PCR Annealing Temperature Calculator
Set the annealing temperature for a PCR reaction from your two primer melting temperatures.
How it works
Ta = min(Tm_forward, Tm_reverse) − offset (offset ≈ 3–5 °C)
The annealing step must be cool enough for both primers to bind, so it is keyed to the lower of the two primer melting temperatures. Setting Ta a few degrees below that Tm gives stable, specific binding; going too low invites mismatched priming and non-specific bands, while going too high can stop the primers annealing at all. Primers with well-matched Tm values (within a couple of degrees) anneal most cleanly.
Worked example
With a forward Tm of 60 °C and a reverse Tm of 58 °C, the lower Tm is 58 °C. Using a 5 °C offset, the annealing temperature is 58 − 5 = 53 °C.
Sources: PCR annealing — Wikipedia
Frequently asked questions
Why base Ta on the lower Tm?
Both primers have to anneal in the same step. Keying to the lower Tm ensures the weaker primer can bind; a temperature tuned to the higher Tm would leave it off the template.
What if my primer Tm values differ a lot?
A large difference (more than ~5 °C) makes it hard to find a temperature that suits both. Redesign one primer so the pair is closer, or use a temperature-gradient PCR to find the best compromise.
Is a fixed offset always right?
No — the 3–5 °C rule is a starting point. Optimize empirically with a gradient run, since polymerase, buffer and template all shift the ideal annealing temperature.
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This calculator is for educational purposes. Double-check important results before acting on them.