313 MOLECULAR BIOLOGY PRACTICAL
AIM
This practical session is designed to provide experience with some common techniques
used in molecular biology:
 Polymerase chain reaction
 DNA quantification
 Restriction enzyme digest
 Gel electrophoresis
To achieve this aim, we will use applications specific to the Skeletal Muscle Research
Group.
BACKGROUND
Transgenic mice:
The Skeletal Muscle Research Group use genetically modified mice to study the effects
of increased gene expression on muscle. These genetically modified mice are called
transgenics and have foreign DNA inserted which causes a gene to be over-expressed
compared with wild-type mice.
One such transgenic mouse strain is Actin/IGF-1. It has a fragment of human IGF-1
DNA inserted next to the α-Actin promoter which is specific to skeletal muscle. By
linking IGF-1 to regulatory elements of highly-expressed α-Actin, IGF-1 also becomes
upregulated. We can genotype mice in a transgenic colony by performing a polymerase
chain reaction (PCR) using primers directed at the human IGF-1 sequence. These
primers will only detect a product in transgenic mice. By including another pair of
primers in the PCR which target DNA elsewhere in the genome – a region which
transgenics and wildtypes share – we have an internal control for the PCR.
Thus the primer mix used for the genotyping PCR contains the following primers:
Actin/IGF-1 – forward
Actin/IGF-1 – reverse
AND
IL-2 forward
IL-2 reverse
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Y1 probe:
The Skeletal Muscle Research Group use a technique called Myoblast Transfer Therapy
to introduce dystrophin into muscle. Normal male donor myoblasts are injected into leg
muscles of female mdx (equivalent of human muscular dystrophy) mice.
The success of the transfer and subsequent survival of the male myoblasts is traced by
hybridizing DNA from female mdx leg muscles with a Y1 probe.
Before a Y1 probe can be made, the Y1 fragment must be excised from the plasmid in
which it was grown. This is done using an EcoRI restriction enzyme digest. EcoRI
detects the sequence GAATTC which occurs at both ends of the Y1 insert within the
plasmid.
Cultured Primary Myoblasts
Male
Female Host
Primary Myoblasts
Y1 Probe
Analysis
Injected Myoblasts
Donor
Skeletal Muscle
DNA Quantitation
(total donor male nuclei)
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LABORATORY SAFETY
Please note that this session involves the use of potentially hazardous chemicals and
equipment. Ethidium bromide, loading dye, hot agarose, high voltage equipment and the
UV box are examples of these.
Your demonstrator will instruct you in dealing with these potential hazards safely prior
and during the lab session.
If you are in any doubt about the use of any chemicals or equipment---please ask.
1. POLYMERASE CHAIN REACTION (PCR) OF TRANSGENIC MOUSE DNA
In this section, we will be genotyping mice from a transgenic colony to determine the
presence or absence of the transgene.


Label 1 tube with ‘M’ - this will be the tube for your master mix.
Label the remaining 3 tubes each with the code of the DNA samples to be
amplified.
Distilled water
10x buffer
25mM MgCl2
5mM dNTPs
Actin primer mix
Taq polymerase
DNA template
TOTAL





1X
16.6µl
2.5µl
1.5µl
1.0µl
1.25µl
0.125µl
2.0µl
25.0 µl
Master mix (4X)
66.5 µl
10.0 µl
6.0 µl
4.0 µl
5.0 µl
0.5 µl
Pipette the components in order as listed under ‘master mix’ into the tube
labelled ‘M’. The demonstrator will pipette the Taq polymerase.
Vortex the master mix.
Aliquot 23 µl of master mix into each of the 3 tubes labelled with sample code.
Vortex DNA and pipette 2 µl of each DNA sample into the tube labelled with the
corresponding code.
Vortex to ensure DNA and master mix are well combined.
Run on thermal cycler using the following conditions:
94oC
2 minutes
o
94 C
45 seconds
55oC
45 seconds
x 32 cycles
o
72 C
45 seconds
72oC
10 minutes
This run will take approximately 100 minutes.
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2. RESTRICTION ENZYME (RE) DIGEST OF Y1 PLASMID
In this section, we will measure the concentration of Y1 plasmid DNA then digest it with
a restriction enzyme.
The spectrophotometer UV light must be on and the ‘Nucleic Acid’ program used.
DNA concentration is proportional to the amount of UV light that is absorbed by the
DNA bases. (Abs260nm value of 1.0 = 50 µg/ml double-stranded DNA)
 Pipette 48 µl of water into a tube, add 2 µl of Y1 plasmid DNA and vortex.
 Pipette 50 µl of water into spectrophotometer cuvette and press ‘Blank’. This sets
the zero or background level.
 Remove water and pipette 50 µl of Y1 plasmid DNA solution into cuvette and
press ‘ReadSamples’.
 Calculate the concentration of DNA in the Y1 plasmid samples (don’t forget to
take the dilution factor into account).
 Work out the volume of Y1 plasmid DNA required for 1 µg.
EcoRI digest:
1 µg Y1 plasmid DNA
10X Buffer
EcoRI enzyme
Distilled water
TOTAL



Positive
1 µl
2 µl
1 µl
16 µl
20 µl
Negative
1 µl
2 µl
17 µl
20 µl
Label 2 tubes – ‘pos’ and ‘neg’.
Pipette the components as indicated above – note that no EcoRI is in the negative
tube.
Vortex tubes to mix and incubate at 37oC for 1 hour.
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3. DETECTION OF PCR AND RE DIGEST PRODUCTS
Make 1.5% agarose gel as follows:
 Measure 50 ml of TAE buffer into bottle.
 Measure 0.75 g agarose and add to TAE buffer.
 Heat in microwave oven (bottle lid must be unscrewed) until mixture bubbles and
is clear.
 Add 1 drop of ethidium bromide (Danger – carcinogen!), swirl gently and pour
into gel casting apparatus and allow to set.
 Pipette 3 µl of blue loading dye into each PCR tube and into each RE tube.
 Load DNA ladder mixture (10 µl), PCR samples (20 µl) and RE samples (20 µl)
onto gel.
 Run at 100 volts for approximately 20 minutes.
 View gel under UV light and record fragment sizes observed.
Score transgenic DNA samples for presence or absence of DNA insert.
What fragment sizes are observed in positive and negative RE digests?
Which band would you use to make a Y1 probe for Southern hybridization?
QUIZ
Why are Interleukin-2 (IL-2) primers included in the PCR primer mix?
What is the role of Taq polymerase in PCR? What property of Taq polymerase enables it
to be used in PCR?
In PCR, what effect does increasing the magnesium concentration have? What effect
does decreasing the annealing temperature have?
Using the simplest formula- work out the melting temperature of the primer
GCAATCCTGGAGTGCAT. Why is the melting temperature of the primer important?
What is the Abs 260/280 ratio for pure DNA?...and for pure RNA?
If a DNA sample has a concentration of 20 µg/ml, how much DNA is in 35 µl?
What is a plasmid?
Why are there so many restriction enzymes?
What does it mean when a restriction enzyme produces sticky ends or blunt ends? Give
an example of a restriction enzyme that generates each.
Why is a DNA ladder used in gel electrophoresis?
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