Important instructions on calculating PCR annealing temperatures

When using Thermo Scientific Phusion or Phire DNA polymerases or master mixes, we recommend calculating primer annealing temperatures using a Tm calculator, which is based on the modified Breslauer's method1. The tables below describe how to use the calculator and a few notes about primer design. (Related: Simplifying primer annealing)

Note: Annealing temperature calculation is not required for Phusion Plus DNA Polymerase due to its universal annealing feature.

Phusion High-Fidelity DNA Polymerase

Primer concentration (nM)Use the actual primer concentration in the calculation. Recommendation for Phusion DNA Polymerase is 500 nM, but it can be varied between 200 nM–1,000 nM.
Salt (mM)Always use the default 50 mM salt concentration in the calculation.
Notes
  • For primers ≤20 nt, use the lower Tm given by the calculator for annealing.
  • For primers >20 nt, use an annealing temperature 3°C higher than the lower Tm given by the calculator.
    Example: If Tms given by the calculator are 66.5°C and 65.0°C, use an annealing temperature of 68.0°C in the actual run.
  • With Phusion Flash DNA Polymerase and Phusion Blood PCR Master Mix, do not perform annealing below 50°C.
  • If the amplification fails with the recommended annealing temperature, use a temperature gradient to optimize the annealing. The annealing gradient should range from the original annealing temperature to the extension temperature (two-step PCR).
  • If high DMSO concentration is used, the annealing temperature determined by the guidelines above must be lowered, as DMSO decreases the melting point of the primers. It has been reported that 10% DMSO decreases the melting temperature by 5.5–6.0°C.2

Phire Hot Start DNA Polymerase

Primer concentration (nM)Use the actual primer concentration in the calculation. Recommendation for Phire Hot Start DNA Polymerase is 500 nM, but it can be varied between 200 nM–1,000 nM.
Salt (mM)Always use the default 50 mM salt concentration in the calculation.
Notes
  • For primers ≤20nt, use the lower Tm given by the calculator for annealing.
  • For primers >20 nt, use an annealing temperature 3°C higher than the lower Tm given by the calculator.
    Example: If Tms given by the calculator are 66.5°C and 65.0°C, use an annealing temperature of 68.0°C in the actual run.
  • With Phire Hot Start DNA Polymerase, use primers with Tm 60°C or higher.
    With Phire Hot Start II DNA Polymerase primers with a lower Tm can also be used.
  • If the amplification fails with the recommended annealing temperature, use a temperature gradient to optimize the annealing. The annealing gradient should range from the original annealing temperature to the extension temperature (two-step PCR).
  • If high DMSO concentration is used, the annealing temperature determined by the guidelines above must be lowered, as DMSO decreases the melting point of the primers. It has been reported that 10% DMSO decreases the melting temperature by 5.5–6.0°C.2

References

  1. Breslauer KJ, Frank R, Blöcker H, Marky LA (1986) Proc Nat Acad Sci 83:3746–3750.
  2. Chester N, Marshak DR (1993) Analytical Biochemistry 209: 284–290.

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