This application calculates the Tm of primers and estimates an appropriate annealing temperature when using different DNA polymerases. How to use this calculator

Important note
If the PCR primer contains desired mismatches, e.g., for creating a mutation or a restriction site, make sure to calculate the Tm only for the correctly matched sequence.

1. Select your DNA polymerase

Platinum SuperFi DNA polymerase
Phusion or Phire DNA polymerase
Taq-based DNA polymerase

2. Select input method

Single pair
Batch

3. Type or paste your sequence

4. PCR Conditions

µM
mM

How to use the Tm calculator

The calculator calculates recommended Tm (melting temperature) of primers and PCR annealing temperature based on the primer pair sequence, primer concentration, and DNA polymerase used in PCR. The calculator also calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient.

The application is designed to calculate Tm according to three different methods.

The modified Allawi & SantaLucia's thermodynamics method (1) is used for Tm and annealing temperature calculation of reactions with Platinum SuperFi DNA Polymerase. The parameters were adjusted on a set of primers seeking to maximize specificity and retain high yield with Platinum SuperFi DNA Polymerase.

The modified Breslauer's thermodynamics method (2) is is used for Tm and annealing temperature calculation of reactions with Phusion or Phire DNA Polymerases.

A separate method is used for Tm and annealing temperature calculation of reactions with Taq-based DNA polymerases.

To use the calculator select your DNA polymerase, type in or paste your primer sequences, and provide your final primer concentration. Tm values, annealing temperature, and other data are automatically generated.

If necessary, use a temperature gradient to further optimize and empirically determine the ideal annealing temperature for each template-primer pair combination. The annealing temperature gradient should start with temperature 6-10°C lower than annealing temperature generated by the calculator and increased up to the extension temperature (two-step PCR).

  1. Allawi, H. T., and SantaLucia, J. (1997). Thermodynamics and NMR of internal G-T mismatches in DNA. Biochemistry, 36(34), 10581-10594.
  2. Breslauer, K. J., Frank, R., Blöcker, H., and Marky, L. A. (1986). Predicting DNA duplex stability from the base sequence. Proceedings of the National Academy of Sciences, 83(11), 3746-3750.