Estimates of the annealing temperature can be calculated in several different ways. These calculated annealing temperatures are a starting point for the PCR experiment, but ideal annealing temperatures are determined empirically. Primer extension Primer extension is usually performed at 72 C, or the optimum temperature of the DNA polymerase . The length of time of the primer extension steps can be increased if the region of DNA to be amplified is long, however, for the majority of PCR experiments an extension time of 2 minutes is sufficient to get complete extension. The number of cycles is usually between 25 and 35.
More cycles mean a greater yield of product.
It is unusual to find procedures which have more than 40 cycles.