PCR Annealing Temperature Calculator
Chemistry & BiologyCalculate the optimal PCR annealing temperature from primer and target melting temperatures.
Optimal Annealing Temperature
Estimated optimal PCR annealing temperature.
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Chemistry & Biology calculators
Frequently Asked Questions
Why use the less stable of the two primers?
A PCR reaction needs both primers to anneal successfully. Using the melting temperature of the less stable (lower Tm) primer ensures the calculated annealing temperature does not exceed what that weaker primer can tolerate.
What happens if the annealing temperature is set too high or too low?
Too high a temperature can prevent primers from binding at all, producing no product. Too low a temperature allows primers to bind imperfectly to unintended sequences, producing nonspecific or extra bands.
Why does target melting temperature carry more weight than primer temperature?
This empirical formula weights the target DNA melting temperature more heavily (0.7 versus 0.3) because successful amplification depends heavily on the primers finding and binding the correct target region specifically, which is more strongly influenced by the target sequence stability.
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