Annealing Temperature Calculator

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Choose whether to supply melting temperatures directly or let the calculator derive them from your primer sequences.
Use the lower of your forward and reverse primer Tm values; that is the limiting primer for specificity.
°C
The melting temperature of the amplified target region. For genomic DNA at physiological GC content, this is typically 70-80 °C.
°C
Rychlik (1990) is the standard empirical formula and is preferred when you have a target Tm. The Tm-5 rule is a quick shortcut when you only have the primer Tm.
Annealing temperature (Ta)Low - possible non-specific bands
52.9°C

Recommended PCR annealing step temperature

Primer Tm58°C
Target DNA Tm72°C
Recommended Ta range (low)49.9°C
Recommended Ta range (high)55.9°C
52.9 °C
Too low<45Low45-55Optimal55-68High68-72Very high72+

Recommended PCR annealing temperature: 52.9 °C

  • Start your gradient PCR at 49.9 °C and end at 55.9 °C to empirically confirm the optimum.
  • Ta is 5.1 °C below primer Tm, providing good specificity while keeping primers efficiently bound.

Next stepRun a gradient PCR over the recommended range, then choose the highest temperature that still gives a clean, bright product band.

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