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PCR Annealing Temperature Calculator

Estimate a PCR annealing temperature from the primers' melting temperatures and the product's length and GC content, using the Rychlik formula and the lower-Tm-minus-5 rule.

Formula

Ta=0.3 Tmprimer+0.7 Tmproduct−14.9T_{\mathrm{a}} = 0.3\,T_{\mathrm{m}}^{\text{primer}} + 0.7\,T_{\mathrm{m}}^{\text{product}} - 14.9
Tmproduct=81.5+16.6log⁡10[Na+]+0.41 (%GC)−675LT_{\mathrm{m}}^{\text{product}} = 81.5 + 16.6\log_{10}[\mathrm{Na}^+] + 0.41\,(\%\mathrm{GC}) - \dfrac{675}{L}
rule of thumb:Ta=Tmlower primer−5 ∘C\text{rule of thumb:}\quad T_{\mathrm{a}} = T_{\mathrm{m}}^{\text{lower primer}} - 5\,^\circ\mathrm{C}
TmprimerT_{\mathrm{m}}^{\text{primer}}
melting temperature of the less stable primer-template pair
TmproductT_{\mathrm{m}}^{\text{product}}
melting temperature of the amplicon

How it works

The annealing temperature is a balance. Too low, and primers bind at partially matching sites and give extra products; too high, and they do not bind enough for efficient amplification. Rychlik and colleagues tested amplifications across a range of temperatures and fitted a relationship between the best annealing temperature and the melting temperatures of the primer and the product.

A simpler rule sets the annealing temperature about 5 °C below the lower primer Tm. The two estimates are given side by side, with a gradient to try around them, because neither replaces testing. Some polymerases, especially high-fidelity ones with their own buffers, specify a different relationship.

Worked example

Primer Tms of 60.5 and 58.7 °C, a 400 bp product of 50% GC, in 50 mM salt.

  1. Tm(product) = 81.5 + 16.6 × log₁₀(0.05) + 0.41 × 50 − 675 / 400 = 78.7 °C.
  2. Ta = 0.3 × 58.7 + 0.7 × 78.7 − 14.9 = 57.8 °C.
  3. Rule of thumb: 58.7 − 5 = 53.7 °C.

An estimated Ta of 57.8 °C, or 53.7 °C by the simple rule; a gradient of 51 to 61 °C brackets both.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Primer Tms come from one method used for both primers.
  • The product's Tm is estimated from length and GC content alone, ignoring its sequence and structure.
  • Standard PCR conditions with a typical buffer.

Common mistakes

  • Applying the formula with Tm values from a vendor's calculator and mixing methods.
  • Using the higher primer Tm. The lower one limits annealing.
  • Skipping the gradient on a difficult template. The formula gives a start, not an optimum.

Related tools

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Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.