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: 89 °C for 1 minute

Annealing: 50 °C for 40 seconds

Elongation: 65 °C for 3 minutes

Can this temperature profile explain the PCR failure?

Answer 1

Answer:

Based on the given cycle:

Denaturation: 89 °C for 1 minute

Denaturation temperature for ds DNA used in the PCR reaction was too low. Due to low temperture, the DNA will not completely denature and amplification efficiency will be low. It is recommended to use a denaturation temperature of 95°C for 20–30 seconds. For DNA templates that contains GC-rich sequences, it is recommended to include a a longer initial denaturation step of 2–4 minutes at 95°C ( prior to PCR cycling) to fully denature the template. A denaturation temperature of  95°C is required to disrupt the hydrogen bonds between complementary bases of double stranded DNA (dsDNA) into single stranded DNA (ssDNA). Due to poor amplification, nothing showed up on the gel.