Important instructions on annealing temperature

When using Phusion, Phire, DyNAzyme or DyNAmo polymerases or kits, we recommend calculating primer Tm with the modified Breslauer's method1. To determine the annealing temperature for the actual PCR run, please see the table below. A few notes about primer design are also included.

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–1000 nM.
Salt (mM) Always use the default 50 mM salt concentration in the calculation.
Notes
  • For primers max 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: Tm's 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 Hot Start DNA Polymerase, use primers with Tm 60°C or higher.
  • With Phusion Hot Start II DNA Polymerase and all non-hot start Phusion DNA Polymerases primers with lower Tm can also be used.
  • With Phusion Flash DNA Polymerase and Phusion Blood DNA Polymerase, 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
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–1000 nM.
Salt (mM) Always use the default 50 mM salt concentration in the calculation.
Notes
  • For primers max 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: Tm's 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 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
Primer concentration (nM) Use the actual primer concentration in the calculation. Recommendation for all DyNAzyme DNA Polymerases is 300 nM–1000 nM.
Salt (mM) Always use the default 50 mM salt concentration in the calculation.
Notes
  • In the actual PCR run use an annealing temperature 5°C lower than the lower Tm given by the calculator.
    Example: Tm's given by the calculator are 66.5°C and 65.0°C => Use an annealing temperature of 60.0°C in the actual run.
  • For primers >20 nt use an annealing temperature 3°C higher than the lower Tm given by the calculator.
    Example: Tm's given by the calculator are 66.5°C and 65.0°C => Use an annealing temperature of 68.0°C in the actual run.
  • 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
Primer concentration (nM) Use the actual primer concentration in the calculation. Recommendation for all DyNAzyme DNA Polymerases is 300 nM–1000 nM.
Salt (mM) Always use the default 50 mM salt concentration in the calculation.
Notes
  • In the actual PCR run use an annealing temperature 5°C below the lower Tm calculated for the primers.
    Example: Tm's given by the calculator are 66.5°C and 65.0°C => Use an annealing temperature of 60.0°C in the actual run.
  • With DyNAmo Flash SYBR Green qPCR Kit a combined annealing and extension step at 60°C works well for most amplicons, if primers are designed to anneal efficiently at 60°C (Tm of the primers is about 65°C). If the primers cannot be designed to anneal at 60°C, the annealing and extension steps can be performed separately. In that case use an annealing temperature 5°C below the lower Tm calculated for the primers.
Primer concentration (nM) Use the actual primer concentration in the calculation. Recommendation for most chemistries with DyNAmo Probe qPCR Kits is between 50 –1000 nM.
Salt (mM) Always use the default 50 mM salt concentration in the calculation.
Notes
  • In the actual PCR run annealing temperature depends on the chemistry used. With TaqMan chemistry primers and probes are usually designed to be annealed and extended at 60°C. Design primer Tm to be about 5 degrees above the annealing temperature and probe Tm about 10 degrees above the primer Tm. For other chemistries, follow the guidelines from the chemistry provider.