Annealing Time and Temperature

Annealing Time

An annealing time of 30-45 seconds is commonly used in PCR reactions. Increase in annealing time up o 2-3 minutes did not appreciably influence the outcome of the PCR reactions. However, as the polymerase has some reduced activity between 45 and 65o C (interval in which most annealing temperature are chosen), longer annealing times may increase the likelihood of unspecific amplification products.

Annealing Temperature

Annealing temperature is one of the most important parameters that need adjustment in the PCR reaction. Moreover, the flexibility of this parameter allows optimization of the reaction in the presence of variable amounts of other ingredients (especially template DNA).