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General Notes on Primer Design in PCR*

annealing efficiency: in general, the longer the primer, the more inefficient the annealing. With fewer templates primed at each step, this can result in a significant decrease in amplified product. The primers should not be too short, however, unless the application specifically calls for it. As discussed below, the goal should be to design a primer with an annealing temperature of at least 50C.

The relationship between annealing temperature and melting temperature is one of the Black Boxes of PCR. A general rule-of-thumb is to use an annealing temperature that is 5C lower than the melting temperature. Thus, when aiming for an annealing temperature of at least 50C, this corresponds to a primer with a calculated melting temperature(Tm) ~55C. Often, the annealing temperature determined in this fashion will not be optimal and empirical experiments will have to be performed to determine the optimal temperature. This is most easily accomplished using a gradient thermal cycler like Eppendorf's Mastercycler gradient.

Melting Temperature (Tm)

It is important to keep in mind that there are two primers added to a PCR reaction. Both of the oligonucleotide primers should be designed such that they have similar melting temperatures. If primers are mismatched in terms of Tm, amplification will be less efficient or may not work at all since the primer with the higher Tm will mis-prime at lower temperatures and the primer with the lower Tm may not work at higher temperatures.

The melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula:

Tmprimer =
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