Aims of this case study
Carry out PCR for 20, 25 and 30 cycles.
Analyse the PCR fragments by agarose gel
electrophoresis.
Find out how the number of cycles affects the
amount of DNA generated.
Observe that the DNA fragments generated
from each plant species are all the same size.
Background information regarding the primers
The primers have been designed to match non-coding
regions of DNA between genes. These regions have a
higher frequency of mutation. Plant species that are
related will tend to have fewer mutations. Species that
are evolutionary distant are likely to have more
mutations. These differences will show up in the size of
fragments obtained through PCR.
3’
5’
5’
3’
Highly conserved
region of DNA
Variable non-coding
region
Primer
Purification of the Extracted DNA
This will remove inhibitors of PCR.
Try to choose areas
that have the same
degree of staining.
Purification of the Extracted DNA
1. Place a clean
cutting/backing board
behind the absorbent
layer.
Place the tip of the
punch over the area to
be sampled, press
firmly and rotate to
remove a paper disc.
Choose an area where
the extract has soaked
through to the back.
Clean the punch between
samples by removing a disc
of paper from an extract-free
area.
Purification of the Extracted DNA
2. Use a cocktail
stick to transfer the
disc from the punch
into a labelled, clear
1.5-ml micro
centrifuge tube.
Use a different
cocktail stick for
each sample.
The next two slides are
about micropipettes
1-ml syringes or narrower ‘micro
syringes’ can be fitted with a disposable
graduated tip.
White tips are marked 2 and 10 μl.
Yellow tips are between 10 and 100 μl.
A short length of silicone rubber tubing
can be used to fix the tip to the syringe
to give an effective seal.
You may be lucky enough to have adjustable
pipettes.
Twist the dial to the desired volume.
Pick up the pipette tip.
Press the plunger to the first, soft, stop.
Insert the pipette tip into the solution to be
transferred.
Slowly release the plunger to retrieve the liquid.
Move the pipette tip to above the desired well.
Press the plunger past the first stop to the second,
hard, stop to transfer the liquid.
Purification of the Extracted DNA
3. Add 150 l of purification reagent
to the disc. Use different tips for
different discs.
4. Close the tube
and flick it to wash
the disc. Ensure
the disc remains in
the liquid.
5. Remove and discard the purification reagent.
6. Repeat steps 3, 4 and 5.
Purification of the Extracted DNA
7. Use a fresh tip to add 150 l of TE-1 buffer to
the disc. Use different tips for different discs.
8. Close the tube
and flick it to wash
the disc. Ensure
the disc remains in
the liquid.
9. Remove and discard the buffer.
10. Repeat steps 7, 8 and 9.
Amplification of Chloroplast DNA (cpDNA)
1. Label the PCR tube.
2.– 4. Add reagents:
4 l sterile deionised water
10 l primer 1 (CHc) (yellow)
10 l primer 2 (CHd) (blue)
PCR beads contain Taq poymerase,
dNTPs, buffers and co-factors.
Amplification of Chloroplast DNA (cpDNA)
5. and 6. Flick the bottom of the PCR tube –
centrifuge if necessary.
7. Use a clean cocktail
stick to transfer the
disc from the micro
tube to the PCR tube.
Ensure the disc is
submerged in the PCR
reagents.
Amplification of Chloroplast DNA (cpDNA)
8. Place the PCR tube in
the thermal cycler.
Alternatively use three water
baths set to the correct
temperatures.
Amplification of Chloroplast DNA (cpDNA)
The PCR programme
1. 94°C for 2 minutes to ensure maximum
separation of the strands.
2. 30 cycles of the following steps:
94°C for 30 seconds
55°C for 30 seconds
72°C for 45 seconds
3. (72°C at final stage for 2 minutes)
9. Samples may be frozen ready for gel
electrophoresis.
Gel Electrophoresis of PCR Products
If a DNA sizing ladder
is available add 2 l of
loading dye to 8 l of
DNA ladder (lilac
micro tube). Mix and
load all 10 l into well
1 in a 1.5% agarose
gel.
This is not on your
work card so don’t
worry.
Diagram: Dean Madden NCBE
Gel Electrophoresis of PCR Products
1. Using a fresh tip, add 2 l of
loading dye to your amplified
sample and mix.
2. Load 10 l of the sample into a
different well in the gel. Note
sample number and well.
3. Repeat step 3 for other PCR
samples.
Gel Electrophoresis of PCR Products
Fit the carbon-fibre electrodes as shown.
Diagram: Dean Madden NCBE
Gel Electrophoresis of PCR Products
Attach the power leads. The samples run from black to
red.
Gel Electrophoresis of PCR Products
You will need
stain, either Azure
A or Fast Blast.
You will also need
a suitable tray,
such as a shallow
ice-cream tub, to
hold the gel and a
piece of plastic to
help move the gel.
Gel Electrophoresis of PCR Products
Using the plastic
card, gently scoop
the gel from the
tank into the tray.
Gel Electrophoresis of PCR Products
4. and 5. Pour Azure A or Fast Blast stain over the
gel and leave for 4 minutes before de-staining.
Remember not to pour the stain down the sink!
Gel Electrophoresis
of PCR
Products
Gel electrophoresis
of PCR
products
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1 2 3
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Lanes 1–3 spinach
Lanes 1 – 3 spinach
PCR
cycles
1 -1–20
20 PCR
cycles
PCR
cycles
2 –2–25
25 PCR
cycles
3–30 PCR cycles
3 – 30 PCR cycles
Lanes
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Lanes
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1 - 1–20
20 PCR
cycles
PCR
cycles
2 – 2–25
25 PCR
cycles
PCR
cycles
3 – 3–30
30 PCR
cycles
PCR
cycles
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