good starting point is
to try three different temperatures (e.g. 54°C, 57°C, and 60°C) and three different Magnesium
concentrations (1.5mM, 2.0mM, and 2.5mM) while keeping all other parameters constant. GC-
rich templates often need a higher annealing temperature and lower Magnesium concentration
(since high salt concentrations will stabilize secondary structures) to amplify well. Very often,
medium GC-rich templates will work at 57°C and 2.0 mM Magnesium, so these might be
conditions that you want to try before starting optimization.
9) A typical PCR program for fragments up to about 300 bp:
95°C 5min, 45x(95°C 15s, Ta 30s, 72°C 15s), 72°C 5min, 4°C
The program takes about 1 hour and 45 min to run. For longer fragments than 300bp, the
extension time at 72°C may need to be extended.
10) Check on 1.5% agarose that you have got a clear, strong product band without excess primers,
primer-dimers or other non-specific products. With 10 pmol of sequencing primer and 15-25 ul of
the PCR product, you could then expect strong, pure signals (single peak heights of ~15-25
units) when testing the products for Pyrosequencing.
Example of PCR reaction mix (2.0 mM MgCl2) for one and ten reactions respectively. The
presented volumes are in microliters.
PCRmix 1x 10
10xPCRbuffer II 5 50
MgCl (25 mM) 4 40
dNTP (2.5 mM) 2,5 25
DMSO 0 0
Primer a (10 uM) 1 10
Primer b (10uM) 1 10
TaqGold 0,3 3
H2O 31,2 312
Sum: 45 450
45 ul PCRmix + 5ul DNA-sample (2 ng/ul)
PCR prog: 95C 5 min, 45x(95C 15s, 54C 30s, 72C 15s), 72C 5 min, 4C
TECHNICAL SUPPORT PHONE +46 (0)18 51 65 65 techsupport@pyrosequencing.com
PYROSEQUENCING AB
VALLONGATAN 1, SE-752 28 UPPSALA, SWEDEN
PHONE +46 (0)18 56 59 00, FAX +46 (0)18 59 19 22
www.pyrosequencing.com
REAGENTS AND SOLUTIONS
Agarose gel, 2% (w/v)

Dissolve 2 g of agarose into 100 ml of 1×TBE buffer (APPENDIX 2A) by warming the solution, e.g., in a
microwave oven. After the agarose has been dissolved, add 5 ml of ethidium bromide. Pour into
electrophoresis tray with combs and allow to solidify at room temperature.
Antibodies
Avidin-Cy5 antibodies (Amersham Bioscience): Reconstitute antibodies using water according to the
manufacturer’s instructions. Dilute 1:200 in blocking solution (see recipe).
Mouse anti-digoxigenin (Sigma): Reconstitute antibodies using water according to the manufacturer’s
instructions. Dilute 1:500 in blocking solution (see recipe).
Cy5.5 anti-mouse antibodies (Amersham Bioscience): Reconstitute antibodies using water according to
the manufacturer’s instructions. Dilute 1:200 in blocking solution (see recipe).
Store antibodies in 100-µl aliquots up to 1 year at −20◦C. Aliquots of working solutions

may be stored for 2 to 3 months at 4◦C protected from the light.
Blocking solution
1.0 g BSA (Sigma; final 1% w/v)
100.0 µl Tween-20 (Sigma; final 0.1% v/v)
20.0 µl 20× SSC (final 4×)
80.0 µl H2O
Store indefinitely at −20◦C
DAPI/antifade medium
Dissolve the entire powder of DAPI (4_,6-diamidino-2-phenylindole; Sigma) in water to a 100 µg/ml
stock solution. Store in 500-µl aliquots up to 1 year at
−20◦C. To combine DAPI in antifade medium, combine the following in order:
5.0 ml PBS 500.0 µl 100 µg/ml DAPI stock (final 1 µg/ml) 0.5 g p-phenylenediamine (Sigma; final 10
mg/ml) Dissolve well then add 45.0 ml glycerol (final 90% v/v) The resulting medium is very viscous.
Transfer to a 50-ml Falcon tube and place on a rotator to ensure proper mixing (30 min). This product is
light sensitive, so wrap the tube with foil. Store in 1-ml aliquots 1 year at −20◦C. New medium must be
made when an increasing amber tint appears. This is also commercially available from Vectashield (Vector
Laboratories).
CAUTION: DAPI is a potential carcinogen and should be handled with caution.
dNTP (dATP, dTTP, dGTP, dCTP) for secondary amplification, 2 mM

10.0 µl 100 mM dATP (final 2 mM)
10.0 µl 100 mM dCTP (final 2 mM)
10.0 µl 100 mM dTTP (final 2 mM)
10.0 µl 100 mM dGTP (final 2 mM)
460.0 µl sterile H2O (final 2 mM)
Store up to 1 year at −20◦C.
dNTP (dATP, dGTP, dCTP, 1.5 mM dTTP) for labeling, 2 mM

7.5 µl 100 mM dTTP (final 1.5 mM)
dNTP (dATP, dTTP, dGTP, dCTP), 10 mM

10.0 µl 100 mM dTTP (final 10 mM)
x-dUTPs
Dilute 1 mM Texas Red 1:5 in sterile H2O (13 ml Texas Red + 52 ml H2O)
Dilute 1 mM spectrum red 1:5 in sterile H2O (14 ml spectrum red + 56 ml H2O)
Dilute 1 mM spectrum far red 1:5 in sterile H2O (14 ml spectrum far red + 56 ml H2O)
Dilute 1 mMspectrum aqua 1:5 in sterile H2O (14 ml spectrum aqua + 56 ml H2O)
Dilute 1 mM spectrum green 1:5 in sterile H2O (14 ml spectrum green + 56 ml H2O)
Dilute 1mMspectrum gold 1:5 in sterile H2O (14 ml spectrum green+56 ml H2O)
Store up to 1 year at −20◦C
1% formalin/PBS/MgCl2
2.7 ml 37% formaldehyde (Sigma; final 1% v/v)
100.0 ml 1× PBS/MgCl2 (see recipe)
Make fresh
Discard in accordance with the regulations of the institution.
70% formamide/2× SSC

35.0 ml formamide (Invitrogen; final 70% v/v)
5.0 ml 20× SSC (final 2×)
10.0 ml H2O
Make fresh
CAUTION: Formamide is a carcinogen and should be handled with caution. It should
be discarded according to biohazard rules of the institution.
HCl, 0.01 M
0.5 ml 1 M HCl (final 0.01 M)
49.5 ml H2O
Store at room temperature until ready for use
Hybridization buffer
500.0 µl high-grade formamide (final 50% v/v)
100.0 µl 20× SSC (final 2×)
100.0 µl dextran sulfate (final 10% v/v)
300.0 µl H2O
Alternatively, this solution may be purchased from DAKO.
Loading dye, 5×
0.125 g bromophenol blue (final 0.25% w/v)
15.0 ml glycerol (final 30% v/v)
35.0 ml H2O
Store for several months to 1 year at room temperature or 4◦C
1× PBS/MgCl2
950.0 ml 1× PBS (APPENDIX 2A)
50.0 ml 1M MgCl2 (final 50 mM)
Store at room temperature until ready for use
Pepsin stock, 10%

100 mg pepsin powder (Sigma; final 10% w/v)
1.0 ml H2O
Store in 20-µl aliquots up to 1 year at −20◦C
RNase A stock solution

10.0 mg RNase A (DNase-free; final 10 mg/ml)
1.0 ml 2× SSC
Dispense into 10-µl aliquots and store at −20◦C
To make an RNase A working solution:
10.0 µl 10 mg/ml RNase A stock solution
990.0 µl 2× SSC
Vortex to mix. Do not freeze-thaw. Discard after use.
20× SSC
175.3 g NaCl
88.2 g sodium citrate
Adjust pH with 10 M NaOH to pH 7
Store for several months at room temperature
0.4× SSC/0.3% NP-40 solution

20.0 ml 20× SSC (final 0.4×)
950.0 ml H2O
3.0 ml NP-40 (final 0.3% v/v)
Add up to 1 liter with H2O
Store up to 6 months at room temperature
Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.
2× SSC/0.1% NP-40 solution

100.0 ml 20× SSC (final 2×)
850.0 ml H2O
1.0 ml NP-40 (final 0.1% v/v)
Add up to 1 liter with H2O
Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.
0.1%Tween-20/4× SSC
1.0 ml Tween-20 (Sigma; final 0.1% v/v)
200.0 ml 20× SSC (final 4×)
799.0 ml H2O
Store at room temperature until ready for use.
1 M HCl
(TCA); however, this recipe is cheaper, easier to prepare, and just as efficient.
Add H2O to 500 ml
BBS (BES-buffered solution), 2×

50 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES; Calbiochem)
280 mM NaCl
1.5 mM Na2HPO4, pH 6.95
800 ml H2O
Adjust pH to 6.95 with room temperature 1 M NaOH
H2O to 1 liter
Filter sterilize through a 0.45-µm nitrocellulose filter (Nalgene)
Store in aliquots at −20°C (can be frozen and thawed repeatedly)
The pH of this solution is critical (pH 6.95 to 6.98). When a new batch of 2× BES buffer is
prepared, its pH should be checked against a reference stock prepared (and tested) earlier.
CaCl2 , 1 M
147 g CaCl2⋅2H2O
H2O to 1 liter
Denhardt solution, 100×

10 g Ficoll 400
10 g polyvinylpyrrolidone
10 g bovine serum albumin (Pentax Fraction V; Miles Laboratories)
H2O to 500 ml
Filter sterilize and store at −20°C in 25-ml aliquots
Dithiothreitol (DTT), 1 M
Dissolve 15.45 g DTT in 100 ml H2O
Store at −20°C
EDTA (ethylenediamine tetraacetic acid), 0.5 M (pH 8.0)

Dissolve 186.1 g Na2EDTA⋅2H2O in 700 ml H2O
Adjust pH to 8.0 with 10 M NaOH (⋅50 ml)
Add H2O to 1 liter
Ethidium bromide, 10 mg/ml

Dissolve 0.2 g ethidium bromide in 20 ml H2O
Mix well and store at 4°C in dark
CAUTION: Ethidium bromide is a mutagen and must be handled carefully.
HBSS (Hanks balanced salt solution)
5.4 mM KCl
0.3 mM Na2HPO4
0.4 mM KH2PO4
4.2 mM NaHCO3
1.3 mM CaCl2
0.5 mM MgCl2
0.6 mM MgSO4
137 mM NaCl
5.6 mM D-glucose
0.02% phenol red (optional)
Add H2O to l liter and adjust pH to 7.4
HBSS can be purchased from Biofluids or Whittaker.
HBSS may be made or purchased without CaCl2 and MgCl2. These are optional components
that usually have no effect on an experiment. In some cases, however, their presence may be
detrimental to a procedure. Consult the individual protocol to see if the presence or absence
of these components is recommended in the materials list.
HCl, 1 M
913.8 ml H2O
HeBS (HEPES-buffered saline) solution, 2×

16.4 g NaCl
11.9 g HEPES acid
0.21 g Na2HPO4
800 ml H2O
Add H2O to 1 liter
Filter sterilize through a 0.45-µm nitrocellulose filter
Test for transfection efficiency and store at −20°C in 50-ml aliquots
An exact pH is extremely important for efficient transfection. The optimal pH range is 7.05
to 7.12.
KCl, 1 M
74.6 g KCl
H2O to 1 liter
MgCl2, 1 M
20.3 g MgCl2⋅6H2O
H2O to 100 ml
MgSO4, 1 M
24.6 g MgSO4⋅7H2O
H2O to 100 ml
MOPS buffer
0.2 M MOPS [3-(N-morpholino)propanesulfonic acid], pH 7.0
0.5 M sodium acetate
0.01 M EDTA
Store in the dark and discard if it turns yellow.
NaCl, 5 M
292 g NaCl
H2O to 1 liter
NaOH, 10 M
Add H2O to 1 liter

10× stock solution, 1 liter:
80 g NaCl
2 g KCl
11.5 g Na2HPO4⋅7H2O
2 g KH2PO4
Working solution, pH ⋅7.3:
137 mM NaCl
2.7 mM KCl
4.3 mM Na2HPO4⋅7H2O
1.4 mM KH2PO4

Solution A: 11.55 ml glacial acetic acid/liter (0.2 M).
Solution B: 19.6 g potassium acetate (KC2H3O2)/liter (0.2 M).
Referring to Table A.2.2 for desired pH, mix the indicated volumes of solutions A
and B, then dilute with H2O to 100 ml.
This may be made as a 5- or 10-fold concentrate by scaling up the amount of potassium
acetate in the same volume. Acetate buffers show concentration-dependent pH changes, so
check concentrate pH by diluting an aliquot to the final concentration.
To prepare buffers with pH intermediate between the points listed in Table A.2.2, prepare
Solution A: 27.2 g KH2PO4 per liter (0.2 M).
Solution B: 34.8 g K2HPO4 per liter (0.2 M).
and B, then dilute with H2O to 200 ml.
This may be made as a 5- or 10-fold concentrate by scaling up the amount of potassium
phosphate in the same volume. Phosphate buffers show concentration-dependent pH
changes, so check concentrate pH by diluting an aliquot to the final concentration.
SDS electrophoresis buffer, 5×
15.1 g Tris base
72.0 g glycine
5.0 g SDS
H2O to 1000 ml
Dilute to 1× or 2× for working solution, as appropriate
Do not adjust the pH of the stock solution, as the solution is pH 8.3 when diluted.
SED (standard enzyme diluent)

20 mM Tris⋅Cl, pH 7.5
500 µg/ml bovine serum albumin (Pentax Fraction V)
10 mM 2-mercaptoethanol
Sodium acetate, 3 M
Dissolve 408 g sodium acetate⋅3H2O in 800 ml H2O
Add H2O to 1 liter
Adjust pH to 4.8 or 5.2 (as desired) with 3 M acetic acid

Solution A: 11.55 ml glacial acetic acid/liter (0.2 M).
Solution B: 27.2 g sodium acetate (NaC2H3O2⋅3H2O)/liter (0.2 M).
and B, then dilute with H2O to 100 ml. (See Potassium acetate buffer recipe for
further details.)

Solution A: 27.6 g NaH2PO4⋅H2O per liter (0.2 M).
Solution B: 53.65 g Na2HPO4⋅7H2O per liter (0.2 M).
and B, then dilute with H2O to 200 ml. (See Potassium phosphate buffer recipe for
further details.)
SSC (sodium chloride/sodium citrate), 20×

3 M NaCl (175 g/liter)
0.3 M Na3citrate⋅2H2O (88 g/liter)
Adjust pH to 7.0 with 1 M HCl
STE buffer
10 mM NaCl
1 mM EDTA, pH 8.0
TAE (Tris/acetate/EDTA) electrophoresis buffer

50× stock solution:
242 g Tris base
37.2 g Na2EDTA⋅2H2O
H2O to 1 liter
Working solution, pH ⋅8.5:
40 mM Tris.acetate
2 mM Na2EDTA⋅2H2O
TBE (Tris/borate/EDTA) electrophoresis buffer

10× stock solution, 1 liter:
40 ml 0.5 M EDTA, pH 8.0 (see recipe; 20 mM)
10 mM Tris.Cl, pH 7.4, 7.5, or 8.0 (or other pH; see recipe)
1 mM EDTA, pH 8.0

50 mM triethanolamine, pH ⋅11.5
0.1% Triton X-100
0.15 M NaCl
Add Triton X-100 as a 10% stock sterilized by Millipore filtration and stored in the dark to
prevent photooxidation (stock is stable 5 years at room temperature).

1 mM EDTA, pH 8.0
150 mM NaCl
TM buffer, 10×
100 mM MgCl2
Tris-buffered saline (TBS)

0.9% (150 mM) NaCl
Tris.Cl [tris(hydroxymethyl)aminomethane], 1 M
Mix and add H2O to 1 liter
Approximately 70 ml of HCl is needed to achieve a pH 7.4 solution, and approximately 42
ml for a solution that is pH 8.0.
decreasing approximately 0.028 pH units per 1°C. Tris-buffered solutions should be adjusted
to the desired pH at the temperature at which they will be used. Because the pKa of Tris is
8.08, Tris should not be used as a buffer below pH ⋅7.2 or above pH ⋅9.0.
TTBS (Tween 20/TBS)

0.1% Tween 20 in Tris-buffered saline (TBS; see recipe)
Composition of Regular Buffers for Stock Solutions
RIPA
50 mM Tris-HCl, pH 7.4
1% NP-40
150 mM NaCl
1 mM EDTA
Protease inhibitors added fresh.
PBS (5X in 500 mls)

20.45 g NaCl
0.465 g KCl
10.142 g Na2HPO4, 7H2O
0.545 g KH2PO4, pH 7.2
1M MgSO4 Stock
24.074 g MgSO4 distilled water to 200 mL, store at RT.
1 M HEPES, pH = 7.0 Stock

119.15 g HEPES (free acid) distilled water to 400 mL. Add solid NaOH a few pellets at a time while mixing
until the pH is ∼6.8; add concentrated NaOH dropwise to achieve pH = 7.0 distilled water to 500-mL
sterile filter and store at 4◦C.
0.5 M EDTA Stock

16.81 g EDTA (sodium salt) distilled water to 90 mL. Adjust pH to 7.0. Bring the volume to 100mL with
distilled water. Store at RT.
0.5 M EGTA Stock

19.02 g EGTA (sodium salt) distilled water to 90 mL. Adjust pH to 7.0. Bring the volume to 100mL with
distilled water. Store at RT.
1M Dithiothreitol (DTT)
1.542 g dithiothreitol distilled water to 10 mL. Disburse into 500-mL aliquots and store at −20◦C.
100 mM MgATP Stock

Check formula weight of the lot of ATP you have and determine the amount required for 10mL of a 100
mM solution. Add 8.5mL distilled water to the determined amount and add 1mL of 1M MgSO4 stock.
Adjust pH to 7.0 distilled water to 10 mL. Disburse into 200-mL aliquots, and store at −20◦C.
100 mM Sodium Orthovanadate

1.839 g sodium orthovanadate distilled water to 8mL in a screw cap tube. Adjust pH to 10 if solution is
yellow. Place in boiling water until clear and recheck pH; repeat as necessary and adjust to final
concentration by checking A265 nm, ext. coeff. = 2925/M/cm and adding distilled water as needed. Store
at−20◦C.
Percentage Solution
A percentage solution is one in which the exact concentration of the solute is known in 100 ml of a liquid or
solution. The concentration may be expressed or determined by weight or volume; it may be written out in
grams and milliliters and expressed as follows: Percentage (w/v) - weight (in grams) in 100-ml volume of
solution Percentage (v/v) = volume (in milliliters) in 100-ml total volume of solution
Examples by Weight
For a 1% (w/v) aqueous solution of sodium chloride, weigh out 1 g of sodium chloride (NaCI) and add to
100 ml of distilled water, or 10 mg of NaCI per milliliter. For a 5% (w/v) aqueous solution of sodium
chloride, weigh out 5 g of sodium chloride and add to 100 ml of distilled water. For 1000 ml of a 5% (w/v)
aqueous solution of sodium chloride, use 10 times the above amounts, that is, 50 g of sodium chloride in
1000 ml of distilled water.
Example by Volume
The percentage of the solution is stated on the label, and a percentage solution should be determined from this
value. For example, the percentage on a bottle of commercial strength hydrochloric acid varies
from 36 to 40%. If you want a 1% solution of HCI, it is erroneousto take 1 ml of 36-40% HCI and add it to 99
ml of distilled water. An acceptable and correct formula for determining the amount of 36% (v/v) HCI needed
to make up 100 ml of a 1% (v/v) aqueous solution of HCI is as follows:
Dilutions for Solutions (Percentage by Volume)
The most accurate formula for making dilutions of solutions is the following:
Percentage (%)you have x unknown volume (ml)
= percentage (%) you want x volume (ml)wanted
Example of Preferred Method
To make up 1000 ml of 70% (v/v) ethanol from 95% (v/v) ethanol,
substitute the known amounts in the above formula:
Although the above method is preferred, you may use a generally

accepted method of subtracting the percentage required from the
percentage strength of the solution that is to be diluted. The difference
will be the amount of water that is to be used.
Example of Subtraction Method
The percentage required is 70% (v/v) ethanol and the solution to be
diluted is 95% (v/v) ethanol. As 95 - 70 - 25, 70 parts of 95% (v/v)
ethanol and 25 parts of distilled water are the amounts to be combined. To make up approximately 1 liter of
70% (v/v) ethanol, you
would simply place 700 ml of 95% ethanol in a 1000-ml graduated
cylinder and fill to the 950-ml mark with distilled water.
Molar Solutions
A molar (mole, molecular) solution is one in which 1 liter (1000 ml)
of the solution contains the number of grams of the solute equal to
its molecular weight (sum of atomic weights).
Examples of Molar Solutions
To make up a 1 M solution of NaCl (sodium chloride)"
1. Obtain the atomic weights of the elements (see a Periodic Table).
Sodium (Na) = 22.997
Chlorine (CI) - 35.459
2. Obtain the sum of the atomic weights from the formula for molecular
weight.
Molecular weight of NaCI = 22.997 + 35.459 = 58.456
3. Weigh 58.46 g of NaC1 and make up to a total volume of 1000
ml with distilled water.
To make up 100 ml of a 1 M solution of NaCl.
1. Same as Step 1 above.
4. Weigh 5.85 g of NaCI and make up to a total volume of 100 ml

with distilled water.
To make up a 0.1 M (1/10 molar) solution of NaCl.

4. Weigh 5.85 g of NaC1 and make up to a total volume of 1000 ml

with distilled water.
Reagents, Buffers, and Indicators
Throughout the monographs in this book, the term reagent solution is used to designate
solutions described in this section. The tests and limits in the monographs are based on the
use of the solutions in the strength indicated below.
Wherever the use of ammonium hydroxide or an acid is prescribed with no indication
of strength or dilution, the reagent is to be used at full strength as described in its mono-
graph. Dilutions are indicated either by the percentage of some constituent or by the vol-
umes of reagents and water mixed to prepare a dilute reagent. Dilute acid or ammonium
hydroxide (1 + x) means a dilute solution prepared by mixing 1 volume of the strong acid
or ammonium hydroxide with x volumes of reagent water.
Unless otherwise indicated, the reagent solutions are prepared and diluted with
reagent water by using standard class A volumetric pipets and flasks. Weights are measured
on a four-place calibrated balance.
For reference purposes, Table 3-1 presents reagents useful in the preparation of buff-
ers for a given pH.
Reagent Water
Throughout the monographs for reagent chemicals, the term water means distilled water
or deionized water that meets the requirements of Water, Reagent, page 716. However, for
specific applications—such as UV determinations or liquid and ion chromatography—
ASTM Type I reagent water (ASTM, 1999) or water for which the suitability has been
determined should be used. In tests for nitrogen compounds, water should be “ammonia-
free” or “nitrogen-free”. Water for use in analysis of ultratrace metals must meet the
requirements on page 718. For some tests, freshly boiled water must be used to ensure free-
dom from material absorbed from the air, such as ammonia, carbon dioxide, or oxygen.
Carbon Dioxide-Free Water. Carbon dioxide-free water can be prepared by purg-
ing reagent water with carbon dioxide-free air (using a gas dispersion tube) or nitro-
gen for at least 15 min or by boiling reagent water vigorously for at least 5 min and
allowing it to cool while protected from absorption of carbon dioxide from the atmo-
sphere; alternatively, fresh 18-M deionized water can be used.
95
96 Part 3: Solutions and Mixtures Used in Tests
Table 3-1. Reagents Useful in Preparing Buffers of a Specific pH Range

pH Range pKa Reagent
0–2 Hydrochloric acid; nitric acid; perchloric acid
0.3–5.3 1.3, 4.4 Oxalic acid, dihydrate; sodium oxalate; potassium tetroxalate,
dihydrate
0.9–2.9, 1.9, 6.2 Maleic acid
5.2–7.2
1.1–1.8 Potassium chloride
1.1–3.1, 2.1, 7.2, 12.4 Phosphoric acid; potassium phosphate, monobasic; potassium
6.2–10.1 phosphate, dibasic; potassium phosphate, tribasic, n-hydrate;
sodium phosphate, monobasic, monohydrate; sodium phosphate,
dibasic, 7-hydrate; sodium phosphate, tribasic, 12-hydrate
1.8–3.8 2.8 Monochloroacetic acid; chloroacetic acid, sodium salt
1.9–6.4 2.9, 5.4 Phthalic acid; potassium biphthalate
2.0–5.4 3.0, 4.4 d-Tartaric acid; potassium tartrate, ½-hydrate
2.1–7.4 3.1, 4.8, 6.4 Citric acid, anhydrous; citric acid, monohydrate; potassium
citrate, monohydrate
2.8–4.8 3.8 Formic acid; sodium formate
3.2–6.6 4.2, 5.6 Succinic acid
3.6–5.6 4.6 Acetic acid; sodium acetate, trihydrate
4.1–6.1 5.1 Hexamethylenetetramine
5.3–7.3, 6.3, 10.3 Carbonic acid; potassium bicarbonate; potassium carbonate,
9.3–11.3 anhydrous; sodium bicarbonate; sodium carbonate, anhydrous;
sodium carbonate, monohydrate
6.5–8.5 7.5 Imidazole
6.8 4.6 (acetic acid); Ammonium acetate
9.3 (ammonium
hydroxide)
6.8–8.8 7.8 Triethanolamine; TRIS hydrochloride
7.1–9.1 8.1 N,N-bis(2-hydroxyethyl)glycine (bicine); 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES)
7.8–9.8 8.8 2-Amino-2-methyl-1,2-propanediol (AMP)
8.2–10.2 9.2 Boric acid; sodium borate, 10-hydrate
8.3–10.3 9.3 Ammonium hydroxide; ammonium chloride
12–14 Potassium hydroxide, 45% solution; sodium hydroxide, 50%
solution
12.4 (saturated solution) Calcium hydroxide
Source: Reproduced courtesy of Mallinckrodt Baker, Inc.

Reagents, Buffers, and Indicators 97
Water (Suitable for LC Gradient Elution Analysis)

Analyze the sample by using the following gradient elution conditions. Use ACS reagent-
grade or better acetonitrile.
Column: Octadecyl (C-18 polymeric phase), 250 × 4.6 mm i.d., 5 m, 12% C load-
ing, endcapped
Mobile Phase: A. Water to be tested
B. ACS reagent-grade acetonitrile
Conditions: Detector: Ultraviolet at 254 nm
Sensitivity: 0.02 AUFS
Gradient Elution:
1. Program the liquid chromatograph to develop a water–acetonitrile gradient as follows:
Time (min) Flow (mL/min) % A (water) % B (acetonitrile)
0 2.0 100 0
20 2.0 100 0
40 2.0 0 100
50 2.0 0 100
2. Equilibrate the system at 100% water. Start the program after achieving a stable
baseline. Repeat the gradient program, and use these results to evaluate the water. No
peak should be greater than 10% of full scale (0.002 absorbance units).
Solutions and Mixtures

Note: Required quantities may be made having the same concentrations as below.
Acetic Acid, 1 N. Dilute 29 mL of glacial acetic acid with water to 500 mL.
Alcohol. Alcohol refers to the monograph for ethyl alcohol, page 308. This is distin-
guished from the monograph for reagent alcohol, page 567, which is a denatured form of
ethyl alcohol.
Alizarin Red S, 1%. Dissolve 0.250 g of alizarin red S in water, dilute with water to 250
mL, filter, and store in glass.
Ammonia–Cyanide, Lead-Free, 2%. In a well-ventilated fume hood, dissolve 2.0 g of

potassium cyanide in 15 mL of ammonium hydroxide, and dilute with water to 100 mL.
Remove lead by shaking the solution with small portions of dithizone extraction solution
until the dithizone retains its original green color; discard the extraction solution. Store the
ammonia–cyanide solution in a polyethylene bottle.
Ammoniacal Buffer, pH 10. Dissolve 90 g of ammonium chloride in 375 mL of
28–30% ammonium hydroxide, and dilute to 500 mL with water. (The pH of a 1 + 10 dilu-
tion with water should be about 10.)
Ammonium Acetate–Acetic Acid Buffer. Dissolve 40 g of ammonium acetate in

water, add 29 mL of glacial acetic acid, and dilute with water to 500 mL. (The pH of a
1 + 10 dilution with water should be about 4.5.)
Ammonium Acetate Buffer. Dissolve 30 g of ammonium acetate in water, and dilute

to 100 mL with water.
Ammonium Citrate, Lead-Free, 40%. Dissolve 40 g of citric acid in 100 mL of water,

and make alkaline to phenol red with ammonium hydroxide. Remove lead by shaking the
solution with small portions of dithizone extraction solution until the dithizone solution
retains its original green color; discard the extraction solution. Store the ammonium cit-
rate solution in a polyethylene bottle.
Ammonium Hydroxide, 2.5% NH3. Dilute 50 mL of ammonium hydroxide with

water to 500 mL.
Ammonium Hydroxide, 10% NH3. Dilute 200 mL of ammonium hydroxide with

water to 500 mL.
Ammonium Metavanadate, 0.25%. Dissolve 1.25 g of ammonium metavanadate in

250 mL of boiling water, cool, and add 10 mL of nitric acid. Dilute with water to 500 mL,
and store in a polyethylene or Teflon bottle.
Ammonium Molybdate, 5%. Dissolve 5 g of ammonium molybdate tetrahydrate in

water, and dilute with water to 100 mL. Store in a polyethylene bottle. Discard if a precipi-
tate forms.
Ammonium Molybdate, 10%. Dissolve 5 g of ammonium molybdate in water, and

dilute to 50 mL.
Ammonium Molybdate–Nitric Acid Solution. Mix thoroughly 50 g of molybdic

acid, 85%, in 120 mL of water, and add 70 mL of ammonium hydroxide. Filter, and add 30
mL of nitric acid. Cool, and pour, with constant stirring, into a cool mixture of 200 mL of
nitric acid and 480 mL of water. Add 0.1 g of ammonium phosphate dissolved in 5 mL of
water, allow to stand for 24 h, and filter through glass wool.
Ammonium Molybdate–Sulfuric Acid Solution, 5%. (For determination of phos-

phate.) Dissolve 5.0 g of ammonium molybdate tetrahydrate in 50 mL of 10% sulfuric acid,
and dilute with water to 100 mL.
Ammonium Nitrate, 10%. Dissolve 10.0 g of ammonium nitrate in water, and dilute
with water to 100 mL.
Ammonium Oxalate, 4%. Dissolve 20 g of ammonium oxalate monohydrate in water,

Ammonium Phosphate, 13%. Dissolve 65 g of dibasic ammonium phosphate in

water, and dilute with water to 500 mL.
Ammonium Thiocyanate, 30%. Dissolve 150 g of ammonium thiocyanate in water,

Aqua Regia. In a fume hood, cautiously mix 5 mL of nitric acid in 15 mL of hydrochlo-

ric acid. Store in a Teflon or glass bottle.
Ascorbic Acid, 5%. Dissolve 5.0 g of ascorbic acid in water, and dilute with water to 100
mL. Prepare fresh solution daily.
Barium Chloride, 12%. (For determination of sulfate.) Dissolve 60 g of barium chlo-

ride dihydrate in water, filter, and dilute with water to 500 mL.
Barium Chloride, 40%. (For determination of sulfate in phosphate salts.) Dissolve 40 g

of barium chloride dihydrate in 80 mL of water, filter, and dilute with water to 100 mL.
Barium Nitrate, 0.1 M. Dissolve 0.654 g of barium nitrate in water, and dilute with
water to 25.0 mL.
Benedict’s Solution. Dissolve 87 g of sodium citrate dihydrate and 50 g of anhydrous

sodium carbonate in 400 mL of water. Heat to aid dissolution, filter if necessary, and dilute with
water to 425 mL. Dissolve 8.65 g of copper sulfate pentahydrate in 50 mL of water. Add this
solution, with constant stirring, to the alkaline citrate solution, and dilute with water to 500 mL.
Bromine Water. (A saturated aqueous solution of bromine.) In a well-ventilated fume

hood, add about 10 mL of liquid bromine to 75 mL of water in a 100-mL bottle so that
when the mixture is shaken, undissolved bromine remains in a separate phase. Store in an
amber glass bottle.
Bromphenol Blue Indicator, 0.10%. Dissolve 0.10 g of the sodium salt form of
bromphenol blue in water, and dilute with water to 100 mL (pH 3.0–4.6).
Bromthymol Blue Indicator, 0.10%. Dissolve 0.10 g of bromthymol blue in 100 mL

of dilute alcohol (1 + 1), and filter if necessary (pH 6.0–7.6).
Brucine Sulfate. Dissolve 0.30 g of brucine sulfate in dilute ACS Reagent-grade sulfuric
acid (2 + 1), previously cooled to room temperature, and dilute to 500 mL with the dilute
acid. If necessary, nitrate-free acid should be prepared as follows: In a well-ventilated fume
hood, dilute the concentrated sulfuric acid (about 96% H2SO4) to about 80% H2SO4 by
adding it to water, heat to dense fumes of sulfur trioxide, and cool. Repeat the dilution and
fuming three or four times.
Cadmium Nitrate, 3%. Dissolve 3.0 g of cadmium nitrate tetrahydrate in water, and
dilute with water to 100 mL.
Chloramine-T. Dissolve 10.0 mg of chloramine-T (sodium p-toluenesulfonchloramide)

trihydrate in 100.0 mL of water. Prepare fresh.
Chromotropic Acid, 0.01%. Dissolve 0.02 g of recrystallized chromotropic acid (see

below) in concentrated, nitrate-free sulfuric acid, and dilute to 200 mL with the acid. Store
in an amber bottle.
To recrystallize chromotropic acid. Add solid sodium sulfate to a saturated aque-
ous solution of chromotropic acid. Filter the resultant crystals using suction with a
Buchner funnel, wash with ethyl alcohol, and air dry. Transfer the crystals to a beaker,
add sufficient water to redissolve, and then add just enough sodium sulfate to repre-
cipitate the salt. Filter as before, wash with ethyl alcohol, and dry at a temperature no
higher than 80 °C. Store in an amber bottle.
Note: If an orange or red precipitate forms in the solution of the sample before
the addition of the reagent, discard the solution and start again. Immediate addi-
tion of the reagent should prevent formation of such a precipitate.
Chromotropic Acid, 1%. Dissolve 1.0 g of chromotropic acid in water, and dilute with
water to 100 mL.
Cobalt Chloride, 6%. Dissolve 5.95 g of cobalt chloride hexahydrate and 2.5 mL of
hydrochloric acid in 20 mL of water, and dilute to 100 mL with water.
Crystal Violet Indicator, 1%. Dissolve 100 mg of crystal violet in 10 mL of glacial ace-
tic acid.
Cupric Sulfate, 6%. Dissolve 6.24 g of cupric sulfate pentahydrate and 2.5 mL of hydro-
chloric acid in 20 mL of water, and dilute to 100 mL with water.
Dichlorofluorescein Indicator, 0.1%. Dissolve 100 mg of dichlorofluorescein in 50

mL of alcohol, add 2.5 mL of 0.1 N sodium hydroxide, mix, and dilute with water to
100 mL.
5,5-Dimethyl-1,3-cyclohexanedione, 5% in Alcohol. Dissolve 5.0 g of 5,5-

dimethyl-1,3-cyclohexanedione in alcohol, and dilute with alcohol to 100 mL.
Dimethylglyoxime, 1%. Dissolve 1.0 g of dimethylglyoxime in alcohol, and dilute

with alcohol to 100 mL.
N,N-Dimethyl-p-phenylenediamine. Add 50 mL of sulfuric acid to approximately

175 mL of water. Cool to room temperature, add 0.25 g of N,N-dimethyl-p-phenylenedi-
amine (p-aminodimethylaniline) monohydrochloride, and dilute with water to 250 mL.
Diphenylamine. Dissolve 10 mg of colorless diphenylamine in 100 mL of sulfuric acid.

In a separate beaker, dissolve 2 g of ammonium chloride in 200 mL of water. Cool both
solutions in an ice bath, and cautiously add the sulfuric acid solution to the water solution,
taking care to keep the resulting solution cold. The solution should be nearly colorless.
Dithizone Extraction Solution. Dissolve 15 mg of dithizone in 500 mL of chloroform

and 5 mL of alcohol. Store the solution in a refrigerator.
Dithizone Indicator Solution. Dissolve 26 mg of dithizone in 100 mL of alcohol.

Store in a refrigerator, and use within 2 months.
Dithizone Test Solution. Dissolve 5 mg of dithizone in 500 mL of chloroform. Keep

the solution in a polyethylene bottle, protected from light and stored in a refrigerator.
Eosin Y, 0.50%. Dissolve 50 mg of eosin Y in 10 mL of water.

Eriochrome Black T Indicator. Grind 0.20 g of Eriochrome Black T to a fine powder
with 20 g of potassium chloride.
(Ethylenedinitrilo)tetraacetic Acid, Disodium Salt, 0.2 M. Dissolve 37.2 g of

(ethylenedinitrilo)tetraacetic acid, disodium salt, dihydrate, in 475 mL of water, and dilute
Ferric Ammonium Sulfate Indicator, 8%. Dissolve 8.0 g of crystals of ferric ammo-
nium sulfate dodecahydrate in water, and dilute with water to 100 mL. A few drops of sul-
furic acid may be added, if necessary, to clear the solution.
Ferric Chloride, 4.5%. Dissolve 4.50 g of ferric chloride hexahydrate and 2.5 mL of
hydrochloric acid in water, and dilute to 100 mL with water.
Ferric Chloride, 5%. Dissolve 50 g of ferric chloride hexahydrate in dilute hydrochloric

acid (1 + 99), and dilute with the dilute acid to 500 mL.
Ferric Nitrate, 17%. Dissolve 17 g of ferric nitrate nonahydrate in 100 mL of water.

Ferric Sulfate, 12%. Add 13 mL of sulfuric acid to 60 g of ferric sulfate n-hydrate. Add
about 350 mL of water, digest on a hot plate (⬇ 100 °C) to dissolve, cool to room tempera-
ture, and dilute with water to 500 mL.
Ferroin Indicator, 0.025 M. Dissolve 0.70 g of ferrous sulfate heptahydrate and 1.5 g
of 1,10-phenanthroline in 100 mL of water.
Hexamethylenetetramine Solution, Saturated. Dissolve about 20 g of hexa-

methylenetetramine in 100 mL of water.
Hydrazine Sulfate, 2%. Dissolve 2.0 g of hydrazine sulfate in water, and dilute with
water to 100 mL.
Hydrochloric Acid, 10%. Dilute 118 mL of hydrochloric acid with water to 500 mL.
Hydrochloric Acid, 20%. Dilute 235 mL of hydrochloric acid with water to 500 mL.
Hydrochloric Acid, 6 M. Dilute 250 mL of hydrochloric acid with water to 500 mL.
Hydrogen Peroxide, 3%. Dilute 10 mL of hydrogen peroxide to 100 mL with water.
Prepare fresh at time of use.
Hydrogen Sulfide Water. In a well-ventilated fume hood, saturate 100 mL of water by

bubbling hydrogen sulfide gas for 1 min. This solution must be freshly prepared. Store in a
fume hood. Securely close container immediately after use.
Hydroxy Naphthol Blue Indicator. Use the mixture with sodium chloride that is com-
mercially supplied, or grind 0.20 g of hydroxy napthol blue with 30 g of sodium chloride.
Hydroxylamine Hydrochloride, 10%. Dissolve 10.0 g of hydroxylamine hydrochlo-

ride in water, and dilute with water to 100 mL.
Hydroxylamine Hydrochloride for Dithizone Test. Dissolve 20 g of hydroxyl-

amine hydrochloride in about 65 mL of water, and add 0.15 mL of thymol blue indicator
solution. Add ammonium hydroxide until a yellow color appears. Add 5 mL of a 4% solu-
tion of sodium diethyldithiocarbamate. Mix thoroughly, and allow to stand for 5 min.
Extract with successive portions of chloroform until no yellow color is developed in the
chloroform layer when the extract is shaken with a dilute solution of a copper salt. Add
hydrochloric acid until the indicator turns pink, and dilute with water to 100 mL.
Indigo Carmine, 0.10%. Dissolve 0.10 g of sample indigo carmine, dried at 105 °C, in
a mixture of 80 mL of water and 10 mL of sulfuric acid, and dilute with water to 100 mL.
Jones Reductor. In a well-ventilated fume hood, cover a 250-g portion of 20-mesh zinc
with reagent water in a 1-L suction flask (see apparatus description below). Pour a solution
containing 11 g of mercuric chloride in 100 mL of hydrochloric acid into the flask; slowly
mix and shake the system for about 2 min. Pour off the solution, and wash the amalgam
thoroughly with hot tap water, and then with reagent water. The column is charged with
six 250-g portions.
Apparatus. Use a dispensing buret, about 22 in. long and 2 in. in diameter,
equipped with a glass stopcock and a delivery tube, 6 mm wide and 3.5 in. long. The
reductor is charged with an 8-in. column of 20-mesh amalgamated zinc (1500 g) and,
on top of this, a 6-in. column of larger (1–2 cm) amalgamated zinc (about 750 g). The
delivery tube is connected to a 1-L flask through a two-hole rubber stopper. One hole
is used as an inlet; the other functions as an outlet for carbon dioxide gas.
Karl Fischer Volumetric Reagent. (Before making Karl Fischer reagent, see the dis-
cussion of commercial reagents starting on page 30.) In a well-ventilated fume hood, dis-
solve 254 g of iodine in 807 mL of pyridine in a 3-L glass-stoppered bottle, and add 2 L of
methanol. To prepare the active reagent, add 1 L of foregoing stock to a 2-L bottle, and cool
by placing the bottle in a slurry of ice pieces. Add carefully about 45 mL of liquid sulfur
dioxide, collected in a calibrated cold trap, and stopper the bottle. Shake the mixture until
it is homogeneous, and set aside for 24 h before use.
Lead Acetate, 10%. Cautiously dissolve 25 g of lead acetate trihydrate in water. If nec-
essary, add a few drops of acetic acid to clear the solution, and dilute with water to 250 mL.
Lithium Chloride, 30%. Dissolve 6.1 g of lithium chloride in water, and dilute with
water to 20 mL. Prepare fresh.
Metalphthalein-Screened Indicator, 0.2%. Dissolve 0.18 g of metalphthalein and

0.02 g of naphthol green B in water containing 0.5 mL of ammonium hydroxide, and dilute
to 100 mL with water.
Methyl Orange Indicator, 0.10%. Dissolve 0.10 g of methyl orange in 100 mL of

water (pH 3.2–4.4).
Methyl Red Indicator, 0.10%. Dissolve 0.10 g of methyl red in 100 mL of alcohol. See
page 443 for a description of the three forms of methyl red (pH 4.2–6.2).
4-(Methylamino)phenol Sulfate, 2%. (For determination of phosphates.) Dissolve

2.0 g of 4-(methylamino)phenol sulfate in 100 mL of water. To 10 mL of this solution, add
90 mL of water and 20 g of sodium bisulfite. Confirm the suitability of the reagent solution
by the following test: Add 1 mL of this reagent solution to each of four solutions containing
25 mL of 0.5 N sulfuric acid and 1 mL of ammonium molybdate–sulfuric acid reagent
solution. Add 0.005 mg of phosphate ion (PO4) to one of the solutions, 0.01 mg to a sec-
ond, and 0.02 mg to a third. Allow to stand at room temperature for 2 h. The solutions in
the three tubes should show readily perceptible differences in blue color corresponding to
the relative amounts of phosphate added, and the one to which 0.005 mg of phosphate was
added should be perceptibly bluer than the blank.
Methylthymol Blue Indicator. Grind 0.20 g of methylthymol blue to a fine powder

with 20 g of potassium nitrate.
Murexide Indicator. Grind 0.20 g of murexide to a fine powder with 20 g of potassium

nitrate.
Nessler Reagent. In a well-ventilated fume hood, dissolve 72 g of sodium hydroxide in

350 mL of water. Dissolve 25 g of red mercuric iodide and 20 g of potassium iodide in 100
mL of water. Pour the iodide solution into the hydroxide solution, and dilute with water to
500 mL. Allow to settle, and use the clear supernatant liquid. Nessler reagent prepared by
any of the recognized methods may be used, provided that it has equal sensitivity. Note:
After testing is done, the solutions should be kept separate as mercury waste, following
local and state laws.
Nitric Acid, 1%. Dilute 5.3 mL of nitric acid with water to 500 mL.
Nitric Acid, 10%. Dilute 53 mL of nitric acid with water to 500 mL.
Oxalic Acid, 4%. Dissolve 20 g of oxalic acid dihydrate in water, and dilute with water to
500 mL.
PAN Indicator, 0.10%. Dissolve 0.05 g of 1-(2-pyridylazo)-2-naphthol in 50 mL of

reagent alcohol.
PAR Indicator, 0.10%. Dissolve 0.10 g of 4-(2-pyridylazo)resorcinol in 100 mL of alcohol.

pH 6.5 Buffer. Dissolve 10.1 g of anhydrous sodium phosphate, dibasic, and 3.05 g of
citric acid monohydrate in water, and dilute with water to 500 mL.
1,10-Phenanthroline, 0.10%. Dissolve 0.10 g of 1,10-phenanthroline monohydrate

in 100 mL of water containing 0.1 mL of 10% hydrochloric acid reagent solution.
Phenol Red Indicator I, 0.10%. Dissolve 0.10 g of phenol red in 100 mL of alcohol,
and filter if necessary (pH 6.8–8.2)
Phenol Red Indicator II, pH 4.7. (For determination of bromide.) Dissolve 33 mg of

phenol red in 1.5 mL of 2 N sodium hydroxide solution, dilute with water to 100 mL (this is solu-
tion A). Dissolve 25 mg of ammonium sulfate in 235 mL of water; add 105 mL of 2 N sodium
hydroxide solution and 135 mL of 2 N acetic acid (this is solution B). Add 25 mL of solution A to
solution B, and mix. Using a pH meter, adjust the pH of this solution to 4.7 if necessary.
Phenoldisulfonic Acid. In a well-ventilated fume hood, dissolve 5 g of phenol in 30

mL of sulfuric acid, add 15 mL of fuming sulfuric acid (15% SO3), and heat at 100 °C for
2 h. Cool, and store in a glass bottle.
Phenolphthalein Indicator, 1%. Dissolve 1.0 g of phenolphthalein in 100 mL of alco-

hol (pH 8.0–10.0).
Potassium Chloride, 2.5%. Dissolve 2.5 g of potassium chloride in water, and dilute
Potassium Chromate, 10%. Dissolve 10.0 g of potassium chromate in water, and

Potassium Cyanide, Lead-Free, 2.5%. In a well-ventilated fume hood, dissolve 5.0 g

of potassium cyanide in sufficient water to make 10 mL. Remove lead by shaking with por-
tions of dithizone extraction solution. Part of the dithizone remains in the aqueous phase
but can be removed, if desired, by shaking with chloroform. Dilute the potassium cyanide
solution with water to 50 mL.
Potassium Dichromate, 10%. Dissolve 10.0 g of potassium dichromate in water, and

Potassium Ferricyanide, 5%. Dissolve 2.0 g of potassium ferricyanide in 40 mL of

water. Prepare the solution at the time of use.
Potassium Ferrocyanide, 10%. Dissolve 4.0 g of potassium ferrocyanide trihydrate

in 40 mL of water. Prepare the solution at the time of use.
Potassium Hydroxide, 0.5 N in Methanol. Dissolve 18 g of potassium hydroxide in

10 mL of water, and dilute with methanol to 500 mL. Allow the solution to stand in a stop-
pered bottle for 24 h. Decant the clean supernatant solution into a bottle provided with a
tight-fitting stopper. Store in a polyethylene or Teflon bottle.
Potassium Iodide, 10%. (For determination of free chlorine.) Dissolve 1.0 g of potas-
sium iodide in water, and dilute with water to 10 mL. Prepare the solution at the time of use.
Potassium Iodide, 16.5%. (For determination of arsenic.) Dissolve 16.5 g of potas-

sium iodide in water, and dilute with water to 100 mL.
Potassium Permanganate, 5%. Dissolve 2.5 g of potassium permanganate in 50 mL

of water.
Silver Diethyldithiocarbamate, 0.5%. Dissolve 1.0 g of silver diethyldithiocarba-

mate in 200 mL of freshly distilled pyridine.
Silver Nitrate, 1.7%. Dissolve 8.5 g of silver nitrate in 500 mL of water. Store in an
amber bottle.
Sodium Acetate, 10%. Dissolve 10 g of sodium acetate trihydrate in water, and dilute
Sodium Borohydride, 0.6%. Dissolve 0.6 g of sodium borohydride and 1.0 g of 50%
sodium hydroxide, and dilute with stirring to 100 mL with water.
Sodium Carbonate, 1%. Dissolve 5.0 g of sodium carbonate, anhydrous, in 450 mL of

water, and dilute with water to 500 mL. Store in a plastic bottle.
Sodium Citrate, 1 M. Dissolve 147 g of sodium citrate dihydrate in 450 mL of water,

Sodium Cyanide, 10%. In a well-ventilated hood, dissolve 10 g of sodium cyanide in

water, and dilute with water to 100 mL.
Sodium Diethyldithiocarbamate. Dissolve 0.25 g of sodium diethyldithiocarbamate

in water, and dilute with water to 250 mL.
Sodium Hydroxide, 10%. Dissolve 50 g of sodium hydroxide in water, and dilute with
water to 500 mL. Store in a polyethylene or Teflon bottle.
Sodium Hydroxide, Ammonia-Free, 6 N. Dissolve 24 g of sodium hydroxide in 100

mL of water.
Sodium Sulfite, 10%. Dissolve 2.5 g of sodium sulfite in water, and dilute with water to
20 mL. Keep in a tightly closed bottle. This solution should be freshly prepared.
Stannous Chloride, 2%. Dissolve 0.50 g of stannous chloride dihydrate in hydrochloric

acid, and dilute with hydrochloric acid to 25 mL. Store in a polyethylene or Teflon bottle.
Stannous Chloride, 40%. Dissolve 20.0 g of stannous chloride dihydrate in 50 mL of

hydrochloric acid. Store this solution in a polyethylene or Teflon container, and use within
3 months.
Starch Indicator, 0.5%. Mix 1.0 g of soluble starch with 10 mg of red mercuric iodide
and enough cold water to make a thin paste, add 200 mL of boiling water, and boil for 1
min while stirring. Cool before use.
Sulfuric Acid, 0.5 N. In a well-ventilated fume hood, slowly add 7.5 mL of sulfuric acid
to 375 mL of water, and dilute to 500 mL.
Sulfuric Acid, 4 N. In a well-ventilated fume hood, slowly add 60 mL of sulfuric acid to

375 mL of water, and dilute to 500 mL.
Sulfuric Acid, 10%. In a well-ventilated fume hood, slowly add 30 mL of sulfuric acid
to 375 mL of water, cool, and dilute with water to 500 mL.
Sulfuric Acid, 25%. In a well-ventilated fume hood, slowly add 70 mL of sulfuric acid
to 375 mL of water, cool, and dilute with water to 500 mL.
Sulfuric Acid, Chloride- and Nitrate-Free. In a well-ventilated fume hood, gently

fume sulfuric acid in a crucible or dish for at least 30 min. Allow to cool, and transfer to a
tightly capped glass bottle for storage.
Tartaric Acid, 10%. Dissolve 50 g of tartaric acid in 400 mL of water, dilute with water
to 500 mL, and filter into a plastic bottle.
Thymol Blue Indicator, 0.10%. Dissolve 0.10 g of thymol blue in 100 mL of alcohol
(acid range, pH 1.2–2.8; alkaline range, pH 8.0–9.2).
Thymolphthalein Indicator, 0.10%. Dissolve 0.10 g of thymolphthalein in 100 mL

of alcohol (pH 8.8–10.5).
Titanium Tetrachloride. In a well-ventilated fume hood, cool separately, in small

beakers surrounded by crushed ice, 5 mL of 20% hydrochloric acid and 5 mL of clear, col-
orless titanium tetrachloride. Add the titanium tetrachloride dropwise to the chilled
hydrochloric acid. Allow the mixture to stand at ice temperature until all of the solid dis-
solves, and then dilute the solution with 20% hydrochloric acid to 500 mL.
Triton X-100, 0.20%. Dissolve 0.20 g of Triton X-100 (polyethylene glycol ether of
isooctylphenol) in water, and dilute with water to 100 mL.
Variamine Blue B Indicator, 1%. Dissolve 0.20 g of variamine blue B in 20 mL of

water, and stir for 5 min.
Xylenol Orange Indicator Mixture. Grind 0.20 g of xylenol orange (either free acid
or sodium salt form) to a fine powder with 20 g of potassium nitrate. Alternatively, use a
solution of 0.1 g of xylenol orange in 100 mL of alcohol (for the acid form) or water (for
the salt form), depending on the solution matrix.
RIPA buffer for mammalian cell lysis:
50mM Tris-cl pH 7.4

150mM NaCl
1% NP40
0.25% Na-deoxycholate
1mM PMSF
1x Roche complete mini protease inhibitor cocktail
1x Pierce phosphatase inhibitor cocktail
Stock solutions used:
100mM Tris-Cl pH 7.4, 300mM NaCl

10% NP40 in water
10% Na-deoxycholate in water (stored at RT in dark)
200mM PMSF in isopropanol
Roche complete protease inhibitor cocktail tablets (cat# 04 693 124 001)
Pierce phosphatase inhibitor cocktail (100x) (cat # 78420)
For 10ml of lysis buffer:
5ml 100mM Tris-Cl pH 7.4, 300mM NaCl

1ml 10% NP40 in water
3.65ml water
250ul 10% Na-deoxycholate in water
1 tablet * Roche complete protease inhibitor cocktail
100ul* Pierce phosphatase inhibitor cocktail
50ul* 200mM PMSF in isopropanol
*add at time of use. PMSF is only good for ~30minutes after dilution into buffer.
Genomic Medicine Biorepository
GMB003
RNA and miRNA Isolation from Human Peripheral Blood

Note: This protocol assumes the investigator is beginning this with one full Yellow-Top (type
A) BD Vacutainer tube of human blood (equals roughly 8 ml) to yield approximately 30 ug of
RNA.
Additional Note: RNA is very easily degraded by ever-present RNAses. Therefore, all of the
tubes and solutions in this protocol must be RNAse-free (autoclaving does NOT inactivate
RNAses). One cannot overemphasize the need for a clean work environment when working with
RNA.
1) Transfer contents of tube into a 50 ml polypropylene conical centrifuge tube.

2) Bring volume to 45 ml with RBC Lysis Buffer (recipe follows protocol).
3) Let stand at room temperature for 10 minutes.
4) Pellet cells at 600 x g (approx 1,400 rpm) for 10 minutes in a room temp centrifuge
(program #3).
5) Carefully decant supernatant.
6) Gently resuspend the pellet in 1 ml of RBC Lysis Buffer and transfer to a 1.5 ml
microcentrifuge tube. – Let stand for 5 minutes.
7) Pellet cells for 2 minutes by centrifuging in a microfuge at room temperature at 3000
rpm.
8) Carefully aspirate the supernatant.
9) Resuspend the pellet in 1 ml of sterile DPBS.
10) Pellet cells as in step 7.
11) Carefully aspirate the supernatant.
12) Add 1200 µl of TRIzol solution to each tube and resuspend the cells. Note: for a full 8 ml
blood tube, the 1200 ul TRIzol solution can be split into 2, 600 µl aliquots and frozen at -
80 C until further processing.
13) Add 0.2 ml of Chloroform (CHCl 3 ) and vortex each tube for 15 seconds, ONE AT A
TIME.
14) Centrifuge the samples at 13,000 rpm for 10 minutes at 4°C.
15) Remove the upper phase and transfer to a clean microcentrifuge tube. Be careful not to
remove any of the white interface when collecting the upper phase of the extraction
16) For the future collection of micro RNA (miRNA), carefully remove ~20% of the volume
of the upper phase from step 16 and place into another clean, labeled, 1.5ml microfuge
tube. Store this aliquot at -80 C until further processing.
17) To the remaining upper phase from step 16, add an equal volume of cold isopropanol and
invert to mix.
18) The samples can be placed in a -20°C freezer to precipitate.
19) Samples are centrifuged at 13,000 rpm for 10 minutes at 4°C. Note: you may be able to
see a small white pellet of RNA at the bottom of the tube after this step.
20) Carefully decant the supernatant, and rinse the pellet with 0.5 ml of ice-cold 75% ethanol.
The 75% EtOH should be prepared RNase-free and stored at -20 C.
21) Centrifuge the samples at 13,000 rpm for 10 minutes at 4°C.
22) Decant the supernatant.
23) Using a pipettor, carefully remove all of the remaining liquid in the bottom of the tube.
Protocol GMB003rB: 20090216NP

Revised and approved 2012JULY26 by B. Sebastian
Genomic Medicine Biorepository
GMB003
24) Allow the pellet to dry for 5 to 10 minutes to remove any remaining ethanol.
25) Dissolve the RNA pellet by adding 20 µl of RNAse-free H 2 O to each sample.
26) RNA should be quantitated within 2 hours of elution. It can be kept at 4 C until that time;
it can also be held temporarily at -20 until permanent storage at -80. Repeated freeze-
thaws are to be avoided, so RNA should be aliquoted for transfer as soon as possible after
quantitation.
10x RBC Lysis Buffer
89.9 g NH 4 Cl
10.0 g KHCO 3
2.0 ml 0.5 M EDTA
Dissolve the above in approximately 800 ml ddH 2 O and adjust pH to 7.3. QS to 1 liter and mix
thoroughly. This solution is stable for 6 months at 2 – 8° C in a tightly closed bottle.
1x RBC Lysis Buffer
Simply dilute the 10x stock solution 1:10 with ddH 2 O. Stable for 1 week at room temperature.
TRIzol Reagent
Invitrogen Life Technologies: Cat No. 15596018
OR
RNA STAT-60 Reagent
Tel-Test: Cat No. CS-111
Other Reagents Needed
Phosphate Buffered Saline (PBS)
Isopropanol (2-propanol)
Ethanol
RNAse-free water
RNAse-Away (a cleaning solution that neutralizes RNAses on bench tops, pipettors, centrifuges,
and other equipment.
Protocol GMB003rB: 20090216NP

Revised and approved 2012JULY26 by B. Sebastian
Recipe for an RNAlater‐like buffer solution:
For 1.5 liters:
935 ml of autoclaved, MilliQ water
700 g Ammonium sulfate
Stir until dissolved
Add 25 ml of 1 M Sodium Citrate
And 40 ml of 0.5 M EDTA
Adjust to pH 5.2 using concentrated H2SO4 (about 20 drops= 1 ml)
Store at RT
15 liters:
7000 g Ammonium sulfate
9.35 liters of water (autoclaved in 20 liter carboy)
250 ml of 1 M Sodium citrate
400 ml of 0.5 M EDTA
~10 ml of concentrated H2SO4
For 60 liters (3 20 L carboys):
37.4 liters of water (~12.5 L per 20 L carboy)
28 kg Ammonium sulfate (9.3 kg per 20 L carboy)
1 liter sodium citrate (333 ml per 20 L carboy)
1.6 liter 0.5 M EDTA (533 ml per 20 L carboy)
~40 ml of concentrated H2SO4 (~13.3 ml per 20 L carboy) to pH 5.2 (use pH paper to check)

FUNCTIONAL GENOMICS CORE UNIVERSITY OF PENNSYLVANIA PETER WHITE, PH.D.
RNA Extraction from

Mammalian Tissues
Reagents
Choose the most appropriate kit for your sample. Consult the Qiagen web site for more
specifics, or call Qiagen technical support (1 (800) 362-7737):
• Qiagen RNeasy® Mini Kit (12): Qiagen (Cat. No. 74104). Yields <100 µg RNA from
0.5 - 30 mg tissue or 1x105 to 1x107 cells.
• Qiagen RNeasy® Midi Kit (12): Qiagen (Cat. No. 75142). Yields <1 mg RNA from 20 -
250 mg tissue or 5x106 - 1x108 cells.
• Qiagen RNeasy® Maxi Kit (12): Qiagen (Cat. No. 75162). Yields <6 mg RNA from
150 mg – 1 g of tissue or 5 x 107 – 5 x 108 cells.
QIAshredder (disposable cell-lysate homogenizers): Qiagen (Cat. No. 79654). Required for
Mini Kit.
14.3 M 2-Mercaptoethanol: Sigma (M3148-100ML)
Buffer RLT (guanidine thiocyanate buffer): 2-Mercaptoethanol (β-ME) must be added to Buffer
RLT (provided with kit – additional 220 ml bottles can be purchased from Qiagen, Cat. No.
79216) before use. β-ME is toxic; dispense in a fume hood and wear appropriate protective
clothing. Add 10 µl β-ME per 1 ml Buffer RLT. Buffer RLT is stable for 1 month after addition
of β-ME. Note: Buffer RLT is not the same as RNAlater.
TRIzol® Reagent (a ready to use mixture of phenol, guanidine isothiocyanate, red dye and other
proprietary components): Invitrogen (Cat. No. 15596-026)
Chloroform: Sigma (Cat. No. C-2432)
RNaseZap® RNase Decontamination Solution, 250 ml: Ambion (Cat. No. 9780)
RNase-free Water: Ambion (Cat. No. 9932 or 9922)
100 % (200 proof) Ethanol: Pharmco (Cat. No. 111ACS200)
70% Ethanol (in RNase-free H2O)
50 ml Falcon Tubes (BD Cat. No. 352070). 9400 RCF rating. Required for Maxi Kit.
15 ml Falcon Tubes (BD Cat .No. 352097). 6000 RCF rating. Required for Midi Kit.
1.7 ml Microcentrifuge Tubes (Denville Scientific, Cat. No. C-2170). Required for Mini Kit.
NOTE: We do not recommend the use of the RNAlater RNA stabilization reagent (this should not be confused with
Buffer RLT). Direct disruption of the tissue or cells in Buffer RLT or Trizol yields the best results in our experience.
If the tissue has been stored in RNAlater, the tissue must first be removed and placed into Buffer RLT and
immediately disrupted.
NOTE: We do not recommend DNase treatment of RNA samples for microarray analysis.
NOTE: If you are not experienced with RNA isolation, please read the literature and tips found on Ambion’s
website: http://www.ambion.com/techlib/basics/rnaisol/index.html.
REVISED 07/01/05 PAGE 1 OF 6

Protocol A – Cells (<107), Islets or Small Amounts of Tissue (<30 mg)

Sample preparation and homogenization
Lysis of the sample in either TRIzol® or Buffer RLT works well; TRIzol however may give
higher yields due to better lysis. Buffer RLT does not contain phenol, but must be used in
combination with the QIAshredder. The user should determine which method works best for
their application.
a. Islets. Prepare the islets according to standard protocols, working quickly, and at the last
step remove any residual wash buffer. Resuspend the islet pellet (nor more than 500
mouse islets) in a 2.0 ml microcentrifuge tube in 1 ml TRIzol or 600 µl of Buffer RLT +
BME. Wear gloves and follow standard RNA handling techniques at all times (see last
page). Once the buffer is added, vortex briefly and immediately proceed to the next step.
b. Cells. To a pellet of cells add 1 ml TRIzol or if using Buffer RLT, use 350 µl (<5 x 106
cells) or 600 µl (<1 x 107 cells). For direct lysis of cells grown in a monolayer, add 1 ml
TRIzol (<10 cm diameter dish) or if using Buffer RLT, use 350 µl (<6 cm diameter
dish) or 600 µl (<10 cm diameter dish), and collect cell lysate with a rubber policeman.
c. Tissue. This protocol should only be followed for use with small amounts of tissue (<30
mg); if using larger amounts of tissue follow Protocol B. The volume of lysis reagent
should be at least 10 fold greater than the volume of tissue. Thus for <30 mg of tissue use
1ml TRIzol or 600 µl Buffer RLT. The tissue must be completely disrupted by
homogenization as detailed in Protocol B, using a homogenizer probe that is appropriate
for small sample volume in a 2 ml microcentrifuge tube. If such a probe is not available
you can attempt to chop the tissue directly in lysis reagent using very fine scissors (Fine
Science Tools, Cat. No. 15012-12). Warning: incomplete lysis will dramatically reduce
yield.
RNA isolation using Qiagen RNeasy® Mini Columns

1. Dependant upon lysis method used, follow the appropriate procedure:
a. For samples processed with TRIzol:
i. Ensure that the sample is completely lysed: if working with cells or islets
vortex well, or if working with tissue ensure complete homogenization.
Samples can be stored at this point at <-70°C for at least 1 year.
ii. Incubate sample for 5 minutes in TRIzol at room temperature.
iii. Add 0.2 ml of chloroform for every 1ml of TRIzol used. Shake
vigorously for 15 seconds and incubate at room temperature for 2-3 min.
iv. Centrifuge samples 5 min. at 12,000 x g at 4°C.
Note: The 4°C spins are essential for phase separation. Room temperature spins may result
in variable phase separation thus resulting in variable RNA yields.
v. Transfer the aqueous phase to a fresh microcentrifuge tube. Proceed
immediately to Step 2.
Note: The aqueous phase is the colorless upper phase that corresponds to ~60% of the
volume of TRIzol used. The interphase should be fairly well-defined.

b. For samples processed with Buffer RLT:

i. Vortex briefly and immediately pipet the lysate directly onto a
QIAshredder spin column placed in a 2 ml collection tube, and centrifuge
for 3 min at maximum speed.
ii. If the sample will be prepared now, proceed immediately to Step 2 (note:
if a small pellet is visible carefully transfer the supernatant to a new
microcentrifuge tube by pipetting before the addition of ethanol).
Alternatively, if the sample will be stored, transfer the supernatant to a
new microcentrifuge tube by pipetting, snap freeze in liquid nitrogen and
store at <-70°C for at least 1 year.
2. Add 1 volume (usually 350 to 600 µl) of 70% ethanol to the cleared lysate, and mix
immediately by pipetting. Do not centrifuge. Continue without delay with step 3.
3. Apply up to 700 µl of the sample, including any precipitate that may have formed, to
an RNeasy mini column placed in a 2 ml collection tube (supplied). Close the tube
gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-
through, but not the collection tube.
Note: If the volume exceeds 700 µl, load aliquots successively onto the RNeasy column, and centrifuge
as above. Discard the flow-through after each centrifugation step.
4. Add 700 µl Buffer RW1 to the RNeasy column. Close the tube gently, and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the column. Discard the
flow-through and collection tube.
5. Transfer the RNeasy column into a new 2 ml collection tube (supplied). Pipet 500 µl
Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 15 s
at ≥8000 x g (≥10,000 rpm) to wash the column. Discard the flow-through, but not
the collection tube.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.
6. Add another 500 µl Buffer RPE to the RNeasy column. Close the tube gently, and
centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to dry the RNeasy silica-gel
membrane.
Note: Following the centrifugation, remove the RNeasy mini column from the collection tube carefully
so the column does not contact the flow-through as this will result in carryover of ethanol.
7. To elute, transfer the RNeasy column to a new 1.5 ml collection tube (supplied).
Pipet 30–50 µl RNase-free water directly onto the RNeasy silica-gel membrane.
Close the tube gently, wait 1 min, and centrifuge for 1 min at ≥8000 x g (≥10,000
rpm) to elute.
Note: Never elute with less than 30 µl water. If the expected RNA yield is >30 µg, repeat the elution
step (step 7) as described with a second volume of RNase-free water. Elute into the same collection
tube.
8. Keep eluted RNA on ice at all times and store at <-70°C.

Simplified RNA Isolation Protocol for Experienced Users

1. Complete sample pre-processing:
a. For samples processed in TRIzol:
i. Lyse / homogenize and incubate sample for 5 minutes in TRIzol at room
temperature.
ii. Add 0.2 ml of chloroform for every 1ml of TRIzol used. Shake
iii. Centrifuge samples 5 min. at 12,000 x g at 4°C. Transfer the aqueous
phase to a fresh microcentrifuge tube. Proceed immediately to Step 2.
b. For samples processed in Buffer RLT:
i. Pipet the lysate directly onto a QIAshredder spin column placed in a 2 ml
collection tube, and centrifuge for 2 min at maximum speed.
ii. Carefully transfer the lysate to a new microcentrifuge tube by pipetting,
avoiding any pellet of cellular debris.
2. Add 1 volume (usually 350 µl to 600 µl) of 70% ethanol to the cleared lysate, and
mix immediately by pipetting.
3. Apply up to 700 µl of the sample to an RNeasy mini column placed in a 2 ml
collection tube. Centrifuge for 15s and discard the flow-through. Repeat as necessary.
4. Add 700 µl Buffer RW1 to the RNeasy column. Centrifuge for 15 s as above.
5. Transfer the RNeasy column into a new 2 ml collection tube. Pipet 500 µl Buffer
RPE onto the RNeasy column. Centrifuge as above and discard the flow-through.
Add another 500 µl Buffer RPE to the RNeasy column. Centrifuge for 2 min to dry
the RNeasy silica-gel membrane.
6. To elute, transfer the RNeasy column to a new 1.5 ml collection tube. Pipet 30–50 µl
RNase-free water directly onto the RNeasy silica-gel membrane. Wait 1 min, and
centrifuge for 1 min to elute.
7. Keep eluted RNA on ice at all times and store at <-70°C.
RNA Isolation with Trizol – Traditional Method (not recommended)
1. Take the aqueous phase from Step 1 above and add an equal volume of ice-cold
Isopropanol. Allow the samples to precipitate at -20°C for 1 hour to overnight.
2. Pellet the RNA by centrifugation at maximum speed for 15 min at room temperature.
3. Decant the supernatant. Wash the pellet in 500 µl of 70% ethanol and spin again 10 min
at maximum speed.
4. Decant the supernatant, removing as much as possible without disturbing the pellet.
5. Briefly air dry RNA pellet and resolubilize in 30 - 50 µl RNAse-free deionized water. Be
sure to vortex or pipette up and down the sample to ascertain that pellet is resolubilized
fully. Store at <-70°C.
NOTE: If tissue is high in RNAses it may be beneficial to resuspend in 100% deionized formamide.

Protocol B– ANIMAL TISSUES (150 to 1000 mg)

Preparation
Clean the homogenizer probe by running it at maximum speed in the probe wash tubes (50 ml
conical tubes) as follows:
I. RNAseZAP®: 30s
II. DEPC Water: 30s
III. 100% Ethanol: 30s
IV. DEPC Water: 30s
Sample preparation and homogenization

The tissue should be placed in at least 10 volumes of TRIzol® or Buffer RLT (or approximately
10 µl reagent per 1 mg tissue). Larger volumes can be used if necessary or desired. Smaller
volumes may lead to RNA degradation during processing or storage. For the purposed of this
protocol we will assume that the weight of tissue used is 1 g. If using 150-500 mg of tissue, use
half the volumes used below - consult the Qiagen RNeasy® Handbook for further details.
1. Arrange appropriately labeled 50 ml conical tubes with 15 ml of TRIzol or Buffer
RLT with 2-Mercaptoethanol in each on ice.
2. Quickly dissect out up to 150 mg to 1 g of tissue and place immediately into tubes
containing cold TRIzol or Buffer RLT on ice.
Note: If working with the pancreas, the animal should be anesthetized and the pancreas removed while the
animal is still living - immediately proceed to step 3, before processing any other samples.
3. Immediately homogenize the tissue using a conventional rotor–stator homogenizer
for at least 45 s at maximum speed until the sample is uniformly homogeneous.
4. Place homogenate on ice and when all samples are complete proceed immediately to
step 7. Alternatively, snap freeze the homogenate in liquid nitrogen and store at <-
70°C for future RNA extraction (this may not be an option with pancreas).
5. Wash the homogenizer probe as above and repeat steps 2 to 4 for each sample. When
finished, ensure that the probe is thoroughly cleaned.
RNA isolation using Qiagen RNeasy® Maxi Columns

6. If the lysate has been stored frozen, thaw quickly and transfer to a new 50 ml tube
(storage at <-70°C reduces the integrity of the tube and they should never be directly
centrifuged).
7. Dependant upon lysis method used, follow the appropriate procedure:
a. For samples processed with TRIzol:
i. Incubate sample for 5 minutes in TRIzol at room temperature.
ii. Add 3 ml chloroform (0.2 ml for every 1ml of TRIzol used). Shake
iii. Centrifuge samples for 15 min at 10,000 x g at 4°C (9000 rpm in Sorval
SLA-600TC rotor).

Note: The 4°C spins are essential for phase separation. Room temperature spins may result
in variable phase separation thus resulting in variable RNA yields.
iv. Transfer the aqueous phase to a fresh tube. Use only this aqueous phase
in subsequent steps and proceed immediately to Step 8.
Note: The aqueous phase is the colorless upper phase that corresponds to ~60% of the
volume of TRIzol used. The interphase should be fairly well-defined.
b. For samples processed with Buffer RLT:
i. Centrifuge the tissue lysate for 15 min at 10,000 x g at 4°C (9000 rpm in
Sorval SLA-600TC rotor). Carefully transfer the supernatant to a new 50
ml tube by pipetting. Use only this supernatant (lysate) in subsequent steps
and proceed immediately to Step 8.
Note: In most preparations a small pellet will form, sometimes accompanied by a fatty
upper layer. Transferring the pellet or the fatty layer may reduce the amount of RNA that
binds to the membrane and cause the spin column to clog. To avoid transferring
contaminants, hold the pipet tip under the fatty upper layer, and don’t disturb the pellet.
8. Add 1 volume (9 to 15 ml) of 70% ethanol to the lysate, and mix thoroughly by
shaking vigorously. Do not centrifuge. Proceed immediately to step 9.
9. Apply half of the sample, including any precipitate that may have formed, to an
RNeasy maxi column placed in a 50 ml centrifuge tube (supplied). Maximum loading
volume is 15 ml. Close the tube gently, and centrifuge for 5 min at 3000–5000 x g
(5000 rpm in Sorval SLA-600TC rotor). Discard the flow-through.
10. Repeat step 9 with the remaining sample from step 8.
11. Add 15 ml Buffer RW1 to the RNeasy column. Close the centrifuge tube gently and
centrifuge for 5 min at 3000–5000 x g to wash the column. Discard the flow-
through.
12. Add 10 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and
centrifuge for 3 min at 3000–5000 x g to wash the column. Discard the flow-through
and replace column in centrifuge tube.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.
13. Add another 10 ml Buffer RPE to the RNeasy column. Close the centrifuge tube
gently, and centrifuge for 10 min at 3000–5000 x g to dry the RNeasy silica-gel
membrane. It is important to dry the RNeasy membrane since residual ethanol may
interfere with downstream reactions.
Note: This centrifugation ensures that no ethanol is carried over during elution. Following the
centrifugation, remove the RNeasy column from the centrifuge tube carefully so the column does not
contact the flow-through as this will result in carryover of ethanol.
14. To elute, transfer the RNeasy column to a new 50 ml collection tube (supplied). Pipet
the appropriate volume of RNase-free buffer of water (0.8 to 1.2 ml) directly onto
the RNeasy silica-gel membrane. Close the tube gently. Let it stand for 1 min, and
then centrifuge for 3 min at 3000–5000 x g.
15. Repeat the elution step (step 14) as described with a second volume of RNase-free
buffer or water. To obtain a higher total RNA concentration, this second elution step
may be performed by using the first eluate (from step 14). The yield will be 15–30%
less than the yield obtained using a second volume of RNase-free water, but the final
concentration will be higher.
16. Store at -80°C.

The Art of Protein Sequence Analysis
Sample preparation by SDS-PAGE and electroblotting onto PVDF

for Protein Sequencing
In order to get the best results out of an N-terminal sequence analysis of samples prepared by SDS-PAGE and
electro-blotting onto PVDF (polyvinylidene difluoride), the following considerations and do's and don'ts are
given below.
Note. N-terminal as well as internal sequence(s) of proteins purified by SDS-PAGE can also be obtained
without blotting (see our brochure for these services).
General remarks
Standard Laemmli [1] or Tris-Tricine [2] gels and blotting procedures can be used, however, initial yields of
sequence analysis may be as low as 5% of the original quantity of protein loaded onto the gel. If sufficient
sample (100 pmol or more) is used, no special precautions are necessary. The do's and don'ts given below
are especially of importance if low quantities of protein material should be sequenced. The most important
precautions to raise the initial yield are listed first; minor improvements are given later.
To minimize N-terminal blocking

Age gels for at least 24 hours. Add thioglycolate (0.1 mM final) to electrophoresis buffer. Minimize chemical
oxidation of amino acids by minimizing activators APS and TEMED. Prepare PAGE gels with the purest acryl
amide available. Treat acryl amide stock with Amberlite (1 g/100ml). Preferentially use PDA (piperazine diacryl-
amide, Biorad)[3] as crosslinker, and HPLC grade glycerol to minimize aldehyde formation.
Much higher initial yields of N-terminal amino acids are obtained if gel systems are used having a running gel
at lower pH (e.g. pH 7.2 [4,5]) then in case of the Laemmli system [6]. Sometimes, pre-electrophoresis with 0.1
mM thioglycolic acid leads to higher initial yields as well [6,7].
Use minimal amount of glacial acetic acid when staining/destaining (max. 1%). If possible, incubate samples
o
for 10 minutes at 37 C in denaturation buffer instead of boiling for 3-5 minutes for possible better Initial Yields.
To minimize losses
Use mini-gels (10 x 10 cm x 0.5-0.75 mm) when only limited protein material (< 100 pmol) is available [8].
Concentrate as much as possible protein per area of PVDF. There are several kinds of PVDF commercial
available (Immobilon Millipore/Waters, PVDF from Biorad, or ProBlott of Applied Biosystems). Problott offers
high binding capacity, and is advised.
About blotting
Proteins should be blotted as soon as possible after electrophoresis, without fixation of the gel. Soak the gel
for 5 minutes in transfer buffer prior to blotting. Meanwhile, pre-wet PVDF membrane with 100% methanol for
a few seconds, and immediately transfer it to the blotting buffer used. Use two layers of PVDF (especially with
Immobilon) for security.
Different transfer buffers can be used. Good results have been obtained with 10 mM CAPS pH 11 /10%
methanol [9] or a 25 mM Tris-HCl/192 mM Glycine buffer pH 8.3 /15% methanol [10] as transfer buffer, but
also with a borax buffer pH 8.25 /15% methanol. In case of proteins with unknown pI's, the first method is the
one of choice (keep pH of transfer buffer above isoelectric point).
Time and power (V/A) to be used during electro-blotting depends on a) type of apparatus; b) the range of Mw's
which are to be blotted; c) type of buffer; d) amount of SDS; and, therefore, should be tested and optimized.
General conditions for 'tank' (wet) blotters and using CAPS buffer: for 0.5 x 10 x 10 cm gels, 50V (about 100-
200 mA) for 30 minutes at room temperature; Tris-Glycine: 40V (300 mA), R.T. for 1-4 hours; for 'semi-dry' 1-
2
2.5 mA/cm for < 30 min.
Note! The electrophoresis conditions given in the article by P. Matsudaira [9] are not suitable (too fast; proteins
remain in gel and in 2nd layer).
Meditech Center L.J. Zielstraweg 1 9713 GX Groningen The Netherlands Tel.: +31 50 3172424 Fax: +31 50 3172420
E-mail: info@eurosequence.nl Internet: www.eurosequence.nl
The Art of Protein Sequence Analysis
If a 'tank-blotter' is used, it is advisable to use p.a. grade methanol for blotting buffers in order to minimize the
quantity of aldehydes which might be present.
Staining
Wash the blot with distilled water prior to staining (3 times 1 minute) to decrease the amount of salts and buffer
components. The electro-blotted proteins are bound very tight to the PVDF membrane and cannot be washed
off with water. Detect proteins preferentially by staining with Coomassie Brilliant Blue (stain max. 5 min.) [9].
First saturate PVDF with 100% methanol for a few seconds. Destain (partially) with 50% methanol. A quick
destaining can be performed by changing the destaining solution several times, and soaking for 1-2 minutes
between changes. A further destaining can be achieved by washing with 90% propanol, however, complete
destaining is not necessary for sequence analysis. Amido Black and Ponseau S can also be used as staining
techniques.
Initial Yields; quantity of protein available for sequencing

Due to partial N-terminal blocking during electrophoresis, low overall initial yields (5-30%, particularly with the
Laemmli system) are routinely found [6]. This should be taken into account when loading a sample on the gel.
SDS-PAGE systems with a running gel pH of 7.2 give higher initial yields (40-60%)[6,5,4]. Signals on a level of
1 pmol (if pure) can be interpreted reliable, although a level of 2-10 pmol is preferred.
The foregoing means, that for an N-terminal sequence analysis of proteins blotted onto PVDF, at least 25 - 50
pmol of protein should be applied onto an SDS-gel; and at least 100 pmol of starting material should be used if
internal sequences have to be determined in case of a blocked N-terminus; e.g. if you want to prepare
samples by means of a CNBr cleavage or Cleveland digestion followed by SDS-PAGE and blotting [7,11].
For fast internal sequence analysis of proteins present in a piece of gel, Eurosequence offers a special service,
which is suitable for 50 pmol quantities (or even less) of protein material. Just send us a piece of gel containing
the protein of interest, and we'll take care of the rest.
Furthermore
The total area of the piece of PVDF containing the protein band should be as small as possible and should not
2
exceed 0.8 cm . Try to concentrate as much as possible protein per area of PVDF.
We are able to sequence PVDF-blotted proteins almost as effective as proteins spotted on polybrene coated
glass filters. Runs with more than 40 positively identified residues have been performed with 100 pmol of
protein.
Literature
1. Laemmli, U.K. (1970) Nature, 277, 680-685.
2. Schäger, H. and von Jagow, G. (1987) Anal. Biochem. 166, 368-379
3. Artoni, G. et al. (1984) Anal. Biochem. 137, 420-428.
4. Shapiro, L.A., Vinuela, E. & Maizel, J.V. (1967) B.B.R.C. 28, 815-820.
5. Swank, R.T. and Munkres, K.D. (1971) Anal. Biochem. 39,462-477.
6. Moos, M. Jr., Nguyen, N.Y. & Liu, T.-Y. (1988) J. Biol. Chem. 263, 6005-6008.
7. M.J. Walsh et al (1988) Biochemistry 27, 6867-6876.
8. Matsudaira, P.T. & Burgess, D.R. (1978), Anal. Biochem. 87, 386-396.
9. P. Matsudaira (1987) J. Biol. Chem. 262, 10035-10038.
10. Towbin, H., Staehelin, T. & Gordon, J. (1979) P.N.A.S. 76, 4350-4354.
11. Williams, K.R. et al (1988) The FASEB Journal 2, 3124-3130.
Version date: June 17, 1997

File: PVDF.eng
Meditech Center L.J. Zielstraweg 1 9713 GX Groningen The Netherlands Tel.: +31 50 3172424 Fax: +31 50 3172420
E-mail: info@eurosequence.nl Internet: www.eurosequence.nl
Arizona Proteomics Consortium Protocol
09/22/09
Silver Staining Method Compatible with Mass Spectrometry

(Adapted from Blum, et al., Electrophoresis 8, 1987 and Shevchenko, et al., Anal Chem, 1996)
Equipment
shaker table
dedicated trays for silver staining
Solutions
Use the highest grade of chemicals/reagents available for all steps.

Prepare Sensitizer, Silver and Developer reagents fresh every time.
Fix 1: 50% methanol/10% acetic acid

Fix 2: 5% methanol/1% acetic acid
Fix 3: 50% methanol
Stop: 6% acetic acid or 40 mM Na2-EDTA
SENSITIZER: 0.02% sodium thiosulfate

20 mg Na2S2O3• 5H2O + 100 ml ddH2O
Reserve a small volume of Sensitizer (2.0 mL) for Developer solution.
SILVER: 0.2% silver nitrate

200 mg AgNO3 + 100 ml ddH2O Keep at 4ºC until used
DEVELOPER: sodium carbonate/sodium thiosulfate/formaldehyde

6.0 g NaCO3 + 50 μl (37% stock) formaldehyde + 2ml Sensitizer (0.02%
Na2S2O3• 5H2O) + 100 ml ddH2O (it may take a moment for the Na2CO3
to dissolve)
Method
Begin protocol immediately following electrophoresis
Following incubation, SILVER must be discarded in hazardous waste container designated

for metals. DO NOT discard in sink.
1. Incubate gel in Fix 1 for 30 min with shaking.

2. Incubate gel in Fix 2 for 15 min with shaking.
3. Rinse gel in Fix 3 for 1min (optional, use for larger gels, 18 –24cm).
4. Wash gel in ddH2O with shaking, at least 3 x 10 min. Staining is best if wash in ddH2O
is extended, e.g. overnight.
5. Sensitize exactly 90 sec in SENSITIZER solution, with shaking.
6. Rinse gel in ddH2O 3 x 30 seconds.
7. Incubate gel in chilled SILVER solution for a minimum of 30 min, with shaking. Gel can
be left in SILVER solution for extended periods (e.g., overnight.).
8. Remove SILVER solution from gel and discard in appropriate hazardous waste container.
9. Rinse gel 3 x 1min **Absolute Maximum**
10. Incubate in DEVELOPER solution, with shaking. Protein stain will become visible
within 1-5 minutes, depending upon the total protein concentration; develop gel to
desired intensity.
11. Stop with 100 mL 6% acetic acid or 40 mM Na2-EDTA, shake for 5 min.
12. Wash gel in ddH2O. Store stained gel in ddH2O at 4ºC.
13. Cut out and destain bands ASAP after staining the gel, follow the silver destaining
protocol.
Destaining Silver Stained Gel Bands Prior to Protease

Digestion
Stock solutions
Use the highest grade of chemicals and water available.
60 mM potassium ferricyanide
198 mg potassium ferricyanide + 10 ml H2O
This solution can be stored in a amber colored glass or plastic bottle for several months.
Mix 1:1 with the 200 mM sodium thiosulfate solution only when ready to destain
bands/spots.
200 mM sodium thiosulfate

316 mg sodium thiosulfate + 10 ml H2O
This solution can be stored in a clear colored glass or plastic bottle for several months.
Mix 1:1 with the 60 mM potassium ferricyanide solution only when ready to destain
bands/spots.
Method
1. Destain the band using 30 mM potassium ferricynide/100 mM sodium thiosulfate

solution until the brown color disappears. Use sufficient solution to cover the
band. The solution may need to be changed if it takes longer than 5 minutes to
destain. DO NOT let the band sit in this solution too long.
2. Remove the destain solution and wash the band for 5-10 minutes at least 5 times
with H2O, the solution should be clear.
3. If the sample will not be digested right away store in water or freeze the gel band.
Protocol for Silver Staining of Gels
Optimized for Mass Spectrometry and Protein Identification
GUIDELINES
Silver staining is used for sensitive detection of proteins Here we describe a silver staining protocol that has been
separated by 1D and 2D SDS PAGE with detection limits from optimized for mass spectrometric analysis. The protocol
0.5-5 ng. Many silver staining protocols and commercial results in confident protein identifications and high
staining kits are not compatible with mass spectrometry sequence coverage by MALDI MS and ESI MS due to a high
due to the use of cross-linking reagents. Commercial recovery of peptides from the stained gel. The protocol has
silver staining kits compatible with mass spectrometry are been tested and documented in many publications (Mortz,
available from different suppliers, including ProteoSilver E et al, Proteomics 1 (11), 1359-63, 2001).
Plus (Sigma) and Dodeca Silver Stain (BioRad).
MATERIALS
Use high quality chemicals and ultra pure water (18.2 Mohm). Agitate the gels and perform work in a fume hood. Wear
gloves at all stages and use clean staining trays with lids to avoid keratin contamination of the gels.
PROCEDURE
1. Run 1D or 2D gel. Incubate the gel in Fixer (40% ethanol, 10% acetic acid, 50% H2O) for 1 hr.
2. Wash the gel in H2O for at least 30 min. NB! Overnight washing with several changes of water will remove all
acetic acid, reduce background staining and increase sensitivity.
3. Sensitize the gel in 0.02% sodium thiosulfate (0.04 g Na2S2O3, 200 ml H2O) for only 1 min. NB! Longer time
will decrease peptide recovery from the gel.
4. Wash gel in H2O for 3 x 20 sec.
5. Incubate gel for 20 min in 4ºC cold 0.1% silver nitrate solution (0.2 g AgNO3, 200 ml H2O, 0.02% formaldehyde
(add 40 μL 35% formaldehyde just before use). NB! Staining is enhanced with cold AgNO3.
6. Wash the gel in H2O for 3 x 20 sec.
7. Place the gel in a new staining tray. NB! Residual AgNO3 on the gel surface and staining tray will increase
background staining.
8. Wash the gel in H2O for 1 min.
9. Develop the gel in 3% sodium carbonate (7.5 g Na2CO3 in 250 ml H2O), 0.05% formaldehyde (add 125 μL 35%
formaldehyde just before use). NB! Change developer solution immediately when it turns yellow. Terminate
when the staining is sufficient.
10. Wash the gel in H2O for 20 sec.
11. Terminate staining in 5% acetic acid for 5 min.
12. Leave the gel at 4 ºC in 1% acetic for storage. Prior to MS analysis the gel is washed in water for 3 x 10 min to
ensure complete removal of acetic acid.
Questions to: info@alphalyse.com

# 2001607
www.alphalyse.com
http://proteomics.cancer.gov
STANDARD OPERATING PROCEDURE

Title: Evaluation using Western Blot
SOP#: M-103
Version #: 1 Author: R. Saul
Date Approved: Feb. 5, 2009 Date Modified:
1. PURPOSE
The purpose of this document is to describe the procedure for performing

Western Blot analysis for the characterization of proteins.
2. SCOPE
This procedure may be used for proteins with molecular weights in the
range of 10KDa to 250KDa. Other procedures must be used for proteins
outside of this range.
3. RESPONSIBILITIES
It is the responsibility of person(s) performing this procedure to be familiar

with lab safety procedures. The interpretation of results must be done by
a person trained in the procedure and familiar with such interpretation.
4. EQUIPMENT
 Criterion™ Blotter assembly includes: Cell assembled with plate

electrodes, Criterion gel holder cassette, fiber pad pack, gel blot
assembly tray, sealed ice cooling unit, roller, magnetic stir bar; BioRad
Cat. #170-4070
 Criterion™ Blotting Sandwiches includes: 0.2µm nitrocellulose transfer
membrane and filter paper; BioRad Cat. #162-0232
 Labcor™ Mini Western Blot staining trays, 35x86x12.7mm; Continental
Lab Products Cat. #731-002 or equivalent
 Magnetic stir plate
 BioRad PowerPac™ HC power supply or equivalent
 Rocking platform; LabLine model 4831 or equivalent
 Water bath or heating block with appropriate insert pre-heated to 90°C
M-103.1 Page 1 of 7
 Epson photo quality scanner or equivalent

 Precision pipettes
 Additional equipment for one-dimensional gel electrophoresis (see
SOP for SDS-PAGE, M-100)
5. MATERIALS
 Pre-cast, 18-lane 4-15% Tris-HCl polyacrylamide gel; BioRad Cat.

#345-0023
 One dimension SDS-PAGE running buffer: 10x Tris/Glycine/SDS
Buffer diluted to 1x with deionized water; BioRad Cat. #161-0732
 Protein molecular weight standards: Dual Color Precision Plus
Protein™ Standards; BioRad Cat. #161-0374
 Coomassie Brilliant Blue 250 staining solution; BioRad Cat. #161-0436
 Transfer Buffer (Tris/Glycine with 20% Methanol): For each liter, add
100mL of 10x Tris/Glycine Buffer (BioRad Cat. #161-0734) to 700mL
deionized water and 200mL of MeOH and pre-chill to 4ºC. Prepare at
least 1.5 liters.
 TMB substrate (1-Step TMB-Blotting); Pierce-Thermo Scientific Cat.
#34018
 Phosphate Buffered Saline (PBS), 10x solution (Fisher Scientific Cat.
#BP399-1) diluted to 1x with deionized water to give 11.9 mM
phosphate, 137mM NaCl, 2.7mM KCl, pH 7.4
 PBS containing 0.1% Tween 20 (PBST): add 1mL of Tween 20 to 1L of
PBS
 Laemmli Sample Buffer; BioRad Cat. #161-0737
 SuperBlock™ Blocking Buffer-Blotting in PBS; Pierce-Thermo
Scientific Cat. #37517
 Powder-free gloves
6. REAGENTS
 Sample Antigen to be tested

 Antibodies corresponding to sample antigens to be tested
 Detection Antibody - Goat anti-mouse antibody conjugated to
horseradish peroxidase (GAM-HRP); Jackson Laboratories Cat. # 115-
035-003 or equivalent
M-103.1 Page 2 of 7
 Mouse IgG, (10.0 mg/mL); Lampire Biological Laboratory Cat.

#7404304 or equivalent
7. PROCEDURE
7.1. Prepare Sample Antigen for PAGE.

7.1.1. For Coomassie staining: Dilute Antigen to 1mg/mL in PBS.
Prepare Antigen for PAGE run by diluting the 1mg/mL prep 1:4
with Laemmli Sample Buffer. Typical dilution scheme: 10µL of
Antigen (1 mg/ml) diluted with 30µL of Laemmli Sample Buffer,
yielding a final concentration of 0.25mg/mL.
7.1.2. For Western blot: Prepare Intermediate Stock of sample
antigen (Sample IS) by diluting antigen with PBS to a
concentration of 0.1 mg/mL. Typical dilution scheme if starting
antigen concentration is 1 mg/mL, add 10µL of antigen to 90µL
of PBS (Sample IS). Then dilute (1:5): one part Sample IS with
4 parts Laemmli sample buffer to yield a final concentration of
0.02 mg/mL. Typical dilution scheme: add 100µL of Sample IS
to 400µL of Laemmli Sample Buffer
7.1.3. Heat Sample Antigens at 90ºC for 5 minutes in a pre-heated
water bath or heating block.
7.2. Prepare mouse IgG control
7.2.1. Prepare Control Intermediate Stock (Control IS) by serially
diluting mouse IgG (10mg/mL) with PBS to a final concentration
of 0.01 mg/mL. Typical dilution scheme: first dilute 10µL of
mouse IgG with 90µL of PBS (1:10 dilution = 1 mg/ml), then
dilute 10µL of the 1:10 solution with 990µL of PBS (1:100
dilution) yielding a final total dilution of 1:1000 (0.01 mg/ml).
7.2.2. Dilute (1:5) one part Control IS (0.01 mg/ml) with 4 parts
Laemmli sample buffer to yield a final concentration of 0.002
mg/mL. Typical dilution scheme: add 100µL of Control IS to
400µL of Laemmli sample buffer
7.2.3. Heat at 90ºC for 5 minutes in a pre-heated water bath or heating
block.
7.2.4. Note: unused Control IgG can be stored at -20ºC
7.3. Run prepared sample antigen (7.2.1) and control IgG (7.2.2) on one-
dimensional SDS-PAGE following the procedure described in SOP M-
100.
7.3.1. A typical SDS-PAGE run for a Western Blot of 3 antibodies
using an 18-lane pre-cast gel is diagrammed below:
M-103.1 Page 3 of 7
7.3.2. Load a typical gel with samples and standards as follows:

7.3.2.1. 10µL of molecular weight standards in lanes 1, 4, 9, & 14
7.3.2.2. 5µL of molecular weight standards in lanes 7, 12 & 17
7.3.2.3. 10µL of the 0.25mg/mL antigen preparation in Lane 2
7.3.2.4. 2.5µL of the 0.02mg/mL antigen preparation in lanes 5,
10 & 15
7.3.2.5. 2.5µL of the 0.002 mg/ml Control IgG preparation in
lanes 6, 11 & 16
7.4. Following completion of the SDS-PAGE run, open cassette housing
and slice gel between Lanes 3 & 4 with razor blade. Place lanes 1-
3 in shallow container and add enough Coomassie Blue R250 stain
to cover the gel. Process gel following the staining procedure
described in SOP M-100.
7.5. Remove the remaining gel from cassette housing (lanes 4-18) and
place in 250mL 1x Transfer Buffer (pre-chilled to 4ºC) for 15
minutes with gentle rocking. Always wear gloves when handling
membranes, filter paper, or gels to prevent contamination.
7.6. Set up transfer apparatus:
7.6.1. Fill the Criterion Blotter tank with 1x Transfer Buffer to about
50% of the fill volume.
7.6.2. Place a magnetic stir bar inside the tank
7.6.3. Place the ice block in the ice block pocket in the back of the cell.
Flip down the lever to hold the ice block in place.
7.7. Gel & membrane sandwich set up:
7.7.1. Pour some chilled 1x Transfer Buffer into each compartment of
the gel/blot assembly tray.
7.7.2. Place the cassette in the back/large compartment of the tray:
Open the cassette so that the red side with handle is vertical
(anode) and the black side (cathode) is lying horizontally and
submerged in 1x Transfer Buffer.
M-103.1 Page 4 of 7
7.7.3. Place a fiber pad on top of the black side of cassette,

submerged in buffer. Push on the fiber pad with gloved finger
tips to thoroughly wet the pad.
7.7.4. Place a piece of filter paper on top of the fiber pad (it should wet
immediately).
7.7.5. Gently place the pre-equilibrated gel on top of the filter paper.
Use roller to remove air bubbles that may be trapped under gel.
7.7.6. Slowly wet the nitrocellulose membrane in the 1x Transfer
Buffer in front compartment by gradually submerging the
membrane starting at one end and continuing until entire
membrane is wet.
7.7.7. Remove wet membrane from the front compartment and place it
on top of the gel, taking care not to trap air. The membrane
should not be moved or adjusted once it touches the gel. Use
roller to roll out bubbles.
7.7.8. Place a piece of filter paper on top of the membrane. Run the
roller gently over the top of the filter paper to remove any air
bubbles trapped in the sandwich.
7.7.9. Wet a second fiber pad in the front compartment of the tray,
again using finger tips to completely saturate the pad. Then
place the wet fiber pad on top of the second filter paper.
7.7.10. Lower the clamp-side of the cassette and lock In the closed
position.
7.8. Blot Transfer
7.8.1. Move the locked cassette into the groove in the blotter tank,
aligning the red side of the card with the red electrode. Make
sure the magnetic stirrer is free to move.
7.8.2. Add remaining 1x Transfer Buffer to the fill level marked on the
tank and place tank on magnetic stir plate. Turn on stir plate to
low setting.
7.8.3. Put lid on transfer tank, plug the cables into the power supply
7.8.4. Set power supply to 100V and press start. Run the blot for 30
minutes.
7.8.5. Upon completion of the run, disassemble the blotting sandwich
and remove the membrane for development (6.9). Clean the
cell, fiber pads and cassette with multiple rinses of deionized
water.
M-103.1 Page 5 of 7
7.9. Blot Development

7.9.1. Place membrane in container containing 100 mL of
SuperBlock Blocking Buffer-Blotting. Incubate for 15 minutes
with gentle rocking.
7.9.2. Remove membrane and slice into 3 segments between wells
8/9 and 13/14 as described in the diagram (6.3.1). The slicing
can be done with a razor blade or scalpel.
 Note: the diagram is just an example and the order and
location will depend on the number of antibodies to be
evaluated and the number of wells used per evaluation. .
7.9.3. Place sliced blot segments into separate mini western blot
staining trays designated for each antibody.
7.9.4. Rinse each blot in tray with 10 mL of PBST.
7.9.5. Dilute antibodies to be evaluated1:5000 in PBST: Add 2µL of
1mg/mL antibody solution to 10 mL of PBST.
7.9.6. Add diluted antibodies to mini trays containing the blots.
7.9.7. Incubate with gentle rocking for 30 minutes.
7.9.8. Rinse each blot 3x with 10mL of PBST.
7.9.9. Dilute HRP-labeled detection antibody 1:7500: add 4µL of
goat anti-mouse HRP antibody to 30mL of PBST.
7.9.10. Add 10 mL of diluted HRP-labeled detection antibody to each
mini tray containing the blots.
7.9.11. Incubate with gentle rocking for 30 minutes.
7.9.12. Rinse each blot 3x with 10 mL of PBST.
7.9.13. Transfer each blot to clean, unused mini trays and rinse with
an additional 10 mL of PBST.
7.9.14. Add 3 mL of TMB substrate and incubate for approximately 5
to 10 minutes, or until the control IgG line has developed.
7.9.15. Stop reaction by rinsing with deionized water.
7.9.16. Remove membranes and allow to air dry.
7.9.17. Scan the final developed blots using a photo quality scanner
such as an Epson Perfection 4490 Photo or equivalent.
M-103.1 Page 6 of 7
8. REFERENCED DOCUMENTS
8.1.1. http://www.jove.com/index/details.stp?ID=759
8.1.2. http://www.afcs.org/reports/v1/BC0001/Protocols/Protein%2
0Transfer%20BIORAD.pdf
8.1.3. Bio-Rad Criterion™ Blotter assembly Instruction Manuals
8.1.4. SOP M-100: SDS-PAGE Evaluation
8.1.5. SOP M-101: Protein Determination by BioRad Protein Assay
M-103.1 Page 7 of 7
Laboratory Biological Safety Manual
And
Standard Operating Procedure
Guidance Document
What information is needed in a laboratory standard operating procedure?

Well developed Standard Operating Procedures (SOPs), or Standard Laboratory
Practices, are essential tools for any laboratory that manipulates biological research
materials. SOP’s serve as a resource to train new lab staff, supplement recurrent training
curriculum, and as a valuable reference in the event of an emergency. The following
components should be considered when establishing minimum best practices in a research
laboratory. However, the value of an SOP only holds merit if it is implemented by all
laboratory workers and enforced by the Principle Investigator.
Purpose
What is the overall purpose of the SOP?
As an example:
This SOP has been developed to outline the hazards involved with research using
lentiviral vectors and how to safely manipulate these materials to avoid any lab acquired
infection (LAI).
Principal Investigator Responsibilities

The Principal Investigator (PI) has the primary responsibility for ensuring that their
laboratory is safe through establishment of the initial risk assessment, administrative
controls, and by ensuring that all work is conducted with appropriate engineering
controls. PI’s must adhere to all applicable guidelines and regulations. The PI is
responsible for the safe use of biological agents in their laboratory.
Laboratory Staff/Student Responsibilities

The laboratory staff and students are responsible for knowing the potential hazards
contained within their respective work areas, in particular the biological material and
appropriate procedures and practices to be used in the laboratory. Laboratory employees
must follow approved laboratory procedures and safety guidelines at all times. For
information regarding minors working in laboratory areas, please contact EHS.
General Emergency Contact Information

The first page of the SOP should include the following information so that it is quickly
and easily accessible:
• Emergency Contact Information for:
o Principle Investigator -
o Lab Supervisor(s) -
o Environmental Health & Safety (EHS) – 301-846-1451
o Occupational Health Services (OHS) - 301-846-1096
o Protective Services – 301-846-1091 (to be used after regular business
hours)
• Locations of:
o Fire Alarms
o Fire extinguishers
o Eyewashes (Note: eyewashes should be flushed monthly)
o Emergency Showers (Note: showers should receive maintenance every 6
months)
o Scrub Kits (Chlorhexidine and Betadine) - available from EHS
Principles of Biosafety
According to the Center for Disease Control publication, Biosafety in Microbiological &
Biomedical Laboratories (BMBL) 5th edition, a fundamental objective of any biosafety
program is the effective management of potentially harmful biological agents through the
use of administrative and engineering controls such as containment. Administrative
controls are management tools that provide staff with a set of guidelines describing how
to safely operate with biological hazards in an environment designed for containment of
the hazard. The term "containment" is used in describing safe methods, facilities, and
equipment for managing infectious materials in the laboratory environment where they
are being handled or maintained. The purpose of containment is to reduce or eliminate
exposure of laboratory workers, other persons, and the outside environment to potentially
hazardous agents. The use of vaccines may provide an increased level of personal
protection. The risk assessment of the work to be done with a specific agent will
determine the appropriate combination of these elements.
Biological Risk Assessment

(refer to http://www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf )
Risk assessment is a process used to identify the hazardous characteristics of a known
infectious or potentially infectious agent or material, the activities that can result in a
person’s exposure to an agent, the likelihood that such exposure will cause a laboratory
acquired infection (LAI), and the probable consequences of such an infection. The
information identified by risk assessment will provide a guide for the selection of
appropriate biosafety levels and microbiological practices, safety equipment, and facility
safeguards that can prevent LAIs. The risk assessment will determine the biological
safety containment level (BSL) at which the work can be safely conducted.
A risk assessment will identify the following:

• Pathogenicity of material: disease incidence and severity
• Route of transmission: parenteral, airborne or ingestion
• Agent stability-how easily the material can be decontaminated
• Infectious Dose
• Concentration of working quantities and infectious organisms per ml of
stock solution
• Origin of material
• Availability of prophylaxis (vaccination)
• Medical surveillance programs and exposure management (post-exposure
prophylaxis)
• Staff skill level and training
Additional risk assessment questions:

• Are procedures being used that may produce an aerosol?
• Are procedures being used that may use needles or sharps?
• Are cultures or cell concentrates purified?
• Are larger volumes being used (>10Liters)
• Has the research material been altered, if so, how, and how does that affect
the hazards associated with the material?
• Is the material attenuated (to reduce the virulence/infectious nature of the
material)?
• How could an exposure event occur?
Biological Safety Containment Levels

(refer to Section IV of http://www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf)
While the majority of the NCI-F biological research laboratories are considered Biosafety
Level 2 (BSL2), BSL2-enhanced or BSL3, a general understanding of the different
biosafety levels and their relevant best practices is recommended.
BSL-1, BSL-2, and BSL-3

Biosafety Level 1 is suitable for work involving well-characterized agents not
known to consistently cause disease in immuno-competent adult humans, and
present minimal potential hazard to laboratory personnel and the environment.
BSL-1 laboratories are not necessarily separated from the general traffic patterns
in the building. Work is typically conducted on open bench tops using standard
microbiological practices. Special containment equipment or facility design is not
required, but may be used as determined by appropriate risk assessment.
Laboratory personnel must have specific training in the procedures conducted in
the laboratory and must be supervised by a scientist with training in microbiology
or a related science.
Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving
agents that pose moderate hazards to personnel and the environment. It differs
from BSL-1 in that 1) laboratory personnel have specific training in handling
pathogenic agents and are supervised by scientists competent in handling
infectious agents and associated procedures; 2) access to the laboratory is
restricted when work is being conducted; and 3) all procedures in which
infectious aerosols or splashes may be created are conducted in
biological safety cabinets (BSCs) or other physical containment equipment.
Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or

production facilities where work is performed with indigenous or exotic agents
that may cause serious or potentially lethal disease through inhalation route
exposure. Laboratory personnel must receive specific training in handling
pathogenic and potentially lethal agents, and must be supervised by scientists
competent in handling infectious agents and associated procedures.
Spills in the Laboratory - Call EHS when a significant spill occurs and if assistance is
needed for clean-up.
Steps to cleaning-up a biological spill:
• Post the area where the spill occurred to avoid the potential for cross
contamination and unnecessary exposure to others in or near the work area.
• Since a spill has the potential to generate an aerosol, let the aerosol settle
(~30min).
• Put on a clean lab coat and gloves and obtain or prepare fresh disinfectant
solution for cleaning-up the spill (freshly prepared 10 % bleach solution or
EPA-Approved Disinfectant). (Refer to EHS Safetygrams ISM-161
http://home.ncifcrf.gov/ehs/uploadedFiles/Ism-161.pdf and ISM-208
http://home.ncifcrf.gov/ehs/uploadedFiles/ISM-208%20(2).pdf )
• Lay paper towels or other absorbent material on top of the spill zone
• Starting from the outermost edge of the spill and working in toward the center
of the spill, pour disinfectant onto the absorbent material and allow sufficient
contact time to render any contaminants inactivated. Contact time is typically
10 to 30 minutes and should be noted on the bottle of disinfectant.
• Properly dispose of waste (including your gloves) in appropriate waste
containers for final disposal.
Decontamination and Virus Inactivation

(Refer to EHS Safetygrams ISM-145 http://home.ncifcrf.gov/ehs/uploadedFiles/Ism-
145.pdf and ISM-208 http://home.ncifcrf.gov/ehs/uploadedFiles/ISM-208%20(2).pdf )
Laboratories should have specific SOP’s for working with and effectively
decontaminating and/or inactivating the following materials:
• HIV and other lentiviruses
• Adenovirus
• Viral vectors (Adeno-, Retro-, Lenti-, etc.)
• All human materials
• Prions
• Recombinant DNA/RNA
• Genetically modified animals
• Biological toxins
• All other BSL-2 and BSL-3 infectious materials
• Any other biological hazards
Training
Laboratory staff should have both instructional and hands-on training for all biological
hazards present in the laboratory. Technicians should demonstrate proficiency in
techniques before being permitted to perform laboratory procedures independently. EHS
provides health and safety training courses, not limited to the following:
• Occupational Safety and Health Administration (OSHA) Bloodborne
Pathogens Standard 29 CFR 1910.1030
• Adenovirus and adenoviral vectors
• Lentivirus and lentiviral vectors
• Diphtheria toxin
• Toxoplasma gondii
• Listeria monocytogenes
Laboratory-specific training should be provided by the PI, lab manager or senior scientist
who has several years experience working with the biological materials and can direct
staff in safe handling of the materials so as to avoid any accidental exposures. All
training sessions should be documented, to include the training session topic, information
covered, instructor, date, and attendees. Depending on the significance of the hazards
involved, curriculum training and proficiency testing may be warranted.
Medical Surveillance
Depending on the biological materials manipulated in the laboratory, vaccinations and/or
other medical surveillance programs may be warranted for employees. Please contact
EHS for assistance with medical surveillance eligibility and enrollment.
Personal Protective Equipment (PPE)
(Refer to EHS Safetygram ISM-139 http://home.ncifcrf.gov/ehs/uploadedFiles/ISM-
139.pdf )
Aside from standard PPE that is required in a BSL2 laboratory, what other PPE is
required to safely manipulate the agent? Does this PPE change if you are manipulating
animals and if so, state this in your SOP. The following is a list of common PPE found in
the laboratory:
• Gloves
• Safety Glasses/Goggles
• Gowns/Aprons
• Laboratory Coat
• Tyvek Suits
• Respiratory protection (fit test and medical clearance required)
• Surgical Mask
• Shoe Covers
• Bonnets
• Face Shields
Biohazard Warning Signs and Posting

Each laboratory must clearly display a sign that provides safety information to visitors
and service personnel. Contact EHS for more information.
Biocontainment and Biological Safety Cabinets (BSC’s) (refer to Appendix A of

http://www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf )
Appendix A presents information on the design, selection, function and use of BSCs,
which are the primary means of containment developed for working safely with
infectious microorganisms.
Please also reference the BSC purchase checklist for approved BSC models for purchase
(http://web.ncifcrf.gov/campus/als/downloads/ChecklistBiologicalSafety.xls).
• Determine if augmentation of laboratory practices is necessary above and

beyond facility biocontainment designation (i.e.- Biosafety Level 2 laboratory
with Biosafety Level 3 practices and procedures, respirator usage, additional
PPE, etc).
• Properly maintained Class I and II BSCs, when used in conjunction with good
microbiological techniques, provide an effective containment system for safe
manipulation of moderate and high-risk microorganisms (Biosafety Level 2
and 3 agents).
• Reference EHS Safetygrams ISM-144, ISM-203, and ISM-206 or contact
EHS for additional training on BSC’s and other engineering control
equipment.
Biological Waste Disposal

174.pdf )
How will the biological material be disposed of?
• Refer to the following EHS website for additional information on medical
waste and autoclave waste disposal.
(http://home.ncifcrf.gov/ehs/ehs.asp?id=66).
• All recombinant material is considered infectious and must be disposed of as
biological waste.
•
Housekeeping
Special practices include: decontaminating work surfaces after completing the work with
the infectious materials, keeping non-research animals out of the laboratory, and
reporting all spills and accidents.
Facility renovations
• Laboratories that are relocating or closing should contact EHS for appropriate
laboratory clearances
• New laboratories scheduled for occupancy require an EHS pre-occupancy
walk through prior to commencement of laboratory operations. Contact EHS
to schedule.
• Either of the above laboratory visits should also be coordinated with any
applicable Facilities Maintenance and Engineering (FME) activities.
Facility decontaminations
(Refer to EHS Safetygrams ISM-123 http://home.ncifcrf.gov/ehs/uploadedFiles/Ism-
123.pdf and ISM-124 http://home.ncifcrf.gov/ehs/uploadedFiles/Ism-124.pdf )
• Contact EHS to coordinate building or laboratory area decontaminations with
an outside contractor
Autoclaving
(Refer to EHS Safetygram ISM-125 http://home.ncifcrf.gov/ehs/uploadedFiles/Ism-
125.pdf )
• EHS monitors autoclave effectiveness and performance on a recurring basis
with B.Stearothermophilus indicators
• Contact EHS to participate in the autoclave monitoring program (301-846-
1451)
Needles and Sharps Precautions

(Refer to EHS Safetygram ISM-146 http://home.ncifcrf.gov/ehs/uploadedFiles/Ism-
146.pdf )
• Substitute plastic-ware when possible
• DO NOT bend, shear, break, recap or remove needle from syringe
• Use sharps containers (don’t fill more than ¾ full), which must be located as
close to work area as possible.
• Non-disposable sharps should be placed in a rigid container for transport to
appropriate area for decontamination, such as by autoclaving
• Only needle-locking syringes or disposable syringe-needle units (needle is
integral to the syringe) should be used
• Syringes that re-sheathe the needle, needleless systems, and other safety
devices may also be used
• Do not handle broken glass directly by hand. Use a mechanical device such
as forceps or protective gloves to pick up and dispose of glass in a glass box
or sharps container
Shipping Biological Material
191.pdf )
• Shipping is regulated by Department of Transportation (DOT) and
International Air Transport Association (IATA)
• A Request for Shipment form must be completed for all shipments available
on the EHS website (http://web.ncifcrf.gov/campus/safety/wizard/).
• Contact EHS (301-846-1451) for questions regarding the hazardous
classification of a shipment
Intra-Facility Transportation of Biological Materials

158.pdf )
• Transportation of biological material from one location to another should be
done with the sample in a primary container, with a secondary sealed and
leak-proof container with a sufficient quantity of absorbent between the
primary and secondary containers. The transport container should also be
• labeled to identify the contents in the event the parcel is misplaced or dropped
and spills.
Security
• Call Protective Services with all issues concerning security (301-846-1091)
• Buildings secured by card key access only
• Containment laboratory entrance and exit
o Restricted access
o Minimize unnecessary access by casual visitors, vendors, or other persons
Select Agents/Toxins
• Research involving Select Agents/Toxins is regulated by CDC/USDA
• Department of Justice security risk assessment and regulatory authorization
and inspections must be successfully completed prior to receipt of any select
agent
• Contact EHS for assistance if you are considering work with select agents and
toxins (301-846-1451)
Additional Resources for Information

Resources for information, consultation, and advice on biohazard control,
decontamination procedures, and other aspects of laboratory and animal safety
management include:
AAALAC International
Association for Assessment and Accreditation of Laboratory Animal Care International
5283 Corporate Drive
Suite 203
Fredrick, MD 21703-2879
Telephone: (301) 696-9626
Fax: (301) 696-9627
Website: http://www.aaalac.org
American Biological Safety Association
American Biological Safety Association
1200 Allanson Road
Mundelein, IL 60060-3808
Telephone: (847) 949-1517
Fax: (847) 566-4580
Website: http://www.absa.org/
CDC Etiologic Agent Import Permit Program

Centers for Disease Control and Prevention
Etiologic Agent Import Permit Program (Mailstop: F-46)
Atlanta, Georgia 30333
Telephone: (404) 718-2077
Fax: (404) 718-2093
Website: http://www.cdc.gov/od/eaipp/
CDC Office of Health and Safety

Centers for Disease Control and Prevention
Office of Health and Safety (Mailstop: F-05)
1600 Clifton Road
Atlanta, Georgia 30333
Telephone: (404) 639-7233
Fax: (404) 639-2294
Website: http://www.cdc.gov/od/ohs/
Select Agents General Information

Website: http://www.selectagents.gov/
FFPE Fixation and Embedding UCHC Research Histology Core
Standard Protocol for Formalin-Fixed Paraffin Embedded Tissue Samples
1. Harvest tissue and rinse in PBS to remove blood

2. Place tissue in at least 10 volumes of buffered formalin (3.7% formaldehyde:10mM
phosphate buffer, ph-7.4) or buffered paraformaldehyde (3.7% paraformaldehyde: 10mM
phosphate buffer, ph-7.4)
- incubate for necessary fixation time
1-2mm slices – 2-3h RT
5-10mm thick – 5h RT
>10mm thick – 2-3h RT + 4oC O/N
3. Rinse tissue 1-2X with PBS, Store at 4oC in 70% ETOH in H2O
4. Prepare tissue processing histology cassettes for samples
- Label PI name in pencil on side
- Label Date/Sample ID on front
5. Prepare transport/storage bucket with 70% ETOH in H2O
6. Trim and transfer individual samples into histology cassette,

clamp closed and immerse in the 70% ETOH in the storage
bucket.
Notes:
 Samples should be placed in the cassettes in the
same orientation that they need to be embedded.
(i.e. the face of the tissue that needs to be cut first
Ver. 02/16/2011
should be placed towards the bottom of the cassette)

 Do not allow individual samples to dry at all – one-at-a-time
 Multiple tissues can fit into one cassette
 Tissues should not be crushed, trim to fit in cassette used (multiple depth cassettes
are available).
 Small tissue pieces < 1-2mm should be placed between blue sponges within
cassette
7. Cover all cassettes with excess 70% ETOH and store at 4oC until submitted to core facility
8. Drop off samples at core facility (L-1017) and complete intake form:
Information required – PI, FRS/Grant Coding to bill to, contact person, contact Phone, email,
processing, slides and stain request, specific requests (eg. We will embed tissues ourselves to
assure specific orientation).
< Leave Forms Here
< Leave Samples Here
9. Histology Core Personnel will contact you when samples are ready or if PI does not have
account available on Research Store Portal.
Ver. 02/16/2011
Materials and Purchasing Information:

Item Catalogue No. Vendor Price
Standard Histology Cassettes (white) 22-272-416 Fisher Case of 1000 for $119.30
Histology Sponges (blue) NC9518995 Fisher Case of 100 for $21.95
Formalin (5gal) 23-245685 Fisher Each for $123.68
Paraformaldehyde, 40%, 100ml NC9810518 Fisher Each for $42.88
Tissue/Sample Containers (500ml) 02-544-226 Fisher Case of 25 for $112.59
Ver. 02/16/2011
RESEARCH HISTOLOGY WORK REQUEST
Date:_______________________
PI:___________________________
Name: ____________________________ Phone Number:________________
FRS Number: _____________________ Email: ________________________
Services Requested:
Paraffin Processing and Embedding: Number of Samples _______________________
Paraffin Sectioning: Number of Slides per Block__________________________
Number of Sections per Slide ________________________
H&E Staining: Number of H&E per Sample/Block ____________________
Which Slide to Stain (first, last, etc…)___________________
Special Staining: Type of Special Stain _______________________________
Which Slide to Stain (first, last, etc…)_________________
How the cassettes are labeled: ____________________________________
Special Instructions or Comments-please provide any additional details below:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
Any questions should be directed to the Histology Core Director:
Dr. Kevin Claffey
Phone: 860-679-8713
Email: claffey@nso2.uchc.edu
Statistics formula sheet This has mean nθ and variance nθ(1 − θ).
The Poisson distribution:
Summarising data λx exp(−λ)
p(x) = for x = 0, 1, 2, . . . .
x!
Sample mean:
n This has mean λ and variance λ.
1X
x= xi .
n
i=1
Continuous distributions
Sample variance:
n n
! Distribution function:
1 X 1 X
s2x = (xi − x)2 = x2i 2
− nx . y
Z
n−1 n−1 F (y) = P (X ≤ y) = f (x) dx.
i=1 i=1
−∞
Sample covariance:
Density function:
n n
!
1 X 1 X d
g= (xi −x)(yi −y) = xi yi − nx y . f (x) = F (x).
n−1 n−1 dx
i=1 i=1
Evaluating probabilities:
Sample correlation:
Z b
g
r= . P (a < X ≤ b) = f (x) dx = F (b) − F (a).
sx sy a
Expected value:
Probability Z ∞
E(X) = µ = xf (x) dx.
Addition law: −∞
P (A ∪ B) = P (A) + P (B) − P (A ∩ B). Variance:

Z ∞ Z ∞
Multiplication law: Var(X) = (x − µ)2 f (x) dx = x2 f (x) dx − µ2 .
−∞ −∞
P (A ∩ B) = P (A)P (B|A) = P (B)P (A|B).
Hazard function:
Partition law: For a partition B1 , B2 , . . . , Bk
f (t)
h(t) = .
k
X k
X 1 − F (t)
P (A) = P (A ∩ Bi ) = P (A|Bi )P (Bi ).
i=1 i=1 Normal density with mean µ and variance σ 2 :
2
Bayes’ formula: 1 1

x−µ
f (x) = √ exp − for x ∈ [−∞, ∞].
2πσ 2 2 σ
P (A|Bi )P (Bi ) P (A|Bi )P (Bi )
P (Bi |A) = = Pk .
P (A) P (A|Bi )P (Bi ) Weibull density:
i=1
f (t) = λκtκ−1 exp(−λtκ ) for t ≥ 0.

Discrete distributions
Exponential density:
Mean value:
X f (t) = λ exp(−λt) for t ≥ 0.
E(X) = µ = xi p(xi ).
xi ∈S
This has mean λ−1 and variance λ−2 .
Variance:
X X Test for population mean
Var(X) = (xi − µ)2 p(xi ) = x2i p(xi ) − µ2 .
xi ∈S xi ∈S Data: Single sample of measurements x1 , . . . , xn .
Hypothesis: H : µ = µ0 .
The binomial distribution:
Method: √
• Calculate x, s2 , and t = |x − µ0 | n/s.

n x
p(x) = θ (1 − θ)n−x for x = 0, 1, . . . , n.
x • Obtain critical value from t-tables, df = n − 1.
• Reject H at the 100p% level of significance if |t| > c, • Calculate
where c is the tabulated value corresponding to col-
s2 = (n − 1)s2x + (m − 1)s2y /(n + m − 2).

umn p.
• Look in t-tables, df = n + m − 2, column p. Let the

Paired sample t-test tabulated value be c say.
• 100(1 − p)% confidence interval for the difference in
Data: Single sample of n measurements x1 , . . . , xn which
population means i.e. µx − µy , is
are the pairwise differences between the two original sets
of measurements.
(r )
1 1

Hypothesis: H : µ = 0. (x − y) ± c s2 + .
n m
Method:
√
• Calculate x, s2 and t = x n/s.
• Obtain critical value from t-tables, df = n − 1. Regression and correlation
• Reject H at the 100p% level of significance if |t| > c,
where c is the tabulated value corresponding to col- The linear regression model:
umn p.
yi = α + βxi + zi .
Least squares estimates of α and β:

Two sample t-test Pn
i=1
xi yi − n x y
Data: Two separate samples of measurements x1 , . . . , xn β̂ = , and α̂ = y − β̂ x.
(n − 1)s2x
and y1 , . . . , ym .
Hypothesis: H : µx = µy .
Confidence interval for β
Method:
• Calculate x, s2x , y, and s2y . • Calculate β̂ as given previously.
• Calculate • Calculate s2ε = s2y − β̂ 2 s2x .
r
s2 = (n − 1)s2x + (m − 1)sy /(n + m − 2).
2

• Calculate SE(β̂) = s2ε .
(n − 2)s2x
x−y
• Calculate t = r . • Look in t-tables, df = n − 2, column p. Let the
1 1

s2 + tabulated value be c.
n m
• 100(1 − p)% confidence interval for β is β̂ ± c SE(β̂).
• Obtain critical value from t-tables, df = n + m − 2.
• Reject H at the 100p% level of significance if |t| > c,
where c is the tabulated value corresponding to col- Test for ρ = 0
umn p.
Hypothesis: H : ρ = 0.
CI for population mean • Calculate 1/2

n−2

t=r .
Data: Sample of measurements x1 , . . . , xn . 1 − r2
Method: • Obtain critical value from t-tables, df = n − 2.
• Calculate x, s2x . • Reject H at 100p% level of significance if |t| > c,
• Look in t-tables, df = n − 1, column p. Let the where c is the tabulated value corresponding to col-
tabulated value be c say. umn p.
√
• 100(1 − p)% confidence interval for µ is x ± csx / n.
Approximate CI for proportion θ
CI for difference in population means
r
Data: Separate samples x1 , . . . , xn and y1 , . . . , ym . p(1 − p)
p ± 1.96
n−1
Method:
• Calculate x, s2x , y, s2y . where p is the observed proportion in the sample.
Test for a proportion
Hypothesis: H : θ = θ0 .
p − θ0
• Test statistic z = q .
θ0 (1 − θ0 )
n
• Obtain critical value from normal tables.
Comparison of proportions
Hypothesis: H : θ1 = θ2 .
• Calculate
n1 p1 + n2 p2
p= .
n1 + n2
• Calculate
p1 − p2
z= r
p(1 − p) n11 + n12
• Obtain appropriate critical value from normal tables.
Goodness of fit
Test statistic
m
X (oi − ei )2
χ2 =
ei
i=1
where m is the number of categories.

Hypothesis H : F = F0 .
• Calculate the expected class frequencies under F0 .

• Calculate the χ2 test statistic given above.
• Determine the degrees of freedom, ν say.
• Obtain critical value from χ2 tables, df = ν.
• Reject H : F = F0 at the 100p% level of significance
if χ2 > c where c is the tabulated critical value.
TE buffer (Tris-EDTA buffer)
Prepared by Ms Alex Aitken
TE is a commonly used buffer solution in molecular biology, especially in procedures involving DNA or
RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that
2+
chelates cations like Mg .
The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.
10 mM Tris, bring to pH 8.0 with HCl, 1 mM EDTA
A stock can be prepared and stored at RT
Component For 500ml

1M Tris pH 8* 5ml
0.5M EDTA pH 8 1ml
dH2O 496ml
Autoclave to sterilize. It is then best to remove a working aliquot and not repeatedly access the stock.
*the pH is usually adjusted to RNA 8.0 for DNA and 7.5 for.
The respective DNA and RNA nucleases are supposed to be less active at these pH values, but pH
8.0 can safely be used for storage of both DNA and RNA.
EDTA further inactivates nucleases, by binding to metal cations required by these enzymes.
Genomic and plasmid DNA can be stored in TE Buffer at 4ºC (39.2ºF) for short-term use, or -20ºC (-
4ºF) to -80ºC (-112ºF) for long-term storage.
Repeated freeze-thaw cycles should be avoided.
To make the component solutions
1M Tris
Tris (hydroxymethyl) aminomethane
MWT 121.4 g/mol
60.57 g in 0.5L mq water
pH to 8.0 using HCl
0.5M EDTA
Diaminoethane tetraacetic acid
MWT 372.2 g/mol
18.6 g in 100ml mq water
pH to 8.0 using NaOH
• EDTA will not be soluble until pH reaches 8.0 – this will take time (hours)
• Use vigorous stirring, moderate heat (if desired) and time
Alex Aitken Page 1 11/10/2012

FIXATION OF TISSUE FOR PARAFFIN EMBEDDING
For general histological staining

1. Cut 2 – 3 mm leaf sections with a razor blade.
2. Fix in 3:1 ethanol:acetic acid (a few ml in a glass vial) for 30’ at room
temperature. Make fixative fresh each week.
3. Remove fixative and add 2 ml 70 % ethanol. Leave for 30’ at room

temperature.
4. Replace 70 % ethanol with fresh 70% ethanol and store indefinitely

at 4oC.
NOTES.
If fixing tissue that is tougher than leaves use FAA as a fixative.
Overnight fixation at room temperature is usually adequate but samples
can be left for longer. Transfer to 50% then 70% ethanol prior to
storage at 4oC.
FAA
50 ml ethanol
5 ml acetic acid
10 ml 37% formaldehyde
35 ml H20
For in situ hybridization or immunolocalization

1. Make fixative fresh each time from paraformaldehyde powder.
Fixative: 4% paraformaldehyde
0.1% Tween 20
0.1% Triton X-100
Add solid to PBS (0.15 M NaCl, 10 mM sodium phosphate pH 7.5).

Stir in fumehood at 60oCto dissolve. Will need to add NaOH pellets
to get solid into solution and then re-pH with HCl. Chill on ice.
2. Cut 2 – 3 mm leaf sections with a razor blade and place in fixative.
Most plant tissues have a cuticle and will thus simply float on the
surface of the fixative. To allow penetration of the fixative, the
tissue must be vacuum infiltrated. To do this, sometimes you have to
weigh down the tissue under the surface, eg. by using a piece of wire
gauze, though the use of detergents often avoids this problem.
Place sample in a vacuum desiccator and apply a vacuum using an oil
pump (a bench type water pump does not provide adequate suction).
Care should be taken to ensure the solution does not boil under the
reduced pressure. Release vacuum slowly to avoid tissue disruption.
Formaldehyde vapour is volatile, so the fixative must be replaced
after the vacuum treatment.
3. Fix tissue overnight at 4oC.
4. Prior to storage, transfer through 30%, 40%, 50%, 60% and 70%
ethanol for an hour each at room temperature.
5. Store in 70% ethanol at 4oC

Total DNA isolation protocol Page 1 of 3
Total DNA isolation protocol

The procedure is suitable for all types of tissues from wide variety of animal, blood
and plant species. All DNA extraction steps are performed at weak acid pH (HEPES
free acid) and optionally with hot chloroform for 'difficult' samples, and at room
temperature.
The following protocol is designed for small and large tissue samples (tissue volume
1 00-200 μl).
Note that isolating genomic DNA not requires gentle mixing because the DNA not be
sheared by vortexing.
CTAB method for DNA extraction protocol

CTAB solution: 2% CTAB, 2 M NaCl, 1 0 mM Na3EDTA, 50 mM HEPES, pH
∼5.3;
1 00 ml: 2 g CTAB, 1 .2 g HEPES-acid, 2 ml 0.5 M Na3EDTA, 40 ml 5 M NaCl;
Chloroform-isoamyl alcohol mix (24:1 );
1 00% isopropanol (isopropyl alcohol, 2-propanol);
70% ethanol;
Fresh 1 xTE (1 mM EDTA, 1 0mM Tris-HCl, pH 8.0).
1 . 2 ml Eppendorf Safe-Lock [http://www.eppendorf.com/int/index.php?

sitemap=2.1 &pb=2e2a6e87de95e790&action=products&contentid=1 &catalognode=9775&productpag
microcentrifuge tube with tissue sample and glass ball (5 or 6 mm) freeze at
-80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz.
2. In 2 ml tube with mechanically disrupted seeds or leaves or herbarium or
blood or DNA solution (CTAB
CTAB purification) add fresh 1 ml CTAB solution
buffer with RNAse A (the sample volume should not exceed 20% of lysis
buffer), vortex very well and incubate the samples at 60-65°C during 30-60
min.
3. Add 700 µl of chloroform, vortex very well (in the MM300 Mixer Mill for 1 min
at 30 Hz); optionally: incubate the samples at 60-65°C during 30 min.
4. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the
upper aqueous layer to a new 2 ml microcentrifuge tube.
5. Add 700 µl of chloroform, vortex very well for 1 minute creating an emulsion
(in the MM300 Mixer Mill at 30 Hz).
6. Spin at maximum speed in a microcentrifuge for 5 minutes.
7. Transferred the upper aqueous layer to a new 2 ml microcentrifuge tube
which contains of 800 µl 2-propanol, vortex well and centrifuge the tubes at
maximum speed in a microcentrifuge for 3 minutes.
8. Discard supernatant and wash pellet by adding 1 .5 ml 70% EtOH, vortex well.
Centrifuge at 1 4,000 rpm for 2 min and discard ethanol.
9. The DNA pellet do not dry and dissolved immediately in 300 μl 1 xTE, pH 8.0
at 55°C for 1 0-20 minutes.
Guanidine thiocyanate method for DNA extraction protocol

GuTC extraction buffer: 2 M guanidine thiocyanate, 1 0 mM Na3EDTA, 50 mM
HEPES, pH ∼5.3;
the final concentration of guanidine thiocyanate may need to optimized for
certain plant/animals tissue from 0.5 to 4 M;
http://primerdigital.com/dna.html 20-10-2015

70% ethanol; 

microcentrifuge tube with tissue sample and glass ball freeze at -80°C, grind
in the MM300 Mixer Mill for 2 min at 30 Hz.
2. Add fresh 1 ml GuTC solution buffer (the sample volume should not exceed
20% of lysis buffer), vortex very well and incubate the samples at 60-65°C
during 30-60 min.
3. Add 700 µl of chloroform, vortex very well for 1 minute creating an emulsion.
Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the
upper aqueous layer to a new 2 ml microcentrifuge tube. Repeat this step.
which contains of 800 µl 2-propanol, vortex well and centrifuge the tubes at
maximum speed in a microcentrifuge for 3 minutes.
at 55°C for 1 0-20 minutes.
CTAB method for DNA extraction protocol from herbarium

CTAB solution: 2% CTAB, 2 M NaCl, 1 0 mM Na3EDTA, 50 mM HEPES, pH
∼5.3;

microcentrifuge tube with herbarium sample and glass ball (6 mm) grind in
the MM300 Mixer Mill for 1 5 min at 30 Hz.
2. Add fresh 1 ml CTAB solution buffer and add 500 µl of chloroform, vortex
very well (in the MM300 Mixer Mill for 3 min at 30 Hz); and incubate the
sample for 1 hour at 60-65°C.
3. Spin at maximum speed in a microcentrifuge for 5 minutes, transferred the
upper aqueous layer to a new 2 ml microcentrifuge tube.
4. Add 700 µl of chloroform, vortex very well (in the MM300 Mixer Mill for 3 min
at 30 Hz) for 3 minute creating an emulsion. Spin at maximum speed in a
microcentrifuge for 5 minutes.
which contains of 900 µl 2-propanol, vortex well.
at 55°C for 20 minutes.
Proteinase K method for DNA extraction protocol

1 . In 2 ml Eppendorf Safe-Lock [http://www.eppendorf.com/int/index.php?

tube with mechanically disrupted animal or plant tissues add fresh 1 ml of
 
extraction buffer (0.8 M guanidine thiocyanate, 1 0 mM EDTA, 5% Tween 20,

0.5% Triton X-1 00, 50 mM HEPES, pH 5.3**) with 200 μg of proteinase K,
vortex very well and incubate the samples at 55°C for several hours or better
overnight at 37°-55°C (the longer the better, until dissolve tissue) with
occasional vortexing.
2. Add 600 µl of chloroform, vortex very well for 1 minute creating an emulsion
(in the MM300 Mixer Mill at 30 Hz); optionally: incubate the samples at 55°C
during 30 min.
4. Transfer the supernatant into a new 2 ml tube containing 800 µl of
2-propanol and 1 00 μl 3M Na-acetate, vortex very well, and centrifuge the
tubes at maximum speed in a microcentrifuge for 4 minutes.
5. Discard the supernatant and add 1 .8 ml of 70% ethanol into tube and vortex
well; centrifuge the tube for 5 minutes at 1 4000 rpm and again discard the
supernatant.
6. Do not dry DNA pellet and dissolved immediately in 300 µl of 1 xTE, pH 8.0
(with RNAse A) at 55°C for 1 0-20 minutes.
* alternative extraction buffers for proteinase K

[http://en.wikipedia.org/wiki/Proteinase_K] :
1 % SDS, 1 0 mM EDTA, 50 mM Tris-HCl, pH 8.0;

3 M guanidine hydrochloride, 1 0 mM EDTA, 5% Tween 20, 0.5% Triton X-1 00, 30
mM Tris-HCl, pH 8.0
TECHNIQUES IN MOLECULAR BIOLOGY – BACTERIAL TRANSFORMATION

The uptake of exogenous DNA by cells that alters the phenotype or genetic trait of a cell is called transformation.
For cells to uptake exogenous DNA they must first be made permeable so the DNA can enter the cells. This state is
referred to as competency. In nature, some bacteria become competent due to environmental stresses. We can
purposely cause cells to become competent by treatment with chloride salts of metal cations such as calcium,
rubidium or magnesium and cold treatment. These changes affect the structure and permeability of the cell wall and
membrane so that DNA can pass through. However, this renders the cells very fragile and they must be treated
carefully while in this state. Competent cells vary in how well they take up DNA. We can express this The amount of
cells transformed per 1 µg of DNA is called the transformation efficiency. Too little DNA can result in low
transformation efficiencies, but too much DNA also inhibits the transformation process. Transformation efficiencies
4 7
generally range from 1 x 10 to 1 x 10 transformed cells per µg of added DNA.

E. coli bacteria are normally poisoned by the antibiotic ampicillin. Ampicillin inhibits synthesis of the
bacterial cell wall (in bacteria like E. coli , found between the inner and outer cell membranes), resulting in bacteria
that are very structurally weak. In the hypotonic media in which these cells grow, cells exposed to ampicillin will swell
and burst or not grow at all. For cells to survive, they must include a means to break down the ampicillin. The
plasmid has an additional gene coding for an enzyme, b-lactamase, that is secreted by cells and in a local area will
hydrolyze the ampicillin. Therefore, by adding ampicillin, only bacteria that contain the plasmid will survive. We also
need to be sure not to allow our transformed E. coli to become overgrown. If the colonies on the LB plates are large
they will break down enough ampicillin so that bacteria without the plasmid will survive and form satellite colonies in
the surrounding region of inactivated antibiotic. Another commonly used antiobiotic is kanamycin. This drug blocks
protein synthesis by binding irreversibly to the ribosome and preventing translation.

An Important Point When Transforming cells – Depending on which strain of bacteria you are using (for
simplicity we either us a strain to maintain DNA or a strain(s) better suited for protein expression) and how they were
made competent, there may be slight but important differences in using the cells for transformation. Therefore it is
important to first check that you are using the proper protocol. DO NOT LET CELLS WARM UP! This will allow the
holes in the cells to seal and your experiment will not work!
Antibiotic Stock Concentration Storage Working Conc (dilution)

Ampicillin (Sodium Salt) 50 mg/ml in water (500X) -‐20° C 100µg/ml (2 µl of stock/ml)
Chloramphenicol 34 mg/ml in EtOH (200X) -‐20° C 170 µg/ml (5 µl of stock/ml)
Kanamycin 25 mg/ml in water (500x) -‐20° C 50 µg/ml (2 µl of stock/ml)
Streptomycin 10 mg/ml in water (200X) -‐20° C 50 µg/ml (5 µl of stock/ml)
Tetracycline HCl 5 mg/ml in EtOH (100X) -‐20° C 50 µg/ml (10 µl of stock/ml)
Most transformation protocols can be divided into these four major steps:
++
1. Preincubation: Cells are suspended in a solution of cations (such as Ca ) and incubated at 0° C. The cations are
thought to complex with the negatively-charged phosphates in membrane lipids in the bacterium. The low
temperature congeals the cell membrane, stabilizing the distribution of charged phosphates and allowing them to
be more effectively shielded by the cations.
2. Incubation: DNA is added, and the cell suspension is incubated further at 0° C. The cations again are thought to
neutralize charged phosphates – this time in the DNA backbone – allowing DNA to adhere or at least be close to
the E. coli cell membrane.
3. Heat shock: The cell-DNA mix is rapidly heated to 37°– 42° C for a short period and then returned to 0° C. The
rapid temperature change creates a thermal imbalance on either side of the cell membrane, creating pores and a
draft that sweeps the plasmid into a small percentage of cells.
4. Recovery/Outgrowth: Nutrient broth is added to the transformation mix which is then incubated at 37° C (ideally
with shaking) before plating on selective media. Transformed cells recover from the treatment, amplify the
transformed plasmid, and begin to express the plasmid’s antibiotic resistance gene before encountering the
antibiotic.
Other Methods of Transformation

E. coli can also be transformed (replacing the heat shock) with a brief but intense electrical pulse in what is
termed electroporation. [For example: brief – 5 millisec, intense – 10,000 V/cm.] Very high transformation
efficiency can be achieved using specialized equipment (an electroporator), typically with disposable cuvettes
outfitted with electrodes. Preparation of 'electro-competent' cells is quite different from that of classically
prepared 'chemically competent' cells, although mid-log phase cells are still typically used. These cells must be in a
very low conductivity medium (few ions) such as 1 mM buffer with glycerol. Electroporation apparently does not
depend on DNA binding to the cell surface. The recovery period is particularly important following electroporation.
Variants of Competent Cell Preparation and Transformation

The 'rapid colony method' of competent cell preparation and transformation omits using mid-log phase cells and
a preincubation. Colonies on a plate are picked and resuspended in CaCl2, and DNA added immediately. Depending
on the antibiotic used for selection, a prolonged recovery period can also be omitted. Therefore, the procedure is
fast, and virtually foolproof for transformations not requiring high efficiency. Transformation efficiencies achieved with
3 4
the rapid colony method (5 x 10 – 5 x 10 transformants per µg supercoiled plasmid DNA), however, can easily be
4 6
~200 times less than those with cells prepared by more labor-intensive classical methods (5 x 10 – 10
transformants per µg DNA). The rapid colony method is perfectly adequate for transformation with purified, intact
plasmid DNA at high concentration. The method is wholly inadequate for transformations with manipulated DNA
(such as following ligations of linearized vector and insert DNAs in a traditional cloning experiment). These
molecules, consisting of relaxed circular DNAs, are 5 – 100 times less efficient at transforming cells than the same
quantity of intact, supercoiled plasmid DNA.
Transformation Parameters are Important

When performing a transformation, it is important to follow directions very carefully, as transformation efficiency
can be affected by many parameters. This is particularly true of the heat shock, which is typically optimized taking
into account the cell type, volume of transformation mix, tube type (shape, material, thickness), length and
temperature of the shock. Heat must be transferred rapidly throughout the cell mix to deliver a sharp shock. Cells
should be kept on ice at all times prior to the heat shock and transferred directly from ice to the water bath, and back
to ice during the procedure.
Antibiotic Selection
Ampicillin (Amp) is a practical, inexpensive antibiotic for routine selection of transformed cells, although some
prefer a sturdier (and more expensive) equivalent antibiotic such carbenicillin. Amp interferes with construction of
the peptidoglycan layer in the cell wall but kills only replicating cells that are assembling new cell membranes.
Therefore, if necessary, a recovery step can be omitted, although it is better to include one. When using other
antibiotics, such as Kanamycin (Kan), a recovery step is required before plating transformed cells. Kanamycin –
which blocks bacterial translation by irreversibly binding the 30S subunit of the ribosome – can kill both replicating
and non-replicating cells that are not expressing the resistance protein.
Satellite Colonies
If plates are incubated too long following a transformation (particularly when using an antibiotic like Amp), small
'satellite' colonies will appear that surround larger transformed colonies. In time, these can grow as large as the
original transformed colonies, and can arise more quickly on plates with many transformants. These are non-
transformed, nonresistant cells that grow in the 'antibiotic shadow' where Amp has been broken down by the large
resistant transformed colonies (the Amp resistance protein is secreted by the cells). Initially, these colonies will be
tiny, but will grow with time. Do not include tiny colonies (particularly surrounding a large colony) in your count of
transformants. It is best to score transformation plates in the morning following transformation before satellite
colonies appear or become large.
Materials & Equipment
Ice bath Bacterial loop or hockey stick Bunsen burner

Plasmid DNA (0.01 - 1.0 mg/ml) SOC liquid growth media Tube racks
37°C and 42°C water baths Ethanol in beaker (1 cm deep) LB agar/Amp/Carb/Kan plates
37°C incubator tube floaties
General Workflow / Protocol for traditional competent cells
• 20 - 100 µl of competent cells (ICE ICE ICE) into prechilled, labeled tubes.
• QUICKLY, mix 1-5 µl of plasmid DNA (10 pg to 100 ng) and mix by tapping of "flicking" the tube with your
finger.
• Incubate the mixture on ice for 20-30 min.
°
• Heat shock at 42 C for 45 sec, immediately place back in ice bucket and incubate for 2 min
• Add 250 µl of SOC and incubate at 37° C with shaking for 45 min. This is called the outgrowth step.
• Plate cells onto appropriate antibiotic containing LB Agar plates. Because it is difficult to predict the level of
transfection (cell health, plasmid quality and quantitity, overall technical handling and size of plasmid) it is
best to spread two plates with a low volume (10-50 µl) and a high volume (200-250 µl) of outgrown cell
culture.
• Spread with hockey stick, beads or inoculating loops. Ensure the entire culture is evenly spread over plate.
You are NOT trying to isolate streak in this procedure. Give plates a few min for liquid to seep into agar.
• Incubate plates at 37° C overnight with the agar upside down.
o
• After ~12-16 hours, remove plates from incubator, seal with parafilm and store at 4 C upside-down.
Lab experimental design:
Plasmid Preparation
• If the plasmid concentration is known, dilute plasmid with water (mol bio grade) to 0.05 mg/ml (aka 0.05
µg/µl).
• Use 1 µl of diluted DNA for each transformant. Calculate and record the mass (ng) of DNA used for the
transformation.
• If the plasmid concentration is unknown, use 1 µl of DNA.
• Record the antibiotic resistance and other information on your plasmid in your notebook.
Tranformation
• Record the type of compenent cells in the notebook - what cell strain, level of competent cells, if commercial
name of company and information on the preparation of cell strain.
• For this class, you will conduct two transformants - one transformant plated for the appropriate antibody (two
volumes - you decide how much for each) and the other will be divided equally onto an LB Agar plate without
antibioitic and another LB plate with an incorrect antibiotic.
Your Procedure/Experimental Design.
• Using the information in this handout and the recorded information on your cell strain, outline a protocol in
your lab book. Check with your instructor before beginning the experiment.
Notes:
SOB is Super Optimal Broth - a rich broth supporting cell growth that contains glucose. SOC is an SOB broth with
glucose. Full name is Super Optimal broth with Catabolite repression. In this case, glucose serves to ensure the
cells use glucose for metabolism leaving other energy sources (amino acids and lipids) for protein expression.
Use of SOC will ensure the antibiotic selective protein can be expressed in transformed cells. Without SOC, much
of the cell resources needed for protein expression will be used for metabolism instead
Outgrowth (aka Recovery) step - important for antibiotic resistance. Transformed cells must express protein to
survive in antibiotic medium/agar plate. In a pinch, Amp or Carb resistant cells can be directly plated onto
antibiotic containing agar. This is because Amp and Carb act by inhibiting enzymes which create the cell wall but
will not stop the cell from making protein needed to degrade the antibiotic. HOWEVER, Kan acts by inhibiting
translation and thus cells will not produce any protein. Cells transformed with Kan resistance plasmids thus need
the outgrowth/recovery period to produce the protein neomycin phosphotransferase II (encoded by the neo gene).
This is the enzyme that degrades Kan. BUT the cell must make the protein from the newly transformed plasmid
before the degredation of Kan can happen. Thus outgrowth is more important for Kan than Amp/Carb resistant
plamids. ... the more you know!!!
Some competent cell preparations (lots of variety on the commercial market) will allow you to skip a heat shock, ice
or even outgrowth step (be careful with Kan). You must read the individual protocol for all commercial steps to
know what is going on and what can be safely skipped.
3
No transformants? Check the antibiotic resistance. Double check the DNA concentration and quality. Degraded or
non-supercoiled plasmid will not transform as efficiently. If the amount of DNA used was high (0.1 to1 µg or
higher), the transformation can be less efficient than if using a lower amount of cells. Also if new or not sure what
is happening, use a positive control - a plasmid that works well, often supplied with commercial preparations.
Post-Lab work
1. Count colonies on transformation plates the next day and calculate the transformation efficiency (see below).
Score your plates in the morning - the later you check, the more satellite colonies will grow and make it difficult to
distinguish between transformants and satellites. If for some reason you do not have time in the morning, at least
remove the plates from the incubator and leave at room temperature until you can count them. (The colonies will
grow more slowly.) Place plates in a drawer rather than on the bench since Amp is light-sensitive.
To aid counting large numbers of colonies (>100), use a Sharpie pen to mark colonies with a dot on the bottom of
the plate as they are counted. Be careful not to miss colonies that are obscured by writing on the bottom of the
plate or are at the edges of the plate.
For plates that appear to have >200 colonies, assuming a fairly even spread of colonies, divide the plate in half (or
quarters) with a ruler and a Sharpie. Count one half (or one quarter) of the plate and multiply by 2 (or 4) to
estimate the total number of colonies. If you use this method, indicate that you have done so in your report.
2. Check your positive and negative control plates for the expected results. For example, a control plate with
competent cells on LB plate (no selection) should be a "lawn." This is especially important if your transformation
did not work as expected. If so, these plates may provide clues as to what went wrong.
3. After counting colonies on your plates, discard all plates in the autoclave bag.
Calculating Transformation Efficiency

a. Determine the total mass (in µg) of plasmid DNA used in the transformation:
concentration of DNA ( µg/µl ) x volume ( µl ) = mass ( µg )
b. Determine fraction of transformation mix spread on the plate
volume of mix spread / total volume of transformation mix = fraction
[Do not include any volume of LB you added at the last minute simply to help spread the cells on the plate.]
c. Determine mass of DNA in volume of mix spread on plate
total mass of DNA x fraction
d. Transformation efficiency is expressed in scientific notation as the number of colonies (transformants) per µg of
supercoiled plasmid DNA.
Complete formula
T.E. = no. of colonies (transformants)
Fraction (of transf. mix vol spread) X total mass of DNA (µg)
or
no. of transformants
[ vol spread (µl) / total vol of mix (µl) ] X conc of DNA ( µg/µl ) X vol of DNA (µl)
4
Iruela-Arispe Lab
University of California, Los Angeles
LIGATIONS
Vector:Insert Ratio
After the vector and insert DNA have been prepared for ligation, determine the concentration
either by spectrophotometer or by agarose gel against a known amount of standard marker.
Test various vector:insert DNA ratios in order to find the optimum ratio for a particular
vector and insert. In most cases either a 1:1 or a 1:3 molar ratio of vector:insert works well.
The following example illustrates to conversion of molar ratios to mass ratios for a 3.0kb
vector and a 500bp DNA insert:
ng of vector X kb size of insert insert

-------------------------------------- X molar ratio of -------
kb size of vector vector
Example:
How much 500bp insert DNA needs to be added to 100ng of 3.0kb vector in a ligation
reaction for a desired vector:insert ratio of 1:3?
100ng vector X 0.5kb insert 3

-------------------------------------- X ---- = 50ng insert
3.0kb 1
For Ligation Procedure follow manufacturer’s recommendations. Remember to include

controls.
Ligation 1 Ligation 2 Ligation 3 Ligation 4
Vector √ √ -- √
Insert √ -- √ √
Buffer √ √ √ √
T4 ligase √ √ √ --
Also for transformation include an additional control for your plate solution (bacteria
alone).

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MICHAELMAS TERM: Genes and proteins; macromolecules in action

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Bacterial signalling and secretion

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