This functional tool calculates the Tm of primers and estimates an appropriate annealing temperature while using different DNA polymerases. It also calculates the recommended Tm (melting temperature) of primers and PCR annealing temperature. 

This calculation is based on factors like the primer pair sequence, primer concentration, and DNA polymerase used in PCR. 

The calculator in addition to the above, calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient.

The modified Allawi & SantaLucia’s thermodynamics method is used here for Tm and annealing temperature calculation. This is based on reactions with Platinum SuperFi, Phusion and Phire DNA Polymerases characteristics. 

The parameters were adjusted on a set of primers seeking to maximize specificity and retain high yields.

If necessary, use a temperature gradient to further optimize and empirically determine the ideal annealing temperature for each of the template-primer pair combinations. 

The annealing temperature gradient should start with a qualifying temperature of 6–10 °C lower than annealing temperature generated by the calculator and increased up to the extension temperature.