Question

You are a researcher working in a molecular laboratory. One day, you need to run a batch of PCRs. You assembled the reagents including Taq polymerase, made the reaction mixtures, and ran them in a thermocycler.

After the PCR program ran to completion, you performed a gel electrophoresis to check the quality of the PCR product. To your dismay, all reactions appeared to have failed! Nothing showed up on the gel.

You reviewed the thermocycler’s log, which displayed the following information about your recent PCR cycles:

Denaturation: 91°C for 1 minute

Annealing: 50 °C for 40 seconds

Elongation: 65 °C for 3 minutes

Can this temperature profile explain the PCR failure? Explain

Answer 1

PCR conditions:
Step I: Initial denaturation = 91 C for 3 min
Step II: Denaturation = 91 C for 1 min
Step III: Annealing = 50 C for 40 sec
Step IV: Extention = 72 C for 3 min
Step V: Go to step II
Step VI: Repeat 30-35 times
Step VII: Final extension = 91 C for 10 min
Step VIII: 4 C for 5 min
Step IX: Stop

In the given reaction set up, the extension is performed at 65 C for 3 min. Taq polymerase shows maximum activity at 72 C.
That could be the reason for the failure of PCR.