This tool 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
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 modified Allawi & SantaLucia's thermodynamics method  is used for Tm and annealing temperature calculation of reactions with Platinum SuperFi, Phusion and Phire DNA Polymerases. The parameters were adjusted on a set of primers seeking to maximize specificity and retain high yields.
To use this 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).
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