Demystifying PCR: fostering scientific literacy through hands-on PCR education

We're finally at the point to start actually amplifying the DNA, but first we need to make sure the reagents are all in the bottom of the PCR tubes. 

1.    Centrifuge briefly to collect all the reaction mix to the bottom of the PCR tubes without any bubbles. Make sure the caps on the tubes are snapped shut - failure to do this will lead to evaporation during the heating and cooling. 

2.   Put the PCR tubes into the thermocycler. The amplification parameters will vary depending on the primers, the size of the target and the machine you are using. Newer machines heat and cool faster, and the newer enzymes and formations are faster at attaching and extending the complementary DNA strands. Our protocol will be similar to the one below for an older PCR machine. Older machines can take time....You might want to let it run over night, if you are starting near the end of the day. 

NB: some machines (7500 ABI) require 0.1 mL tubes not 0.2mL tubes. If you have one of these machines, either specify this when requesting a kit, or transfer each PCR tube from the kit into 0.1 mL tubes. 

In general the phases are similar to this: 

1. An initial Denaturation: usually 94 – 95 °C, 5 min. The two strands of the template DNA unzip.

2. Denaturation for each of the next cycles: 94-95°C, 30 seconds.

3. Annealing: ~55 – 68 °C, 30 seconds. The primer attaches to the single-strand template DNA.

4. Elongation: 72 °C, 30 seconds. Taq polymerase extends complementarynucleotides starting from the primers, creating new strands from the 2 original strands. 

5. Repeat from Step 2, 30-40 times. 

6. A final Elongation step to make sure the strands have fully extended on each strand, 72 °C, 5 min. 

7. Cooling phase at 11°C to hold the samples until they are run on a gel.  

For our primers on our older machine, we will use these specific parameters: 

1. Denaturation: 95 °C, 5 min. 

2. Denaturation: 95°C, 40 seconds.

3. Annealing: 60 °C, 40 seconds.

4. Elongation: 72 °C, 30 seconds. 

5. Repeat from Step 2, 40 times. 

6. A final Elongation step, 72 °C, 5 min. 

7. Hold at 11°C.  

For Newer Machines: 

We also have a newer machine that cycles much faster. for that machine, we recommend skipping the 72°C elongation for these machines with newer Taq formulations, as it extends efficiently during the ramp up to 95°C. This will be much quicker. 

1. Denaturation: 95°C, 5 min. 

2. Denaturation: 95°C, 10 seconds.

3. Annealing: 60°C, 30 seconds.

4. Repeat from Step 2, 40 times. 

5. A final Elongation step, 72°C, 5 min. 

6. Hold at 11°C.  

Next step is electrophoresing your reactions on an agarose gel to see who was the culprit!