The annealing temperature in a PCR is usually very critical for the success of the PCR. The range for the optimal annealing temperature is usually quite narrow. When performing PCR with a Mastermix containing the enzyme polymerase, the mix usually comes with a recommended protocol. The Mastermix simplifies PCR and makes it more straightforward. However, most of the time the annealing temperature in the protocol that comes with the Mastermix will differ from the one published with the primers by a wide margin. In such case, one is usually in a dilemma as to which of the annealing temperature to use.
You should use the annealing temperature that is appropriate for your primers. If you got the primer sequences from a paper and someone published the annealing temperatures they used, definitely try that temperature first and then optimise from there if your PCR doesn't work well.
Go to NEB Tm Calculator ( tmcalculator.neb.com ), enter product group/kit as Taq DNA polymerase, enter your primer sequences and primer concentration, and an appropriate annealing temperature will be displayed. Try using an annealing temperature 1 degree C bellow the displayed annealing temperature. This technique generally works for me if I don't want to optimize and just want to see the results. However in order to optimize you need to perform gradient PCR with a range of temperatures.
Anthony C Ike Ideally You should run the gradient PCR at annealing temperature ±5°C of calculated primer Tm. Number of factors affect the primer annealing and therefore it is ideal to determine the optimum Tm experimentally for your primers.
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