PCR with Phusion®, Phire®, Q5®, and Similar Polymerases 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, including 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, so they stabilize 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 being used, the annealing temperature should be about 6-12°C above the primer Tm as calculated by our software.
polymerases are fused to a processivity-enhancing dsDNA-binding domain, so they stabilize 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 ...
