BIOCHEMISTRY

PRACTICAL
MANUAL &
TUTORIALS
5. Prepare 9 sets of standards, 1 set per workstation and 1 set for instructor standard
curve
Label seven microtubes #1-7, and aliquot 25 ul of each standard into the appropriately
labeled tubes as shown below

Tube Label Standard (mg/m!)

0.125
2 0.250
0.500
4 0.750
5 1.000
1.500
2.000

6. Prepare the standard curve samples for the instructor standard curve.
Note: Be careful not to touch the optically clear sides of the cuvettes. Any fingerprints will
interfere with the light path.
Label 8 cuvettes (blank, #1-7) and prepare standards.
Mix 1x dye reagent by inverting before pipeting
Pipet 1 mi of 1x dye reagent into 8 cuvettes

Pipet 20 ul of 1x PBS into blank cuvette

Pipet 20 ul of each standard into the corresponding cuvette
Mix samples by pipeting up and down or by covering with Parafilm and inverting
3 times
Read absorbance of samples on spectrophotometer within 1 hour

Instructor's Manual
Table 3. Estimated vs. Quantitated Protein Concentrations
Estimated Protein Quantitated
Sample
Concentration Spectrophotometer
Protein
(mg/ml) (from
Table 1) (mg/ml) Concentration
(fromTable 2)_
Sample A
SampleB
Data and Analysis:
1. Record the standard Curve absorbance data from the
spectrophotometer report at the common workstation in Table 4.

Table 4. Standard Curve Absorbance Values

Sample A595 Concentration
(mg/ml)D
Std. #1 0.125
Std. #2 0.250
0.500
Std. #3
Std. # 4 0.750
Std. #55 1.000
Std. #66 1.500
Std. #7 12.000

2 Create a standard curve by plotting the A595 values of the known

standards (from step 1) on the y-axis versus the concentrations in
mg/ml on the x-axis. Plot the data points on linear graph paper, and
draw a line of best fit.

3. Read the concentration of the unknown samples by reading across

from the absorbance of the unknown samples until you intersect with
the standard curve and then read the concentration. Record these data
in Table 5 below.

4. Adjust the final concentration of the unknown samples determined

in step 4 by multiplying the concentration by the dilution factor used.

For example, milk diluted 1:50 gives a reading of 0.224 absorbance

units, which gives a concentration of M mg/ml. The final concentration
of milk is M x 50=_ _mg/ml
 (3

5. Determine the final concentration of the unknown samples and

record in Table 5 below.
Table 5. Final Concentration of Unknown Samples
Sample A595 Concentration Dilution Final
Read from Factor Concentration(mg/ml)
Standard
Curve
(mg/ml)
Sample
A
Sample
B
6. Compare your results to your estimates in Table 1, and enter the
data in Table 5.
 Enzymes
o Enzymes are typically proteins (some nucleic acids have also
been found to be enzymes) that act as catalysts, speeding up
chemical reactions that would take far too long to occur on their
own.
Enzymes speed up the vast majority of the chemical reactions
thatoccur in cells.
o Reactions that break down molecules (such as those involved in
digestion and cellular respiration) and those that build uP
molecules (such as the ones involved in photosynthesis and DNA
replication) all require enzymes.
oEach type of enzyme has a specific shape that compliments the
structure of its substrate.
o The substrate is the molecule or molecules that the
enzyme
Converts into product.
o The substrate fits into an indentation in the
called the active site.
globular protein
o The shape and chemical
properties of this active site are critical
to the enzyme's function.
o Cellobiose in solution is composed of
two glucose molecules
covalently connected by ab1-4 linkage.
Cellobiase has pocket that fits the cellobiose molecule.
a
Cellobiase helps stabilize the cellobiose in a shape so that
the bond between the two glucose molecules can be
broken.
Once the b 1-4 bond in cellobiose has been
two
broken, the
glucose molecules are released from the cellobiase,
and the enzyme is free to bind to another
cellobiose and begin the cycle again. molecule of
o Many chemical reactions that enzymes speed up can occur at a
much slower rate without the
enzymes.
o Enzymes are "picky" about the conditions at which they work
best.
o The temperature and pH
must be ideal for the enzyme to
catalyze reactions efficiently.
Cellobiase Enzyme
o In this laboratory experiment, you will be studying cellobiase.
o Cellobiase is involved in the last step of the
process of breaking
down cellulose, a molecule made up of bundled long chains of
glucose that are found in plant cell walls, to glucose.
o This is a natural process that is used
by many fungi as well as
bacteria (some present in termite guts, others in the stomachs
 of ruminants and also in compost piles) to produce glucose as a
food source.
o Breaking down the cellulose from plants into sugar is also an
important step in the creation of ethanol for fuel.

Cellobiase Substrates
o The natural substrate for the enzyme cellobiase is cellobiose
(Figure 6).
o This is a dissacharide composed of two beta glucose molecules.
o When scientists study enzyme function, it is best if there is an
easy way to detect either the amount of substrate that is used
up or the amount of product that is formed.
o Solutions of cellobiose (substrate) and glucose (product) are
clear, and there are not many simple, inexpensive, fast methods
to detect these molecules quantitatively.
o When cellobiose is bound by cellobiase, the cellobiase breaks
apart the b 1-4 bond that connects the two glucose moleculees
.

and then releases two glucose molecules.

o So, to make this reaction easier to follow, an artificial substrate,
p-nitrophenyl glucopyranoside, will be used.
o This artificial substrate can also bind to the enzyme and be
broken down in a manner similar to the natural substrate
cellobiosse
When the artificial substrate, p-nitropheny! glucopyranoside, is
broken down by cellobiase, it produces glucose and P
nitrophenol.
o When p-nitrophenol is mixed with a solution that is basic in pH
(such as the stop solution provided in the kit), it will turn yellow.
o The amount of yellow color is proportional to the amount of p-
nitrophenol present.
o The yellow color, is detected by a simple colorimetric
quantitative method
o And for every molecule of p-nitrophenol present, one molecule of
p-nitrophenyl glucopyranoside is broken apart.
o For the cellobiase reactions being run, another advantage of
using a basic solution to develop the color of the p-nitrophenol is
that the basic pH will also denature, the enzyme and stop the
reaction
 o By looking at small increments of time, you will be able to
determine whether the rate of the enzyme is constant or
whetherit slows down toward the end as the amountof
substrate decreases.

Pre-lab Questions
1. What type of molecule is an enzyme?
2. Why is an enzyme's shape important to its function?
3. How does an enzyme speed up chemical reactions?
4. What is the name of the enzyme involved in this laboratory
experiment?

Activity 1: Determine the Reaction Rate in the Presence or

Absence of an Enzyme
o In this activity, you will compare the rate of breakdown of p-
nitrophenyl glucopyranoside into glucose and p-nitrophenol in
the presence and absence of cellobiase.
o Enzymes are molecules that increase the rate of a reaction, but.
are not used up in the reaction.
o Because the enzyme can keep processing the substrate over and
over again, very few molecules of enzyme are needed relative to
the number of molecules of substrate.
o Because it is difficult to add really small volumes, your instructor
has diluted the enzyme with a buffer solution- this will allow
you to easily add the required volume that still contains a very
small number of molecules of enzyme.
o However, to ensure that the buffer in which the enzyme was
diluted does not affect the rate of formation of the prodüct, a
control reaction containing just the buffer will be.run alongside
the reaction containing the diluted enzyme.
o To the first reaction tube, you will add enzyme into a solution of
substrate and determine the initial rate of reaction (product
formation).
o The second reaction, which is the control reaction, will have the
same buffer added to the same substrate, but does not include
enzyme.
o This way, you will be able to compare the breakdown rate of p-
nitrophenyl glucopyranoside to glucose and p-nitrophenol in the
presence of enzyme and the presence of a control buffer.
Student Workstation(4 Students) Quantity per Station (
Activity 6:Test the ability of mushroom extracts to increase the reaction rate
Mortar and pestles 1
Weight boats or weigh paper 1
Filter paper, cheese cloth, or strainer 1

Optional Accessories Quantity

100-1,000 gl adjustable micropipet (catalog #166-0508EDU, 166-0553EDU)
100-1,000 ul pipet tips (catalog #223-9350EDU) 8
SmartSpecTM Plus spectrophotometer (catalag #170-2525EDU), Vemier SpectroVis
spectrometer, or similar
Waterbath, 120 V (catalog #166-0504EDU)
Digital dhry bath, 120 V (catalog #166-0562EDU)
DyNAChill cooler (catalog #166-0564EDU)
Mini incubation oven, 120 V (catalog #166-0501 EDU)
Mini centrifuge, 120 V (catalog #166-0603EDU) 1

Refills Available Separately

Biofuel Enzyme kit temperature sensitive reagents bag (catalog #166-5036EDU) containing
enzyme (cellobiase), substrate (p-nitrophenyl glucopyranoside), standard (p-nitrophenol),
2x stop solution, 10x resuspension buffer, extraction buffer
1.5 mi standard disposable polystyrene cuvettes (catalog #223-9955EDU)
Conical centrifuge tubes, package of 50 (catalog # 166-0475EDU)
Disposable plastic transfer pipets, non-sterile, 500 (catalog #166-0480EDU)
1.5 ml EZ MicroTM test tubes, pkg of 500 (catalog #223-9480EDU)

3
Initial Setup for All Activities
Note: Solutions such as 1.5 mM substrate, low concentration enzyme, stop solution, and
of each soluion listed in
resuspension buffer will be used for multiple activities. The volume
six activities. In the setup
the intial setup for all activities is the amount required for a
information for each individual activity, the minimum amount of reagent needed for that one
activity is listed.
Also, the set of 5 standards will be reused for each activity for quantitative or qualitative
determination of the amount of product formed. Do not discard these solutions. The
cuvettes can be covered with Parafilm and stored at 4°C between laboratory periods if
necessay.
Finally, the cuvettes used to measure reaction time points, DPTPs, and the 15 ml conical
tubes used for reactions during each activity will be reused by the students for subsequent
activities. These should be washed out thoroughly with deionized or distilled water between
each activity and saved for later use. For ease of reuse of these items, we recommend that
students label them using laboratory tape rather than writing directy on the surfaces using
a marker.

Material Neededfor Advanced Preparation Quantity

1.5 mM substrate 120ml
Low concentration enzyme 40ml
1x stop solution 120 ml
1x resuspension buffer 64 ml
Colorimetric standards (S1-S5) 8 ml each
15 ml conical tubes 32
Cuvettes 40
Serological pipettor and pipets
Marking pen
Procedure (Estimated time-1 hour)
1. Label eight 15 ml conical tubes "1.5 mM Substrate"'. Add 15 ml of 1.5 mM substrate to
each tube.
2. Label eight 15 ml conical tubes "Enzyme". Add 5 ml of low concentration enzyme to
each tube.
3. Label eight 15 ml conical tubes "Stop Solution". Add 15 ml of 1x stop solution to each
tube.
4. Label eight 15 ml conical tubes "Buffer". Add 8 ml of 1x resuspension buffer to each
tube.
5. Label eight cuvettes S1. Add 1 ml of S1 standard to each cuvette.
6. Label eight cuvettes $2. Add 1 ml of S2 standard to each cuvette.
7. Label eight cuvettes S3. Add 1 ml of $3 standard to each cuvette.
8. Label eight cuvettes S4. Add 1 ml of S4 standard to each cuvette.
9. Label eight cuvettes S5. Add 1 ml of S5 standard to each cuvette.

Instructor's Manual

13
Protocol1
1. Locate the 15 ml conical tubes labeled "Stop Solution", "1.5 mM
Substrate", "Enzyme" and "Buffer"
o Label each of the tubes with your initials.

2. Label five cuvettes E1-E5 (for five time points).

o Label only the upper part of the cuvette face.

3. Label the two remaining cuvettes "Start" and "End" on the upper
part of the cuvette.
o The cuvettes will serve as control time points at the start and
end of the reaction and neither cuvette will contain enzyme.

4. Using a clean plastic pasteur pipettetete, pipettetete 500 l of stop

solution into each of the seven labeled cuvettes.
o The stop solution is a strong base, so avoid getting it on your
skin or clothes.
o Rinse the PASTEUR PIPETTETE well with water and save it for
future activities.

5.
Locatetwo empty 15 ml conical tubes. Label
and the other "Control".
one "Enzyme Reaction"

6. Using a clean PASTEUR PIPETTETE, pipettete 2 ml of 1.5 mM

substrate into the 15 ml conical tube labeled "Enzyme Reaction"
o Use the samee PASTEUR PIPETTETE and pipettete 1 ml of 1.5
mM substrate into the conical tube labeled "Control".
o Rinse the PASTEUR PIPETTETE well with water and save it for
future activities

7. Label one PASTEUR PIPETTETE "E" for enzyme and the other "C"
for control.
o Only use the labeled "E" for the
PASTEUR PIPETTETE labeled
tube and
EE
enzyme reaction tube
enzyme and the PASTEUR PIPETTETE
labeled"C" for the control reaction tube.

Read and understand steps 8-11 fully before proceeding. These

steps are time sensitive!
8. Using the PASTEUR PIPETTETE labeled "C", pipettete 500 l of
buffer into the 15 ml conical tube labeled "Control" and gently mix.
o Once you have mixed the buffer with the substrate, removve
500 ul of this solution and add it to your cuvette labeled "Start".
 (uA
9. Using the PASTEUR PIPETTETE labeled "E", pipettete 1 ml of
Reaction".
enzyme into the 15 ml conical tube labeled "Enzyme
o Gently mix, then START YOUR TIMER.
o This marks the beginning of the enzymatic reaction.

10. At the times indicated below, use the PASTEUR PIPETTETE

labeled "E" to remove 500 l of the solution from the Enzyme
Reaction" tube and add it to the appropriately labeled Cuvette
containing the stop solution.

Table 1. Time intervals

Cuvette Time
E1 1 min
E2 2 min
|E3 4 min
E4 6 min
E5 8 min
11. After all the enzyme samples have been collected, use the
PASTEUR PIPETTETE labeled "C" to remove 500 ul of the solution from
the "C" reaction tube and add it to the cuvette labeled "End".

12. Rinse out all PASTEUR PIPETTETEs with copious amounts of water
and save them for later activities.
o After you have finished your analysis, rinse out your reaction
(conical) tubes and cuvettes with copious water and save them
for later activities.
Note: Do not discard the unused stock solutions or cuvettes
containing standards. They will be used for the next activity.
 Genomic DNA extraction
Why Extract Genomic DNA?
o Genomic DNA isolation is the first step'for many downstream
procedures such as cloning, PCR, and restriction endonuclease
digestions.
o These applications require the isolation of high quality genomic
DNA.
o Genomic DNA (gDNA) is found in prokaryotic (bacteria) and
eukaryotic (animal, plant, and yeast) cells.
o In eukaryotic cells gDNA is closely associated with DNA binding
proteins(histones).
o Therefore, when isolating DNA, one would have to separate the
DNA from proteins and other components that are also found
inside the cell.
o In addition, the DNA must be extracted carefully to ensure that it
remains intact during the purification process.

The basic steps in DNA extraction are:

1. Grow (or collect) cells/tissues from which genomic DNA is to be
extracted.
2. Lyse or break open cell membranes and cell walls (if applicable) by
chemical or physical means.
3. Remove cellular debris.
4. Remove remaining cellular proteins.
5. Purify DNA.

o All of the steps listed above have variations depending on the

cell type and the purity of the DNA needed.

Purifying Genomic DNA from Plants

o Isolation of DNA from plants has complications not seen in other
organismns.
o Plants are multicellular eukaryotic organisms with rigid cell walls
composed of multiple layers of cellulose.
o The cell wall maintains shape and gives rigidity to plant cells.
I t s presence complicates DNA extraction.
o Plant polysaccharides, such as the cellulose from the cell wall,
co-localize with DNA during the purification process.
o In addition, certain plants, such as many conifers and fruit trees,
contain a high concentration of compounds called polyphenols.
o When such plants are broken down with lysis buffer, the
polyphenols bind to the DNA, making it useless for further
experiments.
 ( A )

from plants with high levels of polyphenols, including

grape species, many fruit trees, and conifers.
PVP binds to the polyphenols, preventing them from
complexing with the DNA, and the high salt reduces the
cO-precipitation of polysaccharides with the DNA.

Common Methods of DNA Extraction

o After cell lysis and centrifugation to remove cellular debris,
proteins and RNA are still present in the supernatant along with
the DNA.
o
o
There are several methods to remove these contaminants.
It is important to remember that during the purification processS,
the lysate should never be vortexed or mixed vigorously, since
this would result in breaking or shearing of the large molecules
of genomic DNA into smaller pieces.
o Intact, full-length DNA molecules are required for subsequent
steps, such as PCR
o If DNA becomes sheared, the gene of interest may be broken
into multiple pieces, and therefore may not be amplifiable.

The following are examples of common techniques that are used for
purifying genomic DNA:
Silica-binding-based purification- DNA binds strongly to silica in
the presence of high concentrations of chaotropic salts, such as
guanidine, that disrupt hydrophobic interactions.
Ion exchange chromatography- Purification of DNA with ion
exchange chromatography uses a positively-charged resin or other
matrix, usually in a glass or plastic chromatog1 phy column. DNA and
RNA are both negatively charged and bind to the positively charged
matrix through ionic interactions.
.Organic extraction - DNA can be purified in a two-step process of
enzyme treatment and organic extraction. Proteins in the cell lysate
are degraded by treatment with proteases, enzymes that break
proteins into small pieces.
RNA Removal RNA is often removed from gDNA preparations by
treating the sample with enzymes called ribonucleases (RNases) that
selectively degrade RNA without damaging the DNA. Ribonuclease A,
a nuclease that cleaves only single-stranded RNA molecules, is
commonly used.
Concentrating the Extracted DNA
o Many protocols result in a DNA solution that is too dilute for
experimental purposes, thereby requiring further concentration
of the DNA.
o The most common methods use either ethanol or isopropanol in
the presence of a high salt concentration.
o DNA precipitates in the presence of high salt concentrations (for
example, NaCl) and either ethanol or isopropanol.
The cations in the salt neutralize the charge on the phosphate
backbone of the DNA and allow DNA molecules to come close
together.
o Normally, in aqueous solution, the strong negative charges of
DNA molecules repulse each other.
o The ions do not bind strongly to DNA in aqueous solution, but in
the presence of an organic solvent, such as an alcohol, DNA-
cations form a tight complex and precipitate out of solution.
o If there is enough DNA present, a white precipitate should be
visible, but DNA may very well be present even if a precipitate is
not observed.
o The precipitated DNA is pelleted by centrifugation, after which
the pellet is washed at least once with 70-80% ethanol to
remove any remaining salt.
Note that 100% ethanol cannot be used for the wash
because salt will not dissolve in pure ethanol.
Nor can the ethanol concentration be <70%, as the DNA
might become resuspended with the salt and be lost.
o After the washes, the DNA pellet is dried to evaporate any
remaining ethanol and then resuspended either in water or the
desired buffer.

Measuring DNA Concentration, Yield, and Purity

o Once purified, the concentration, yield, and purity of DNA can be
determined by measuring the absorbance of the DNA solution
A260 using a spectrophotometer.
solution with an A260 of 1 contains approximately 50 ug/ml of
DNA.
o The purity of the DNA can also be estimated with spectroscopy.
A pure preparation of DNA has a ratio of A260: A280 of 1.8. If the
A260:A280 is <1.8, the DNA is probably contaminated with protein (if
the ratio is >1.8, the DNA may be contaminated with RNA)
.Note that the genomic DNA isolated using the nucleic acid extraction
module contains RNA. In order to get a reliable concentration
measurement, contaminating RNA can be first removed by treating
with RNase A.
Each student team requires the following items for DNA extraction from2
plants. Aliquotting reagents for individual student workstations is up to the
instructor's discretion.

Materials Needed for Each Workstation Where Provided Quantity

Razor blades or scalpels Instructor's own

Microcentrifuge tubes, 1.5 ml Kit 2

Microcentrifuge tubes, multicolor, 2 ml Kit

Capless collection tubes, 2 ml Kit

Micropestles Kit

Sterile water (at 70°C) Kit 200

Lysis buffer (prepared with DTT) Kit 2 ml

1x wash buffer (with 95-100% ethanol added) Kit 5 ml

Aurum Mini columns, purple Kit

1,000 ul adjustable micropipet Instructor's own

Tips for 1,000 ul adjustable micropipet Instructor's own As needed

200 ul adjustable micropipet Instructor's own 1

Tips for 200 l adjustable micropipet Instructor's own As needed

Marking pen Instructors own

70% ethanol Instructor's own 2 ml

Plant samples Collected by students 2

Tube rackS Instructor's own

Represents the amountto be aliquoted for each workstation if the instructor chooses to do so.

19
Advance Preparation for Day 2

Objectives Obtain plant material

Warm the sterile water and prepare for use
during the elution step

Prepare lysis buffer

Prepare the wash buffer

Time required 30 min

Procedures
1. Obtain plant materials. The best results will be obtained from young,
fresh leaves. However other parts of plants have been used with
success. It is important not to put the fresh plant tissue in the freezer.
If tissue needs to be stored-store at 4°C for as short a time as possible
and ensure tissue is kept moist.
2. Set water bath to 70°C.
3. Place tubes of sterile water in the water bath at 70°C. The warmed
water is going to be used for eluting the genomic DNA.
4. Prepare lysis buffer.
Add 0.3 g of DTT to 20 ml of lysis buffer for a final concentration of
100 mM DTT. Once DTT has been added to the lysis buffer, it should
be stored at -20°C and is stable for up to 2 months. If longer storage
is required, prepare only enough lysis buffer for the current laboratory
activity and store the remaining lysis buffer at room temperature and
the remaining DTT at 4°C.
5. Prepare wash buffer.
Add 95-100% ethanol to the 5x wash buffer. The amount of ethanol to
add is indicated on the wash buffer label. The wash buffer can be
stored at room temperature for up to 1 year once ethanol has been
added.
6. Prepare 70% ethanol
Add 14 ml of 95-100% ethanol to 6 ml of distilled/deionized water.

20
Genomie DNA
Isolation - Quick
Guide
Stage 1
1. Label one 1.5 ml flip-top micro
centrifuge tube with your initials and
plant name for each plant sample.

2. Pipet 200 ul of lysis solution into

each tube. Lysis sokution

3. For each plant weigh 50-100 mg of

plant material and record the weight.

4. For each plant, use a razor blade or

scalpel to chop the plant material into
1-2 mm pieces. Add the chopped
material to the lysis solution:

5. For each plant, use a micropestie to.

grind the plant material for at least
31min.

6. Once a homogenous lysate has

been generated (i.e. 'chunks' of plant
naterial are no longer visible), add
500 ul of lysis solution to the lysate
and grind further if homogeneity has
not yet been achieved.
Lysis solution

15
 o If a specific restriction site occurs in more than one location on a
DNA molecule, a restriction enzyme will make a cut at each of
those sites, resulting in multiple fragments.
o Therefore, if a linear piece of DNA is cut with a restriction
enzyme whose specific recognition site is found at two different
locations on the DNA molecule, the result will be three fragments
of different lengths.
o If the piece of DNA is circular and is cut with a restriction
enzyme whose specific recognition site is found at two different
locations on the DNA molecule, the result will be two fragments
of different lengths.
The length of each fragment will depend upon the location of
restriction sites on the DNA molecule.
o Because they cut DNA, restriction enzymes are the "chemical
scissors" of the molecular biologist.
o Whena particular restriction enzyme "recognizes" a particular
recognition sequence (four- or six- base pair (bp)) on a
segment of DNA, it cuts the DNA molecule at that point.
o Like all enzymes, restriction enzymes function best under
specific buffer and temperature conditions.
See REN theory

Agarose Gel Electrophoresis

o Agarose gel electrophoresis separates DNA fragments by size.
o DNA fragments are loaded into an agarose gel slab, which is
placed into a chamberfilled with a conductive buffer solution.
o A direct current is passed between wire electrodes at each end of
the chamber.
o Since DNA fragments are negatively charged, they will be drawn
toward the positive pole (anode) when placed in an electric field.
o The matrix of the agarose gel acts as a molecular sieve through
which smaller DNA fragments can move more easily than larger
ones.
o Therefore, the rate at which a DNA fragment migrates through
the gel is inversely proportional to its size in base pairs.
o Over a period of time, smaller DNA fragments will travel farther
than larger ones.
o Fragments of the same size stay together and migrate in. single
bands of DNA.
o These bands will be seen in the gel after the DNA is stained.
o See Agarose theory
 (10

Kit Inventory Checklist

This section ists the equipment and reagents necessary to conduct the Forensic DNA
Fingerprinting laboratory. We recommend that students be teamed up- wo to four studaents
per workstation.

Kit Components Number/Kt (V)

1. Crime Scene (CS) DNA with buffer, lyophilized, 60 ug 1 vial
2. Suspect 1 (S1) DNA with buffer, yophilized, 60 ug 1 vial
3 Suspect 2 (S2) DNA with bufer, lyophilized, 60 yg vial
4. Suspect 3 (S3) DNA with buffer, lyophilized, 60 u9 1 vial
5. Suspact 4 ($4) DNA with butfer, lyophilized, 60 ug 1 vial
6. Suspect 5 (S5) DNA with bufer, lyophilized, 60 ug vial
7. EoRLPs, restriction enzyme mix, lyophilized, 3,000 units 1 vial
8. Sterle water, 2.5 m vial
9 Hindll lambda digest (DNA size marker), 0.2 ug/ul, 100 pl 1 vial
10. DNA sample loading dye 1 vial
11. Fast Blast DNA stain, 500x, 100 ml 1 vial
12. Colored micro test tubes, 2.0 m 60
13. Clear micro test tubes, 1.5 mi 30
14. Agarose powder, 5g
15. Electrophoresis buffer, 50x TAE, 100 ml
16. Foam micro test tube holders 16
17. Gel staining trays

Required Accessories Number/kKit (

Adjustable micropipet, 2-20 ul, catalog #166-0506EDU 1-8
Ppet tps, 2-200 5 racks of 200, catalog #223-9347EDU
Horizontal electrophoresis chamber, catalog #166-4000EDU 1-8
Power supply, catalog #164-5050EDu 1-4
Adjustable micropipet, 20-200 ut, catalog #166-0507EDU
Adjustable micropipet, 100-1000 u, catalog # 166-0508EDU
Pipet tips, 100-1000 u, 5 racks of 200, catalog #223-9350EDUU
Pemanent markers
Microwave oven or hot plate
Distlled water
500 mi Erlenmeyer flask for microwaving agarose
500 ml fiask or beaker for dilusting DNA stain
ce bucket with ice
Laboratory tape (not Scotch 3M brand or similar tape)

Optional Accessories Number/Kit

37"C water bath, catalog #166-0504EDU or
Mini incubation oven, catalog #166-0501EDU
Gel suppont film (50 sheets), catalog #170-2984EDU
Microcentrifuge, catalog #166-0602EDU
or mini centrifuge 1
Rocking platfom, catalog #166-0709EDU

2
 Lesson 2 Electrophoresis of DNA Samples
Materials Neededfor Each Workstation Quantity (
Agarose gel electrophoresis system 1

Agarose gel
Digested DNA samples
Hindll lambda digest (DNA marker)
DNA sample loading dye
Permanent marker
Pipet tips, 2-20 ul 13
Adjustable micropipet, 2-20 ul 1
Waste container
Gel support film (if applicable) 120 ml per 2 stations
Fast Blast DNA stain, 1x or 100x*
Large containers for destaining (if applicable)" 1-3 per 2 stations
Foam micro test tube holder 1 a
Power supply
Gel staining tray 1 per 2 stations
Electrophoresis buffer (1x TAE) 275 ml per station

Instructor'sWorkstation
Microcentifuge
or mini centrifuge (optional) 4
Rocking platform (optional) 1

Post-Lab Activity: Analysis of Results

Materials Neededfor Each Workstation Quantity
Millimeter ruler
Semilog graph paper
Gel support film (if applicable)"

Instructor's Workstation
None required

Depending on whether the quick or overnight staining will be follwed.

Instructors Manual

12
Lesson 2 Agarose Gel Electrophoresis and Visuallzation of DNA
Fragments
Advance Preparation

Objectives Propare Hirlll lambda digast (DNA marker) and alilquot (optional)
Aliquot sample DNA loading dye (optional)
Prepare the electrophoresls chamber
Dlute Fast Blast DNA strain to 1x (for overnight staining)
or 100x concentration (for quick stalning)
Set up student and teacher workstations
Time required 45 minutes
What is required Hindlll lambda digest (DNA marker)
Sample loading dye
Electrophoresis chambers, casting trays, and combs
Electrophoresis buffer (1x TAE)"
Fast Blast DNA stain, 500x

Procedures
1. Prepare Hindll lambda digest (DNA marker) and aliquot (optional). Add 20l of DNA
sample loading dye to the stock tube containing the Hindil lambda DNA marker. Heat
the marker to 65°C for 5 minutes, then chill on ice -this results in better separation of
the marker bands. Label clear micro test tubes "M". Aliquot 15 pl of the DNA markers
containing oading dye to 8 clear micro test tubes labeled "M".
2. Aliquot DNA sample loading dye. Label eight clean micro test tubes "LD" for loading
dye and aliquot 50 ul of sample loading dye into each tube. Distribute one tube to each
team.
3. Prepare the electrophoresis chamber. When the agarose gel has solidified, sample
loading and electrophoresis can begin.
a. When placing the gel tray into the electrophoresis chamber, make sure that the
sample wells are at the black cathode end. DNA samples wil migrate toward the
red anode end during electrophoresis. Make sure the tray is fully seated in the
chamber.
Prepare therequired volume of 1x TAE buffer, if you have not prepared it already.
b.
C.
Submerge the gel under about 2 mm of 1x TAE buffer.
d. Prepare samples for gel loading. See laboratory protocol in the student section.
Note: Power requirements vary depending on gel thickness, length, and concentration, and
on type of electrophoresis buffer used. For this exercise we recommend using a constant
voltage of 100 V for 30 min. See Appendix D for a faster electrophoresis protocol which
allows the gel to be run in 20 min.

4. Prepare Fast Blast DNA stain

a. To prepare 100x stain (for quick staining), dilute 100 ml of 500x Fast Blastwith
400 ml of distlled or deionized water in an appropriately sized flask or bottle and
mix. Cover the flask and store at room temperature until ready to use.
b. To prepare 1x stain (for avemright staining),dilute 1 mi of 500x Fast Blast with 499 ml
of distilled or deionized water in an appropriately sized fask or botte and mix. Cover
the flask and store at room temperature until ready to use.
0.25x TAE butfer is used for fast gel etectrophoresis. Aefer to Appendix D for detailed infomation.

Instructor's Manual

17
 Molten 1% agarose in 1x TAE (See Advance Prep)
37°C water bath or incubator (optional) 1 per class
Microcentrifuge 1 per class

Observations
1) Describe the samples of DNA (physical properties)
DNA?
2) Is there any observable difference between the samples of
3) Describe the appearance of the restriction endonuclease mix.
4) Combine and react.

1. Using a new pipette tip for each sample, pipette 10 ul of the

enzyme mix "ENZ" to each reaction tube as shown below.
a. Pipette up and down carefully to mix well
Note: Change tips whenever you switch reagents, or, if the tip touches
any of the liquid in one of the tubes accidentally. When in doubt,
change the tip! DNA goes in the tube before the enzyme. Always add
the enzyme last.
Now your DNA samples should contain:

DNA Samples (10 pl |EcoRI/PstI Total

Reaction Volumme
each) Enzyme Mix

Crime Scene [CS] 10l 20

Suspect 1 [S1] 10 l 20 ul
Suspect 2 [S2] 10 ul 20
Suspect 3 [S3] 10 g 20 ul
Suspect 4 [S4] 10 l 20 ul
Suspect 5 [S5] 1 10 ul |20 ul
2. Mix the tube contents
3. Tightly cap on each tube.
4. Mix the components by gently flicking the tubes with your finger.
5. If there is a centrifuge available, pulse the tubes for two s conds
to force the liquid into the bottom of the tube to mix and
combine reactants. (Be. sure the tubes are in a BALANCED
arrangement in the rotor).
6. Incubate the samples.
a. Place the tubes in the foam micro tube holder and incubate
them at 37°C for 45 minutes
 2

Glycerol:
I s a trihydroxy alcohol

Simple compound with 3 Hydroxyl groups

Free O-H group is POLAR!!

One monomer which is a building block of a lipid

Can form acylglycerol (simple lipid type) by forming an
ester linkage with a fatty acid
Stearic acid
Is a fatty-acid
Fatty acid is along chain aliphatic carboxylic acid
A common
component of several lipids
Most contain even number of carbon atoms (in range of Ci2
to Cao)
C-C and C-H are nonpolar bonds
One POLAR group (Carboxylic group - COOH)
Can form acylglycerol (simple lipid type) by forming an

ester linkage with a trihydroxy alcohol

Butter (fats) and Olive oil
Fully formed lipids
NONPOLAR
At normal temperatures, fats are solids and oils are liquids

Combination ofa fatty acid and a trihydroxy alcohol where

all the 3 Hydroxyl groups of the alcohol from ester
linkages
with fatty acids is called TRIACYLGLYCEROL

2
 Biochemistry 2
Practical: Lipid analysis lI

1. Alkali hydrolysis

2 Lipase hydrolysis

1. Alkali hydrolysis

Reter to your text book and read the section explaining saponification. Note that

the process occurs in the

naturally body through the action of the enzyme lipase.

1 Combine the foliowing in a test tube:

small piece fat

3 mL 20% KOH

Add a boiling stone and heat carefully flame

over a (bunsen burner)
3 Boil for approximately 3 min

Cool under running tap water (until the solution solidifies)

5. Add 2 mL H,0 and mix well (NOTE the foam...)
6 Explain what has happened during this experiment
BIOCHEMISTRY PRATICAL

LIPID ANALYSIS II

Triacylglycerols are not components ofmembranes

Accumulate in adipose tissue (primarily fat cells)

Provide a means of storing fatty acids

FUNCTION: Serve as concentrated stores of metabolic energy

Ester linkages of fatty acids hydrolyzed within an organism by t

enzyme lipases

Reaction can be reproduced outside the organisms

Catalysts are acids and bases

SAPONIFICATION: reaction of glyceryl estér with NaOH and

KOH to produce soap (which is the corresponding salt of the long-

chain fatty acid) and glycerol.

Soaps used with hard water react with calcium and magnesium ions

in the water

Forms a precipitate (scum left on inside of bath)

 (122

RESTRICTION ENZYMES
First break through leading to recombinant DNA technology was the
discovery of microbial enzymes that cuts dsDNA
Refered to as restriction enzymes (REN's) or restriction
endonucleases
These enzymes recognize and cleave specific sequences about 4-6 bp

long
Sequences recognized by REN's are palindromic
NAMELY, sequence reads the same from L to R and R to L
e.g. the bacterium Hemophilus aegypticus produces an

enzyme named Haell that cuts DNA wherever it encounters the

sequence

5'GG/CC3
3'CC/GG5

5 GG CC 3
3' CC GG 5

.The forward slash indicates where the sequence is cut

. This specific base sequence is known as the "recognition
sequence" for Hindll.

More than 900 REN's

Some sequence specific and some not, have been isolated from

over 230 strains of bacteria

Restriction enzymes generally have names that reflect their origin
Nucleases are further described by addition of the prefix "endo" or

"exo" to the name:

"endonuclease" applies to sequence specific nucleases that break
nucleic acid chains somewhere in the interior
Nucleases that function by removing nucleotides from the ends of the

molecule are called "exonucleases."