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BIOCHEMISTRY

BIOL 2362
FURTHER METABOLISM AND GENE EXPRESSION
LABORATORY MANUAL
Academic Year (2011-2012)

Department of Life Sciences


University of the West Indies

TABLE OF CONTENTS

Page
LAB PREPARATION & REPORT SUBMISSION

CITATION OF REFERENCES

LAB SKILLS ASSESSMENT SHEET

1. PRACTICAL # 1a
Isolation, Quantification and Purity Determination of DNA
from Rat Liver and Kidney and Cows Blood

2. PRACTICAL # 1b
Determination of the Total DNA and RNA Content
in Rat Liver, Kidney and Brain

13

3. PRACTICAL # 2
Induction and Estimation of -Galactosidase Synthesis
In Escherichia coli

21

APPENDIX 1: Notes on Graphing

34

Lab Submission Data Sheets

LAB PREPARATION & REPORT SUBMISSION


FLOWCHART
As part of your preparation for the lab session, you are required to come to the lab with a
comprehensive flowchart outlining the procedures and your plan to undertake the lab
session in question. On the top of the page of the flowchart you should have:
The volume of each reagent you are required to take.
Major notes/reminders
Expected trends/results for a given experiment.
Questions (if any) about the procedure
Absence of your flowchart will result in an immediate deduction of marks.
IN-LAB ASSESSMENT
Your lab skills and ability to produce accurate results are assessed in the in-lab
assessment. Every lab session, both you and your partner will be assessed. The final mark
will be acquired by summing the totals of all the assessment and scaling it down to an
appropriate mark which is then included in your final lab mark.
LAB REPORT SUBMISSION
Biochemistry lab reports are submitted in two parts: a Pre-lab and a Post-lab. The prelab consists of the Title, Aim, Theory and Procedure and accompanying References. The
Post-lab focuses on your Results and Calculations and Discussion and must also be
referenced. Follow the instructions below strictly.
BIOCHEMISTRY PRE & POST LABS SHOULD BE TYPED (PREFERABLY) ON
LETTER SIZE PAPER OR WRITTEN ON FOLDER/BINDER PAGES. A bonus of
0.5 marks is awarded as an incentive for a well presented lab report that strictly follows
the guidelines below.
PRE-LAB SUBMISSION
NAME:

LAB PARTNER:

ID#

DATE (of lab session):

Title Of Lab This is given on the lab handout (e.g. Titration Curves)
Aim:

This states the objective(s) of the lab, the aim of the experiment
e.g. To determine., To investigate.

Theory:

The theory should pertain to the objectives of the lab. It is a


concise introduction that gives information needed to understand
the subject and objectives of the experiment. It clearly states the
questions that the experiment sets out to answer or the hypothesis
3

being tested, along with the experimental approach that was used. It
puts the experiment into perspective. YOUR INTRODUCTION
MUST BE REFERENCED. Failure to reference your work will
result in loss of marks.
Procedure:

The procedure must be presented in narrative style in the past tense.


DO NOT ITEMISE OR PUT IN PUT IN POINT FORM. Include a
list of materials and reagents used at the beginning of this section.

References:

See pgs 5 & 6 of this manual. FOLLOW INSTRUCTIONS


RIGOROUSLY!

POST-LAB SUBMISSION
NAME:

LAB PARTNER:

ID#

DATE (of lab session):

Results: The results section presents the data and observations that bear upon the
objectives of the experiment. Results are presented in tables (PLEASE DO
NOT INSERT DATA SHEETS FROM THE LAB SESSION!), figures,
graphs, calculations, diagrams, and sometimes very brief, accompanying notes.
The following is a guideline:
Tables Must:
1. Be numbered
2. Have a title (this should fully describe what the table is about e.g. Table 1: Results of
Protein Calibration Curve Construction)
1. Be properly drawn (i.e. comprehensive layout of variables)
2. Incorporate all data (e.g. volume of reagents used, absorbance values, amount of
protein in mg etc.)
Calculations:
1. Only do a sample calculation (e.g. if you are constructing a protein calibration curve,
using increasing volumes of standard protein solution, you are expected to show a
calculation showing how much protein there is in a chosen aliquot of solution. You
are not expected to show the working for each aliquot that you pipette out. This is
redundant as the same method is employed.
Alternatively you may be expected to determine the protein content of a specific mass
of tissue. You may be required to do this for several different types of tissues,
however, provided that the dilution factors and processing steps are the same,
you are only required to show the working for one tissue type. Of course even if you
do not show the working, you will include all calculated values in a summary table.
4

2. You must show all steps in your calculations. Marks will be lost if you do not show a
logical flow of steps.

Graphs Must:
1. Be numbered
2. Have a title (this should be fully descriptive e.g. Graph #1: Protein calibration curve
showing how protein content (mg) varies with absorbance at 750 nm)
3. Be neatly drawn (if hand drawn use a sharp pencil point, avoid leaving stray marks
etc.)
4. Possess a scale (e.g. on horizontal axis 1 cm represents 1 unit/mg protein etc.)
3. Have labeled axes (e.g. vertical axis Absorbance at 750 nm; horizontal axis
Protein content in mg)
Discussion:
The discussion is a very important part of the lab script. It demonstrates a
students analytical skills and gives insight into how well the student understands
the lab exercise. This is where you interpret your results and compare it to
findings previously reported in literature.
The discussion should:
1.
2.
3.
4.
5.
6.
7.
8.
9.

Answer the questions raised in your Aims/Objectives.


FOCUS ON YOUR RESULTS!!!!!
Show significant trends in the results
Point out the significance of the results/trends by comparing your findings to known
theoretical values or theories.
State and discuss whether the results obtained conform to the theoretical expectations
and if they do not suggest possible explanations.
Account for sources of error and indicate why certain precautions may have been
taken. (e.g. tissue samples kept on ice)
Indicate why certain reagents were used (if the addition of such reagents are
important for the particular reaction).
BE REFERENCED to support your findings.
Give suggestions for improvements in experiments.

Additional Discussion:
Answer these questions separately from the discussion.
*References:
See pgs 5 & 6 of this manual. FOLLOW INSTRUCTIONS RIGOROUSLY!
FAILURE TO COMPLY WITH THE ABOVE INSTRUCTIONS WILL RESULT
IN LOSS OF MUCH NEEDED MARKS!

LAB GRADES

Pre-labs are to be submitted on the day of the lab in question and post labs one week after
the lab date. Each part is individually marked. The mark for a given lab is produced by
summing the totals of the pre and post labs and scaling it down to 10. The marks for all
your lab sessions are then summed and this is added to the scaled-down average of your
in-lab assessments. This final mark is then scaled to 20%, which is your final lab mark
(unless otherwise stated).
Once a week after the submission date has passed, labs will not be accepted unless a very
reasonable excuse is offered.
Your lab grade accounts for 20% of your total course grade. There MAY be a practical
exam at the end of the semester. Otherwise, assessment is based on lab reports,
performance in the lab (e.g. lab manipulation skills, lab conduct, adherence to safety
regulations etc.) and any additional lab exercises (e.g. pop quizzes). Satisfactory
performance in the practicals is a prerequisite for a passing grade. Candidates who do not
obtain an overall passing grade (8 out of the 20%) for the practicals will be required to
repeat the entire course.

NB PLAGIARISM IS TREATED VERY HARSHLY (whether the source of


materials is the Internet or a colleagues report). If you are discovered plagiarizing
you will lose at least 2-3 marks out of 10 for the lab in question. Repeat offenders
will lose even more marks. The TA and demonstrators are very experienced in
detecting plagiarism, thus, it would be very unwise of you to attempt it.

COURSE ASSESSMENT
The grade awarded for biochemistry is calculated as follows:
Final examination
In-course Assessment

60%
40%

The in-course assessment comprises of two (2) written term examinations at 10% each
and performance in the practicals worth 20%.

General Guidelines For A Good Lab Report

Layout

Fully labeled
tables, diagrams
and figures

Within word limit


Legible, justified
report

Inappropriately
labeled Tables,
Diagrams and

Figures
Exceeds word
limit
Script is difficult
to read
Tables,
Diagrams and
Figures lack

titles

Exceeds or is
severely under
word limit

Illegibly written

Objectives

Materials &
Methods

Introduction

Concise
Based on objectives
All essential
background
ALL goals
information
Past Tense
clearly
Contains necessary
stated
diagrams/
No tables
biochemical
reactions
References cited in
text
Has some main
points
Some
Missing some
objectives
equations or
missing
diagrams
Few in text
references

Past Tense
Has tables

Verbose
Poorly
Lacks important

constructed background points


and
Plagiarism detected
missing
No diagrams/

objectives
reactions
No in-text references

Results

Discussion

References

Concise
All results/
Observations are
analyses are
well explained &
clearly shown &
reinforced by
properly tabulated expected or
Includes all
Correct & easy to published results
cited text
follow sample
Includes
Written as
calculations
suggestions for
indicated in
Clearly
improvement and
manual
documented
important
observations/
precautions noted
diagrams
In-text references
cited
Incoherent layout
of results
Results are
Reference
cited in text
Difficult to follow & partially explained
missing some
but not
sample
included in
Inferences are not
calculations
supported by cited
reference list
references
or vice versa
Incomplete
observations/
diagrams

Results are

missing
In point form
Incorrect & difficult
multiple
to follow sample
tenses

calculations
Includes
Poorly described
tables

observations/
diagrams

Verbose
Trends are

reported but not


explained
Too much focus on
precautions

No references
cited in text

Not written as
stated in
manual
no reference
list

Grade

GoodExcelle
nt
Fair

Weak

BIOL 2362

B. CITATION OF REFERENCES FOR THE BIOLOGICAL SCIENCES


Because all the sources of information provided in your biochemistry labs (introduction
and discussion) were not your original work, it must ALL be referenced. The
Chicago Manual of Style describes methods that are frequently used by the Biological
Sciences. We in Biochemistry will accept the author-date system of referencing.
AUTHOR DATE SYSTEM
MAKING A CITATION WITHIN THE TEXT:
At every point in the text at which reference is made to a particular
document, include the author's surname and the year of publication in
parentheses.
e.g. In a recent study (Smith 1982), it was shown that .....or alternatively
the reference can be found at the end of the sentence e.g. ..as previously
described (Smith 1982).
If the author's name occurs in the sentence, give the year of publication
alone.
e.g. Smith (1982), discusses the .....
If there are two or three authors, give the surnames of all individuals. If there
are more than three, give the surname of the first followed by et al.
e.g. Smith, Lopez and Martin (1982) reported that....
e.g. In an earlier study (Smith et al. 1982) .....

CITING REFERENCES IN A BIBLIOGRAPHY:


Bibliography should be arranged in alphabetical order by author's surname
followed by year of publication.
Use the same punctuation.
The following lists examples of citations from different sources.
1. Reference to a book:
Author(s)/editor(s). Year of publication. Title of publication in italics. Edition (if not 1st).
Publisher, place of publication. (page no. optional)
Example:
Fletcher, R. and Voke, J. 1985. Defective colour vision. Hilger, Bristol.
2. Reference to a chapter in a book:
Author(s) of chapter. Year of publication. Title of chapter in italics. In: Author(s)/editor(s)
of book. Title of book. Edition (if not 1st). Publisher, place of publication.
Example:

BIOL 2362

Parsons, T.R. 1980. Zooplankton production. In: Barnes, R.S.K. and Mann, K.H. eds.
Fundamentals of aquatic ecosystems. Blackwell, Oxford.
3. Reference to a journal (periodical) article:
Author(s) of article. Year of publication. Title of article. Title of periodical in italics,
volume number (issue number): first page - last page.
Example:
Tedder, T. and Isaacs, C. 1989. Isolation of cDNA's encoding the CD19 antigen of human
and mouse B lymphocytes. J. Immunol. 143(3): 712-717.
4. Reference to an internet source:
Author's name. Title of document, in quotation marks. Title of complete work (if
relevant), in italics or underlined. Date of publication or last revision. URL, in angle
brackets. Date of access in parentheses.
Citing a personal site
Joseph Pellegrino, "Homepage," 12 May 1999, <http://www.english.eku.edu/
pellegrino/default.htm> (12 June 1999).
Citing a professional site
Gail Mortimer, The William Faulkner Society Home Page, 16 September 1999,
<http://www.utep.edu/mortimer/faulkner/main faulkner.htm> (19 November
1997).
National Association of Investors Corporation, NAIC Online, 20 September 1999,
<http://www.better-investing.org> (1 October 1999).

BIOL 2362

LAB SKILLS ASSESSMENT SHEET


Names:
Cupboard #

Date:

Total
Punctuality
Late (-5)
Safety
Was not wearing gloves (-2)
Improperly worn lab coat (-2)
Inadequate foot ware (-2)
Pours toxic waste down the sink
(-5)
Pours non-toxic waste into waste bottle (-3)
Pools all wastes together, toxic and harmless and then disposes (-3)
Instrument Usage
Holds pipette incorrectly (-3)
Often slams spec cover (-3)
pH meter electrode not replaced in pH buffer (-4)
pH of solution taken without stirring or agitating (-4)

Leaves used tip on pipette (-2)


Spills sample in spec (-4)
pH meter electrode placed on desk (-4)

Efficiency
No Flowchart (-10)
Makes one or few careless mistakes (-3)
Makes careless mistakes which renders entire experiment useless (-5)
Has no plan, concentrations/volumes of solutions worked out in advance (-5)
Lab Etiquette & Courtesy
Group takes too much reagents (-3)
Water bath near dried out (-2)

Water bath more than half filled (-2)


Flasks and Tubes not labeled (-2)

Accuracy & Understanding


Student does not understand what he/she is doing (-3)
Some results are off (-1)
All results off (-3)
Cleanliness
Tips on desk (-3)
Pipettes on bench when not in use (-1)
Places dirty utensils in cupboard (-5)
Leaves equipment on/plugged in (-3)

Miscellaneous paper, parafilm on desk (-2)


Left dirty utensils on desk at end of lab (-3)
Leaves workspace dirty/wet (-3)

Breakage

BIOL 2362

DEPARTMENT OF LIFE SCIENCES


BIOL 2362
PRACTICAL # 1a
Isolation, Quantification and Purity Determination of DNA from Rat Liver and
Kidney and Whole Cows Blood
Student Learning Outcomes
You should be able to:
Extract DNA from various animal tissues
Quantify DNA using UV spectrometry
Determine purity of various DNA samples
Comment on the integrity of DNA after agarose gel electrophoresis
Isolation and quantification of DNA is an important first step in many analytical techniques
in molecular biology, genetics and biochemistry. In this lab, DNA will be extracted from
three animal tissues using two different techniques followed by quantification and purity
determination.
DNA isolation from animal organs involves homogenization which macerates the tissues and
frees the nuclei from the cells. Components in the isolation buffer usually dissolve the
membranes, remove contaminants (mainly protein) and prevent DNA degradation. Once a
relatively pure supernatant is acquired, DNA is precipitated with high salt concentrations and
ethanol or isopropanol. With whole blood, separation of the red blood cells must proceed first
before the addition of reagents to lyse and precipitate DNA from the white blood cells.
This lab will be written up and submitted along with the following lab.
REAGENTS for Liver and Kidney DNA Isolation
Isopropanol (place in -20C freezer)
3M sodium acetate, pH 5.2 (Molecular biology grade)
10% SDS
1M Tris HCl, pH 8.0
0.5M EDTA
5M NaCl
(10:1) TE, pH 7.4
5mL 1M Tris HCl, pH 8
1mL 0.5M EDTA
Add 450mL sterile distilled water, adjust pH, make up to 500mL
Proteinase K (prepare fresh)
Add 5mL (10:1) TE to100mg Proteinase K. Mix to dissolve.
Lysis Buffer
1mL (10:1) TE
8mL 5M NaCl
0.4mL 0.5M EDTA
Make to 100mL with sterile distilled water.
REAGENTS for Whole Blood DNA Extraction

Buffer A
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BIOL 2362

0.32M sucrose
10 mM Tris HCl
5mM MgCl2
0.75% Triton-X
Adjust pH to 7.6
Buffer B
20 mM Tris-HCl
4 mM Na2EDTA
100 mM NaCl
Adjust pH to 7.4
N.B. All solutions should be sterile. Buffer A should be autoclaved prior to addition of
Triton-X-100. Sterile filtering of solutions instead of autoclaving is a better option.

PROCEDURE for Isolation of DNA from Liver and Kidney


1. Sacrifice and remove the liver and kidneys from one rat. Weigh organs and quickly
place on ice.
2. Add 3mL/g lysate buffer to organs and homogenize. Record volume.
3. For every mL of homogenate above, add 15ul of 20mg/mL Proteinase K. Vortex.
4. Add 160uL of 10% SDS per mL of supernatant recorded in step 2. Solution may
become viscous at this point. Vortex well.
5. Place in a shaking water bath at 45C for 1 hr.
6. For every mL homogenate, add 350uL of 5M NaCl. Vortex. Record volume.

YOUR WORK STARTS HERE


7. In a clean glass test tube, take 0.6mL of each of the liver and kidney homogenate and
add 2.4mL Lysis Buffer to each. Vortex well.
8. Centrifuge both tubes at 3000 rpm for 10 minutes at room temperature.
9. Carefully pour each supernatant into individual clean, labeled test tubes. Add 0.3mL
3M sodium acetate to each tube and mix. Place on ice for 10 minutes.
10. Add 5mL of pre-cooled isopropanol from the -20C freezer. It is VERY
IMPORTANT that the isopropanol is very cold.
11. Invert to mix. A visible cloudy mass should appear. This is your DNA.
12. Centrifuge at room temperature for 1 min to pillet DNA.
13. Pour off supernatant and add 3mL TE buffer (10:1). Label properly, parafilm tube and
give to your demonstrator for storage storage.

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PROCEDURE for Isolation of DNA from Whole Blood


1. Collect 40mL of cows blood and spin at 15000 rpm for 8 minutes.
2. Decant supernatant into the 2.5% bleach solution provided (waste) and add 5mL of
Buffer A and 10mL of cold sterile water to the pellet. Vortex gently or invert tube to
thoroughly re-suspend pellet and leave to incubate on ice for 3 minutes.
3. Centrifuge the re-suspended pellet at 5000 rpm for 15 minutes at 4oC. Discard
supernatant into 2.5% bleach solution.
4. Re-suspend pellet in 2 ml of buffer A and 6 ml of water (washing step).
5. Spin at 5000 rpm for 15 minutes at 4oC. The pellet should be white to cream in
colour. If pellet is significantly red, repeat washing step again.
6. Add 5 ml of Buffer B and 500 l of 10% SDS to pellet. Re-suspend pellet by
vortexing vigorously for 30-60 seconds. Then add 50 l of Proteinase K solution
(20mg/ml).
7. Leave to incubate for two hours at 55oC in a water bath. Remove samples and leave
to cool to room temperature (or leave for 2-3 minutes on ice). Add 4 ml of 5.3 M
NaCl solution. Vortex gently for 15 seconds.
8. Spin at 4500 rpm for 15-20 minutes at 4oC. Pour off supernatant into a fresh tube.
Take care not to dislodge pellet. To the supernatant, add an equal volume of cold
isopropanol (stored at -20oC). Invert 5-6 times gently to precipitate DNA.
9. Centrifuge for 1 min 12,000 rpm to pillet DNA. Wash with 1 ml of 70% ethanol.
Centrifuge as before and invert tube on tissue. Leave DNA to dry for 15-20 minutes at
room temperature. Re-suspend in 3mL of TE. Label properly, parafilm tube and give
to your demonstrator for storage.
YOUR WORK CONTINUES HERE IN THE FOLLOWING LAB SESSION
1. Centrifuge all samples at 3000rpm for 10 minutes at room temperature to remove all
traces of debris. Pour off supernatant (stock DNA) into a clean labeled test tubes.
2. Accurately pipette 250uL of your liver and kidney samples into separately labelled
tubes and make the volumes of each to 3mL with TE buffer. Do the same with 0.5mL
of your cows blood DNA. SAVE ALL SOLUTIONS. Some of each of your
samples will be run on an agarose gel. This will be done for you.
3. Using the appropriate cuvette, blank spectrophotometer with TE. Measure and record
the absorbance of your diluted liver, kidney and blood samples above at 260nm and
280nm.

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4. Pipette 2mL of each your liver, kidney and blood stock DNA into separately labeled
test tubes and add 1.5mL 20% (v/v) TCA solution to each. Vortex.
5. Heat all mixtures in a boiling water bath for 15 minutes. Leave to cool at room
temperature.
6. Determine the DNA and RNA concentrations of your liver, kidney and blood samples
as shown on pg 15 step 13 of the manual. DO NOT DILUTE THE EXTRACTS
HERE (lab 1 a) FOR THE RNA PROCEDURE.

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DEPARTMENT OF LIFE SCIENCES


BIOL 2362
PRACTICAL # 1b
Determination of the Total DNA and RNA Content in Rat Liver, Kidney and Brain
Student Learning Outcomes
You should be able to:
To quantify DNA and RNA via visible spectrophotometry using a destructive method.
PRINCIPLE
Many methodologies have been developed for extracting, characterizing and quantifying
DNA and RNA. Nucleic acids are often identified by their distinctive absorption spectra.
All nucleic acids absorb intensely in the ultraviolet (UV) range, a result of the conjugated
double bonds in both purines and pyrimidines. In this experiment, however, the extracted
nucleic acids will be partially degraded and converted to coloured compounds that can be
measured spectrophotometrically in the visible range.
The extraction method used is based on the method developed by W.C. Schneider (1957)
which involves cell disruption followed by extraction with trichloroacetic acid and
ethanol. The assay procedures are based on colour reactions with the pentose moieties in
the extracted nucleic acids.
A.

The estimation of DNA by the Diphenylamine reaction:

The diphenylamine reaction is specific for 2-deoxypentoses in general and is therefore


specific for DNA. The reagent contains acetic and sulphuric acids. When heated with
DNA, the acids cleave some of the phosphodiester bonds and hydrolyze the glycosidic
linkages between sugars and purines. This straight chain form of deoxypentose is
converted to the highly reactive -hydroxylevulin aldehyde, which reacts with
diphenylamine to give a blue complex. The intensity of the blue colour, which is
measured at 600nm, is directly proportional to the concentration of DNA in the sample.
RNA does not react in the diphenylamine assay.
B.

The estimation of RNA by the Orcinol reaction:

This reagent contains concentrated hydrochloric acid. When RNA is heated with acid in
the presence of ferric chloride (FeCl3.6H2O) as a catalyst, the acid cleaves some of the
phosphodiester bonds and hydrolyzes the glycosidic linkages between sugars and purines.
The hot acid also converts the ribose to furfural, which, when in the presence of ferric
ions, reacts with orcinol to produce green-coloured compounds. The orcinol reaction is
not as specific as the diphenylamine reaction as all pentoses, including the deoxyribose of
DNA, will react to some extent.
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For a given DNA and RNA solution of the same concentration, the colour intensity
produced by the orcinol reaction of DNA is one tenth that of the reaction with RNA.
Therefore the calibration curve for orcinol cannot be used to directly determine the
concentration of RNA in the TISSUE EXTRACT, as this contains a mixture of DNA and
RNA. The absorbance due to DNA is easily estimated once the concentration of DNA in
the extract has been determined. This will be 10% of the absorbance of the same
concentration of RNA on the calibration curve. Once the absorbance due to DNA is
obtained, subtract this from the absorbance reading for the extract. Using the remaining
absorbance value, determine the concentration of RNA from the calibration curve for the
orcinol reaction.
Prelab Questions:
1.

What does treatment with cold TCA and hot TCA accomplish?

2.

What is the purpose of adding 95% ethanol?

3.

What does the RNA to DNA ratio reflect about the constituent cells?

REAGENTS
1.
Diphenylamine reagent
Dissolve 1.0g of diphenylamine in 100mL of glacial acetic acid and add 2.75mL
of concentrated H2SO4, reagent quality.
2.

Orcinol reagent
Mix together 0.2g of orcinol, 60mL conc. HCl and 0.2mL 10% (w/v) ferric
chloride solution (must be fresh).

3.

Standard nucleic acid solutions


(a)
DNA solution 1mg/mL
(b)
RNA solution 0.2mg/mL

4.

0.1M KCl

5.

5% TCA

6.

10% TCA

7.

20% TCA

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CAUTION!
STRONG ACIDS ARE USED IN THIS PROCEDURE:
DO NOT PIPETTE BY MOUTH
USE GLASS CUVETTES
USE GLOVES AT ALL TIMES
DO NOT POUR REAGENTS INTO THE SINK. THEY WILL EXPLODE ON
CONTACT WITH WATER. POUR ALL REAGENTS INTO THE PROVIDED
WASTE BOTTLE (CONTAINING 1M NaOH), IMMERSED IN THE ICE BATH
LOCTED ON THE CENTRE BENCH.
IT IS ADVISED THAT PROTECTIVE GOGGLES SHOULD BE WORN.
IF ACID SPILLS ON YOUR SKIN, WASH IMMEDIATELY WITH SOAP AND
THEN WATER

PROCEDURE:
A.

Calibration curve for DNA

1.

Pipette 2mL of the stock DNA solution to a test tube and then add 1.5mL of a
20% (v/v) TCA solution.

2.

Heat the mixture in a boiling water bath for 15 minutes. Leave to cool at room
temperature for 2 minutes and then quickly cool the tubes under the tap. This is
your DNA-acid hydrolysate.

3.

Set up 8 test tubes as follows:


(a)
Add 0.1, 0.2 (x2), 0.3, 0.5(x2) and 0.8mL of the DNA-acid hydrolysate to
labeled test tubes and then bring the final volume to 1.0mL with distilled
water.
(b)

Add 2mL of the diphenylamine reagent to each tube.

(c)

Prepare a blank containing 0.7mL of water, 0.3mL of 20% TCA and 2mL
of diphenylamine reagent.

(d)

Mix all tubes thoroughly and boil for 10 minutes in a waterbath

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(e)

Cool the tubes and measure the absorbance at 600nm after setting the
spectrophotometer at zero with the reagent blank. Plot Absorbance at
600nm against DNA content (mg).

B.
1.

Calibration curve for RNA


Pipette 2mL of the stock RNA solution to a test tube and add 1mL of a 20% (v/v)
TCA solution.

2.

Heat the mixture in a boiling water bath for 15min. This is your RNA-acid
hydrolysate.

3.

Set up 8 test tubes as follows:


(a)
Add 0.1, 0.2 (x2), 0.3, 0.4(x2) and 0.5mL of the RNA-acid hydrolysate to
labeled test tubes and bring the volume to 1mL with distilled water.
(b)

Add 2mL of the orcinol reagent to each tube.

(c)

Prepare a blank to contain 0.7mL of 5% TCA and 0.3mL water and 2mL
of orcinol reagent.

(d)

Mix all tubes thoroughly and then boil for 10 minutes in a waterbath.

(e)

Cool and measure their absorbance at 660nm against the reagent blank.
Plot absorbance at 660nm against RNA content (mg).

C.

Estimation of DNA and RNA in Rat Liver, Kidney and Brain Tissues

1.

Kill one rat (200-250g-body weight) and remove the liver, kidney and brain and
weigh them.

2.

Homogenize the tissue in 0.1M KCl (1 g tissue : 10mL of 0.1M KCl)

YOUR WORK STARTS HERE


3.

Pipet 2mL of the homogenate in a glass small-rimmed test tube of approximately


12mL capacity.

4.

Add 5mL of ice-cold 10% TCA to the centrifuge tube. Mix well by gentle
inversion (place a foil cap over the test tube to protect your finger).

5.

Centrifuge at 1300g for 5minutes.


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6.

Using a Pasteur pipette, remove and discard the supernatant. If there is a solid
layer floating on top, save this by inserting the Pasteur pipette beneath the layer
in order to aspirate the supernatant.

7.

Add another 5mL of ice-cold 10% TCA to the pellet and resuspend with the
Pasteur pipette. Centrifuge and aspirate as described in steps 5 and 6 leaving two
drops of liquid above the pellet.

8.

With a Pasteur pipette, disperse the pellet in the liquid above it. Then add 10mL of
95% ethanol (room temperature) to the dispersed pellet.

9.

Centrifuge at 1300g for 5 min. Decant and discard the supernatant.

10.

Repeat steps 8 and 9.

11.

Add 5.0mL of 5% TCA (room temperature) to the centrifuge tube and disperse the
pellet with a Pasteur pipette. Place the tube in a boiling water bath for 10 minutes
agitating every few minutes.

12.

Centrifuge at 1300g for 5 minutes and carefully decant the supernatant in a clean
test tube marked A (12 mL capacity). MEASURE THE VOLUME OF THE
SUPERNATANT. This nucleic acid extract will be used in the assays.

13.

Determine the DNA and RNA concentrations in the acid hydrolysates as follows:
(a)

In the case of DNA determination, Transfer 0.5 and 1.0mL aliquots of


the nucleic acid extract to labeled test tubes. Bring the volume to 1mL,
where necessary, with distilled water. Add 2.0mL of diphenylamine
reagent and treat as you do for the DNA calibration curve. Do the same
with your samples from lab 1a.

(b)

In the case of RNA determination, dilute the nucleic acid extract 1 in 4


with water. DO NOT DILUTE SAMPLES FROM LAB 1a. Transfer
0.2mL and 0.4mL of the diluted extract to labeled test tubes. Bring the
volume to 1mL with water. Add 2mL of orcinol reagent and treat as you
do for the RNA calibration curve.

TREATMENT OF RESULTS AND WRITE UP


LAB #1a

17

BIOL 2362

1. Given that an absorbance reading at 260nm of 1 (OD2601) = 50ug/mL double stranded


DNA determine the concentration of DNA in your samples on pg 3, step 3.
2. Determine the 260nm/280nm ratio for each sample (pg 3, step 3). What is the
significance of this value? Comment on your results.
3. Determine the concentration of each tissue of your DNA samples. For liver and
kidney, express this result in mg/g and blood mg/mL whole blood.
4. Of what practical application is DNA isolated from this method of use in
biochemistry and molecular biology/genetics?
5. Comment, make inferences and explain your results seen on the agarose gel.

18

BIOL 2362

LAB #1a & 1b Tabulate the data for questions 2 and 3. Clearly distinguish between
the samples prepared in Lab 1a and 1b.
1.

Determine the colour contribution of DNA to the orcinol reaction of the extract.
(DNA reacts one-tenth as much as the same concentration of RNA). From the
calibration curve for the orcinol reaction, find the absorbance value for an RNA
concentration that is the same concentration as the concentration of DNA in the
extract. Take one-tenth of that absorbance as the absorbance component due to
DNA and enter the values.

2.

Calculate the DNA and RNA concentration (mg/g tissue) for all your samples.

3.

Determine the total DNA and RNA in each organ for all your samples.

4.

Do the different tissues contain different amounts of DNA and RNA? In which
tissue is the RNA concentration the highest? In which tissue is the DNA
concentration the highest? Explain the significance of your results.

5.

Discuss the advantages and disadvantages of both procedures (Lab 1a and 1b) as
quantitative methods to determine DNA concentrations.

6.

Compare the values acquired for liver and kidney tissues (mg/g) in Lab 1a and
Lab 1b. Explain your differences. Comment on your results.

REFERENCES:
1.
2.

Roger, L.P., Adams, John T Kowler & David P Leader. The Biochemistry of the
Nucleic Acids, 10th Edition.
Harpers Biochemistry

19

BIOL 2362

BIOL 2362
DATA SHEET: LAB # 1 a & b
DETERMINATION OF THE TOTAL DNA & RNA IN RAT LIVER, KIDNEY AND
BRAIN TISSUES
NAME:.LAB PARTNER:
LAB#1a
ABSORBANCE READINGS FOR DNA FROM LIVER, KIDNEY & BLOOD
Tissue

Tissue Mass (g)/


Volume (mL)

260 nm

600 nm
660 nm
0.5mL 1.0mL 0.5mL 1.0mL

280 nm

Liver
Kidney
Blood

LAB #1b
A.

CALIBRATION CURVE FOR DNA


Tube

1
(blank)
mL DNA-acid
--hydrolysate
A600nm
0.00
DNA content
(mg)

B.

0.1

0.2

0.2

0.3

0.5

0.5

0.8

0.00

CALIBRATION CURVE FOR RNA


Tube

1
(blank)
mL RNA-acid
--hydrolysate
A660nm
0.00
RNA content
(mg)

0.1

0.2

0.2

0.3

0.4

0.4

0.5

0.00

20

BIOL 2362

21

BIOL 2362

C.

ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BLOOD (1a)

LIVER
Mass (g)/Volume
(mL) of Tissue
A600nm
(DNA)
A660nm
(RNA)

D.

BRAIN

---0.5 mL
1.0 mL
0.2 mL
0.4 mL

ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN (1b)

LIVER
Mass of tissue (g)

----

Final volume (mL)


of homogenate
A600nm
(DNA)

----

A660nm
(RNA)

E.

KIDNEY

BRAIN

KIDNEY

0.5 mL
1.0 mL
0.2 mL
0.4 mL

DNA Results FROM LAB 1a & 1b HOMOGENATES


Protocol

Concentration of Nucleic Acid (ug/g or ug/mL)


Liver
Kidney
Blood
Brain

Lab 1a DNA
Lab 1a
Homogenates Lab 1a RNA
Lab 1b DNA
Lab 1b
Homogenates Lab 1b RNA

22

BIOL 2362

DEPARTMENT OF LIFE SCIENCES


BIOL 2362
PRACTICAL # 2
Induction and Estimation of -Galactosidase Synthesis in Escherichia coli.
Student Learning Outcomes
You should be able to:
Prepare and assay an enzyme of bacterial origin
Indirectly determine the level of gene expression by assaying enzyme activity
INTRODUCTION:
Enzymes synthesized by microbes can be divided into two categories:
(a)

Constitutive enzymes: synthesized irrespective of the presence of the substrate for


the enzyme or a precursor of the substrate in the growth medium.

(b)

Inducible enzymes: synthesized in detectable amounts only in the presence of


compounds (termed inducers) in the growth medium, which are usually substrates (or
structurally related analogues) for the enzymes.

Note: Synthesis of inducible enzymes can often be repressed by the end product of the
metabolic pathway.
Enzyme-catalyzed reactions may be followed in many ways depending on the nature
of the system under study. For example, one can monitor the rate of disappearance of
a reactant or the formation of a product, the rate of gaseous exchange, the effects of
coenzymes etc.
The activity of the enzyme -galactosidase can be readily estimated through
colourimetry. It is an inducible enzyme found in the bacterium, Escherichia coli,
specified by the Z gene. The enzyme catalyses the hydrolysis of the disaccharide,
lactose, releasing -D-glucose and -D-galactose. The assay method involves the use
of an artificial chromogen substrate, o-nitrophenyl--D-galactopyranoside (ONPG),
the hydrolysis of which yields o-nitrophenol (ONP). In alkaline solution, ONP has an
intense yellow colour with an extinction maximum at 420 nm.
Reagents
1. LB Broth (1L)
Composition: 10g Tryptone (or peptone), 5g Yeast Extract, 10g NaCl, pH 7.4.
Sterilize for 20 min in an autoclave.
2. Mineral Salts Medium (1L)
Composition: 8g K2HPO4, 3g KH2PO4, 0.1g MgSO4.7 H2O, 1g (NH4)2SO4, 0.1% Yeast
Extract, 5% glucose (or lactose), pH 7.0. Sterilize glucose (or lactose) separately for 20 min.
EXPERIMENTAL

23

BIOL 2362

A.

Preparation of a calibration curve for estimation of o-nitrophenol.

1.

Prepare a standard solution of ONP in distilled water, containing 1 mol ONP/


mL.

2.

Using this standard solution set up a series of tubes containing varying amounts of
ONP and a constant amount of alkali as outlined in the table below.

TUBE NO.
1
2
3
4
5
6
7

ONP SOLN. (mL)


0.25
0.50
0.75
1.00
2.00
2.50
0.00

Na2CO3 (mL)
1.00
1.00
1.00
1.00
1.00
1.00
1.00

Distilled Water (mL)


3.75
3.50
3.25
3.00
2.00
1.50
4.00

3.

Mix the contents of tubes thoroughly, measure absorbance at 420nm using the
contents of tube 7 as a reagent blank.

4.

Plot a calibration graph relating absorbance at 420nm to ONP concentration


(moles/mL).

Q. 1

Is Beers Law obeyed over the ranges examined?

N.B.

YOU ARE TO WORK IN GROUPS OF 4. Simply work with the group


directly opposite to you.

B.

GALACTOSIDASE INDUCTION IN E. coli

(i)

Preparation of Cell Suspensions

E. coli cells have been grown in LB Broth (25 mL) overnight. The suspension was
centrifuged and the cells resuspended in 2 mL sterile water. 1 mL was added to glucose
media (50 mL) and 1 mL to lactose media (50 mL). These were then left to grow
overnight (or approximately 18 hours). Note: these quantities quoted are for each pair of
students.
24

BIOL 2362

At this point you will begin your experiment.


5.

Label two centrifuge tubes glucose A and lactose A.

6.

Add 10mL of the appropriate culture to the tubes.


Harvest the cells by
centrifuging at 3000 rpm for 20 min. Discard supernatant.

7.

Resuspend the cells in each tube using 10 mL sterile water.

8.

Repeat the centrifugation and resuspension steps above.

9.

Centrifuge again for a final time, discard the supernatant.

10.

For the tube labeled glucose A, resuspend the pellet in 2 mL of sterile water. For
the tube labeled lactose A, re-suspend the pellet in 6 mL of sterile water.

11.

From each of these tubes, take 1 mL of suspension and place into two new test
tubes labeled glucose B and lactose B respectively.

12.

To the new tubes (labeled B)add 1 mL toluene. Vortex the toluene tubes for 3
min.

(ii)

Estimation of -galactosidase activity in the different preparations.

13.

Pipette 4 mL of 0.1 M phosphate buffer (pH 7.2), into each of four new test tubes
labeled glucose A, lactose A, glucose B and lactose B,

14.

Add 1.5 mL 0.05M ONPG as substrate.

15.

Place the test tubes in a water bath at 37 C and allow the contents to attain this
temperature (wait 5min).

16.

Start the reaction by adding 1 mL of the respective enzyme preparation A or B.


Note the time of the start of the reaction.

17.

Shaking the tube beforehand, remove 0.5 mL samples at specific time intervals (0,
5, 10, 20, 30, 40, 50, 60 min).

18.

Pipette immediately into tubes containing 0.5mL 1 M sodium carbonate and


adjust the volume to 5 mL with distilled water.

19.

Mix the contents thoroughly by inverting the tube five times.

25

BIOL 2362

20.

Clarify by centrifugation for 3 min.

21.

Carefully transfer the supernatant to the cuvette and read the absorbance at 420
nm. Use 0.5 mL 1 M sodium carbonate and 4.5 mL water as your blank.

26

BIOL 2362

Treatment of data
Determine the activity (units/mL) of your four stock samples.
Calculate and compare the units/ml of enzyme in each of your stock samples.
Also plot ONP concentration (umol/ml) against time (mins) and draw tangents at the
linear portions of the curves to determine U/ml (initial velocity) for each sample.
Discuss your results fully.

REFERENCES:
1.

Lederberg, J. (1950). J. Bact. 60. 381.

2.

Hestrin, S., Feingold, D. S., & Schramm, M. Methods in Enzymology. Vol. I. Ed.
P. Colowick & N. O. Kaplan. p. 241.

3.

Kaempfer, R.O.P. and Magasanik, B. (1967). J. Molec. Biol. 27. 475.

4.

Robert F. Boyd (1988) General Microbiology. 2nd Ed. Times Mirror, Mosby
College Publishing p. 237-238.

27

BIOL 2362

DEPARTMENT OF LIFE SCIENCES


BIOL 2362
DATA SHEET: LAB # 2
INDUCTION AND ESTIMATION OF -GALACTOSIDASE IN E.coli

NAME:..LAB. PARTNER:..

A.

Calibration curve for the estimation of o-Nitrophenol


TUBE
1
2
3
4
5
6
7*

[ONP] moles/mL

A420nm

*reagent blank

B.

Galactosidase Induction in E.coli

Time (min.)
0
5
10
20
30
40
50
60

Glucose A

Absorbance at 420nm
Glucose B
Lactose A

28

Lactose B

BIOL 2362

APPENDIX 1: NOTES ON GRAPHING


A. DRAWING GRAPHS BY HAND
A graph is a plot of two quantities. One is something that you control and is called the
independent variable. Usually it goes on the x-axis. The other quantity that changes as
you change the independent variable is called the dependent variable. This goes on the
y-axis.
For example if you put varying volumes of a protein solution in a series of test tubes and
then measure the absorbance, the variable you control is the quantity of protein, so it goes
on the x-axis. The absorbance is what changes as you change the protein quantity, so it
goes on the y-axis.
Always use graph paper when drawing graphs.
UNITS AND EXPONENTS
Assume you did a protein determination and recorded the following data:
Protein Content (mg)
0.001
0.003
0.005
0.007
0.008

Absorbance reading at X nm
0.05
0.16
0.225
0.33
0.375

The best thing to do is to change the units to easier numbers to be plotted. For example,
multiply the values by 1000 so that you obtain readable values like 1, 3, 5, 7 and 8. Then
on your graph you can use exponents to reflect this. The x-axis would now be labelled
mg x 1000 or mg x 103. Additionally if your values are very large numerically, one can
simply find the log10 value and plot points using a log scale. Remember to take the
antilog for any value obtained from the graph that will be used in further calculations.
VARIABLE RANGE
In order to determine an appropriate variable range; subtract the lowest data value from
the highest data value. Do each variable separately.

29

BIOL 2362

SCALE
Never forget to insert the scale for both axes, as the two may vary. Additionally the scale
must be constant. If you define 1cm on the x-axis to be 1g then that scale must be
maintained.
LABEL AXES
Ensure that both axes are clearly labelled. Axes titles should always include the name of
the variable as well as the unit the variable is measured in e.g. Protein content (g). NB
please insert arrowheads at the end of the axes to indicate the direction of progression.
PLOTTING DATA POINTS
Data points for a single data series can be plotted using any one of the following symbols:
x, +, ,,,,, etc. In order to plot more than one data series on a graph, one must use
different symbols to distinguish between different lines (one can also use lines of
different colours). Insert a key to distinguish between different data series.

DRAWING THE LINE


To make a graph you must know something about the system and what you expect to see.
For example, in a protein assay, you expect a straight line. You must then draw the best
straight line possible through the points. This can be achieved through a computer
program (which draws the line for you) or by using a calculator with a statistics function
to tell you the slope of the line, or even by drawing it yourself. Never simply connect the
dots!
If the points on the scatter graph lie within a narrow band, i.e. there is a good correlation
between the two aspects of data, it may be possible to draw a line of best fit that shows
graphically the trend of the correlation. The line of best fit is an approximation or
estimate and it usually goes through the mean of each set of data that you draw. See
Figure 1 below. Note the line of best fit does not necessarily have to pass through the
origin, nor any of the points actually plotted. In biochemistry, however, especially in the
case of calibration curves, the line must at least pass through the origin.
When the relationship is expected to be linear, the best line is determined by linear
regression. This is a mathematical process in which the line is placed to minimise the
area between the points and the line. This is also called the least squares method.

30

BIOL 2362

Figure 1. (a) scatter plot (b) line of best fit. NB that the best-fit line does not necessarily pass
through any of the points plotted.

BAD DATA POINTS


You are the one to decide when you have a bad data point. The computer does not make this
decision for you. Table 1 shows a data series that has been plotted on the graph shown in
Figure 2. Notice that 5 of the 6 points fall perfectly on a straight line. However one point (30,
0.05) is way off. Your first action should be to redo the 30-g tube rather than report data of
that calibre. If that is not possible, simply draw the line through the 5 perfect points and
ignore the bad point.

Table 1: Absorbance vs. mg Protein


mg protein
Absorbance
0
0.00
10
0.10
20
0.20
30
0.05
40
0.40
50
0.50

Absorbance @ 750 nm

0.5

0.4

0.3

0.1

0
0

13

25

38

Protein content (ug)

31

50

BIOL 2362

Figure 2. Graph with a bad data point

USE THE WHOLE GRAPH


It is best to use as much of the graph as possible. Also, the closer the line is to a 45
angle, the more accurate you will be when you interpolate an unknown from it. DO NOT
extrapolate the line beyond the last data point especially in the case of calibration
curves. Refer to Beer-Lamberts Law and the section on Spectrophotometry.

TITLE:
Your graph must be numbered and have a descriptive title. For example, Graph #1:
Protein Calibration Curve.

B.

GRAPHING WITH COMPUTERS

There are many graphing programs available but Excel is the most popular choice at this
level. Therefore we will use Excel to demonstrate how you can analyse data using
spreadsheets and graphing programs.
Loading the Spreadsheet
The first step is to set up the spreadsheet. With Excel you have a basic grid of columns,
which are labelled A-Z, and rows, which are numbered one to several hundred. Each
combination of a number and letter is called a cell. You start by putting data into the cells.
Cells can hold data in the form of numbers or letters. If you wanted to plot absorbance vs.
mg of protein for the Lowry assay, you might have data that looks like Table 2.
When you load this into Excel, you could put the column labels (mg Protein and
Absorbance into the spreadsheet. The program will talk you through the creation of the
graph, including labelling the axes. It is best to put the values that will be on the x-axis
into the left-hand column, as it simplifies the graphing process. Figure 3 shows what the
spreadsheet would look like.

Table 2: Absorbance versus mg Protein for the Lowry Assay


mg Protein
Absorbance
0
0.00
10
0.10
20
0.21
32

BIOL 2362

30
40
50

0.29
0.40
0.52

33

BIOL 2362

Figure 3. Excel spreadsheet.

Creating the Graph


Once you have loaded the data into the spreadsheet, you are ready to create the graph.
First you need to select the data that will be put into the graph, so click on the A1 cell and
drag down and over until all of your data is highlighted. Then you click on the wizard
button. The first dialog box gives you a choice of chart types, such as bar graph, pie
graph, scatter plot and so on. Normally you will want to do the XY scatter plot, so this
will be used as the example.
There is a potential danger in using the computer to make the graph! The default value
for many programs would yield straight lines connecting the dots. THIS IS WRONG IN
BIOCHEMISTRY!
You want the best fit line of some type. In your case it would be a linear regression line.
Choose the option XY scatter that does not attempt to put any line over the points. The
lines will be put in later. Click on Next to continue the dialog. The next box (box 2 of
4) asks about chart source data. One you have correctly selected the data from your
column, you can go on to the following box by clicking on the Next
icon. This brings you to box 3 of 4 in the dialog process. This is the chart-formatting step
where you decide how you want the graph to look. There is a title: tab you can select
and then add in the graph title and the title of both axes. There is a gridline tab that
34

BIOL 2362

determines the number of visible gridlines. The fourth dialog box gives you the option of
putting the graph as an object on the spreadsheet itself or making it a separate page.
SELECT THE LATTER.
Remember to always select for a graph that goes through the origin when plotting
calibration curves.
To avoid creating the default connect the dots graph, we selected a graph that had no
line. You therefore still need to connect the line. Create the line using the pulldown
menus on the top bar. Choose chart and then add trendline choice from the pulldown
menu. The box that pops up gives you a picture of possible types of lines, including linear
regression and polynomial. Select linear regression for linear data.
Non-Linear graphs
Such graphs would start off the same way but you would use the chart wizard differently
to customise your graph. A common example would be when analysing enzyme kinetics
data.

35

BIOL 2362

BIOL 2362
DATA SUBMISSION SHEET: LAB # 1a & b
DETERMINATION OF THE TOTAL DNA & RNA IN RAT LIVER, KIDNEY AND
BRAIN TISSUES
NAME:.LAB PARTNER:
LAB#1a
ABSORBANCE READINGS FOR DNA FROM LIVER, KIDNEY & BLOOD
Tissue

Mass (g)/
Volume (mL)

260 nm

600 nm
660 nm
0.5mL 1.0mL 0.5mL 1.0mL

280 nm

Liver
Kidney
Blood

LAB #1b
F.

CALIBRATION CURVE FOR DNA


Tube

1
(blank)
mL DNA-acid
--hydrolysate
A600nm
0.00
DNA content
(mg)

G.

0.1

0.2

0.2

0.3

0.5

0.5

0.8

0.00

CALIBRATION CURVE FOR RNA


Tube

1
(blank)
mL RNA-acid
--hydrolysate

0.1

0.2

0.2

0.3

0.4

0.4

0.5

36

BIOL 2362

A660nm

0.00

RNA content
(mg)

0.00

37

BIOL 2362

H.

ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN TISSUES

LIVER
Mass of tissue (g)

----

Final volume (mL)


of homogenate
A600nm
(DNA)

----

A660nm
(RNA)

I.

BRAIN

KIDNEY

0.5 mL
1.0 mL
0.2 mL
0.4 mL

ESTIMATION OF DNA & RNA IN RAT LIVER, KIDNEY AND BRAIN (1b)

LIVER
Mass of tissue (g)

----

Final volume (mL)


of homogenate
A600nm
(DNA)

----

A660nm
(RNA)

J.

BRAIN

KIDNEY

0.5 mL
1.0 mL
0.2 mL
0.4 mL

DNA Results FROM LAB 1a & 1b HOMOGENATES


Protocol

Concentration of Nucleic Acid (ug/g or ug/mL)


Liver
Kidney
Blood
Brain

Lab 1a DNA
Lab 1a
Homogenates Lab 1a RNA
Lab 1b DNA
Lab 1b
Homogenates Lab 1b RNA

38

BIOL 2362

DEPARTMENT OF LIFE SCIENCES


BIOL 2362
DATA SUBMISSION SHEET: LAB # 2
INDUCTION AND ESTIMATION OF -GALACTOSIDASE IN E.coli

NAME:..LAB. PARTNER:..

C.

Calibration curve for the estimation of o-Nitrophenol


TUBE
1
2
3
4
5
6
7*

[ONP] moles/mL

A420nm

*reagent blank

D.

Galactosidase Induction in E.coli

Time (min.)
0
5
10
20
30
40
50
60

Glucose A

Absorbance at 420nm
Glucose B
Lactose A

39

Lactose B

BIOL 2362

40