Primer Tm Calculator

How to calculate primer Tm

1. Paste the forward primer and, if you have one, the reverse primer, written 5' to 3'.
2. Set the primer, sodium, magnesium and dNTP concentrations of your PCR mix.
3. The Tm of each primer appears with its length, GC content, the difference between the two and a starting annealing temperature.

Frequently asked questions

Which method does it use?

Nearest-neighbor thermodynamics with the SantaLucia (1998) parameters. Magnesium is counted as extra sodium, Na⁺ + 120 × √(Mg²⁺ − dNTPs), following von Ahsen et al. (2001).

What annealing temperature should I use?

A common starting point is 5 °C below the lower of the two Tm values; a gradient PCR finds the best one. Some polymerases, such as high-fidelity enzymes in their own buffers, come with their maker's Tm calculator and rules, which give different numbers.

How close should the two Tm values be?

Within about 5 °C, so both primers bind well at the same annealing temperature.