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