8. It?s your first week working in the lab and unfortunately you program the PCR

Question

8. It?s your first week working in the lab and unfortunately you program the PCR machine incorrectly so that your first three PCR experiments do not work as expected. However, from each of your mistakes you learn more about how PCR works! For each of the following scenarios, state what you think the results will be and briefly explain your reasoning. a) The denaturation step was performed at 72 degree C. b) The annealing step was performed at 96 degree C. c)The annealing step was performed at 24 degree C.

Explanation / Answer

The denaturation step should not be 72oC because the it is the step which decide the temperature of DNA melting, so it should be 93-98oC as between this temperature range H-bonding between the DNA bases would be disrupted to give single-stranded DNA molecules.

The annealing step should not be performed at 96 or 24oC. In fact, it should be low enough to allow for hybridization of the primer to the strand but at the same time high enough for the hybridization to be specific. So, the temperature range should be 50-65oC.

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