FAQ: How should I determine the appropriate annealing temperature for my reaction?

Please use NEB’s Tm Calculator to determine the appropriate annealing temperature for your primer pair and NEB polymerase/buffer of interest.

Unlike other calculators, the NEB Tm Calculator takes into consideration buffer components that affect melting temperatures and empirical observations when calculating the optimal annealing temperature. 

Other online calculators may underestimate the best Q5 polymerase annealing temperature.

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Efficient PCR is a dynamic balancing act of chemicals and reactants that promote specific primer interaction with its compliment in the template at the selected annealing temperature. While annealing temperatures are constant values selected by the scientist, melting temperatures between each primer and the template can differ from amplicon to amplicon.


Note: this section specifically discusses annealing of an oligonucleotide primer to a DNA template.

During the denaturation step of PCR, high temperature separates template dsDNA into ssDNA, revealing complex nucleotide sequences that permit annealing (binding, hybridization, association) of a complimentary single-stranded oligonucleotide primer at a lower temperature.

  • The annealing temperature (TA) is the temperature used during the primer annealing step of a PCR, which is dependent on primer melting temperature.
  • The melting temperature (TM) of a primer is the temperature at which 50% of the primer is bound to its perfect complement and 50% is free in solution due to dissociation ("melting") from its compliment.

Why using the correct annealing temperature is important for successful PCR
The annealing temperature of a reaction is usually lower than the melting temperature to ensure primer hybridization to the template.

  • If the annealing temperature is too high, the primer will not anneal to the template and amplification will not proceed.
  • If the annealing temperature is too low, nonspecific binding of the primer(s) to the template or each other (primer dimers) can occur, causing:
    • Increased likelihood of nonspecific product formation.
    • Decreased formation of the intended product due to inefficient reaction conditions.

PCR reactants that influence primer melting temperature and reaction annealing temperature
Melting temperatures are not constant values in a PCR and are influenced by a number of factors:

  • Primer length and proportion of guanine and cytosine relative to adenine and thymine (% GC content)
    • Dictates the amount of hydrogen bonding between the primer and its compliment.
      • The more hydrogen bonding (higher Tm) of a primer to its template, the more energy needed to break those bonds (higher temperature).
  • Primer concentration
    • The melting temperature of primers in a PCR is determined by the DNA species in molar excess, which should be the primers.
  • Magnesium and dNTPs
    • The free concentration of magnesium ions [Mg2+] determines the melting temperature of a DNA duplex, but magnesium can be sequestered by the reactants and products of the PCR.
      • The positive charge of magnesium chelates the negatively charged phosphates of dNTPs, primers, and ssDNA.

Reduction of electrostatic repulsions (between primer and ssDNA phosphates) increases primer

  • Concentration of monovalent cations (Na+, K+)
    • Monovalent cations support DNA duplex stability, similarly to magnesium ions.
      • Monovalent cations and magnesium ions compete for DNA binding.
        • Increasing monovalent cation concentration decreases magnesium binding to DNA.