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 in your own words (4 points)

Answer 1

This is general method of PCR, where in step

1.     During thermocycling a 15–30 second denaturation at 95°C is recommended.

2.     Annealing:

The annealing step is typically 15–60 seconds. Annealing temperature is based on the T_m of the primer pair and is typically 45–68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated T_m.

3.     Extension:

The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended.

4.     Cycle number:

Generally, 25–35 cycles yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.

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In the given experiment,

There is no two step denaturation. May be a reason of failure.

Second, there is no repeated cycles in the program may be a reason of failure.

Third, the product has to be kept on 4 degree at the end.