BIO430: PCR Laboratory Exercise (Part I)
Protocol Overview
The strategy for this experiment uses nested PCR to amplify portions of the GAPC gene from
gDNA of the plant of interest. A series of control reactions will also be run, including
Arabidopsis gDNA and a plasmid, pGAP, with a portion of the GAPC gene as positive controls
and a no-template negative control. In the initial round of PCR, a set of blue primers using
degenerate (less specific) sequences will amplify the GAPC gene from the gDNA. Then, in the
second round of PCR (the nested PCR), a more specific set of yellow primers will amplify
GAPC from the initial PCR products. It is very important not to reverse the order in which the
primers are used or to mix the two primer sets together in the PCR reactions.
Materials Needed
PCR master mix (2x)
Initial GAPDH PCR primers, blue
gDNA — previously extracted from 2 plants
Arabidopsis gDNA (diluted to 5 ng/μl)
pGAP control plasmid DNA
Sterile water
20 μl adjustable-volume micropipet and filter tips
PCR tubes
Microcentrifuge tube
Marking pen
Ice bath
Tube rack
Quantity
120 μl
4 μl
6 μl
6 μl
100 μl
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Procedure for Initial PCR (First-Round PCR)
Plan the first round of PCR. You will perform one initial PCR for each of the two plant gDNA
samples you have extracted, two positive controls (one using control gDNA and the other using
pGAP plasmid DNA), and one negative control with sterile water instead of DNA, for a total of
5 PCR reactions. Generate a table to record the label on each PCR tube, the DNA template, and
the primers used to amplify the DNA (see below).
1. Referring to your table, label your PCR tubes with your initials and the tube label.
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2. Place the PCR tubes on ice.
3. Ensure all the reagents are thoroughly mixed, especially the gDNA. Mix tubes containing
reagents by flicking to ensure the gDNA is homogeneously distributed. Before opening the
tubes, spin in a microcentrifuge for 5–10 seconds to force contents to the bottom of the tube (to
prevent contamination).
4. Pipet 20 μl of 2x MM into each PCR tube.
5. Add 15 μl of sterile water to each tube.
6. Referring to your initial PCR plan, use a fresh pipet tip to add 5 μl of the appropriate DNA
template to each tube and gently pipet up and down to mix reagents. Use a fresh filter tip each
time. Recap tubes.
7. When your instructor tells you to do so, place your PCR tubes into the thermal cycler.
The PCR reaction will run for the next several hours using the following Initial GAPDH PCR
program:
Initial denaturation: 95°C for 5 minutes
Then 40 cycles of:
Denaturation: 95°C for 1 minute
Annealing: 52°C for 1 minute
Extension: 72°C for 2 minutes
Final extension: 72°C for 6 minutes
Hold: Forever 15°C
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