Can I configure SnapGene to show primer Tm values suitable for PCR polymerases such as Phusion®, Phire®, and Q5®?
For most PCR polymerases, the optimal annealing temperature for the PCR reaction is about 0–5°C below the Tm for the primers.
For some PCR polymerases such as Phusion®, Phire®, and Q5®, the optimal annealing temperature is about 6‑12°C above the primer Tm. The reason is that these polymerases are fused to a processivity–enhancing dsDNA-binding domain that stabilizes the primer-template complex.
The approach recommended by NEB and Thermo Scientific is to use older thermodynamic parameters to calculate the Tm for polymerases such as Phusion®. Those older parameters are less accurate, and they tend to give Tm values about 6–9°C higher than the newer parameters. NEB then recommends using an annealing temperature 0-3°C above the inaccurately high Tm values, or about 6–12°C above the actual Tm.
The SnapGene team has decided not to provide inaccurate Tm values. Instead, we recommend that when a polymerase such as Phusion® or Phire® or Q5® is used, the annealing temperature should be about 6–12°C above the primer Tm as calculated by our software.