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Primer Melting Temperature (Tm) Calculator

Estimate PCR primer melting temperature with the Wallace rule or a salt-adjusted formula, plus GC% and annealing temperature.

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Tm is the temperature at which half the primer is hybridised; the annealing temperature is a suggested PCR starting point.

About this tool

Enter a primer sequence to estimate its melting temperature (Tm), the temperature at which half of the duplex is single-stranded. The sequence is uppercased and reduced to A, C, G, T and U before the bases are counted. Everything is computed in your browser.

The basic method uses the Wallace rule for short primers (under 14 nucleotides): Tm = 2 × (A + T) + 4 × (G + C). For primers of 14 bases or more it switches to the GC-based formula Tm = 64.9 + 41 × (GC − 16.4) / length. The salt-adjusted method applies Tm = 81.5 + 0.41 × GC% − 675 / length + 16.6 × log10([Na⁺]), where the sodium concentration is entered in millimolar, to account for buffer conditions.

A suggested annealing temperature of about Tm − 5 °C is provided as a practical starting point for PCR, along with the primer's GC% and length. For example, the 8-mer ATGCATGC (4 A/T and 4 G/C) gives a Wallace Tm of 2×4 + 4×4 = 24 °C.

Frequently asked questions

When is the Wallace rule used?
The basic method applies the Wallace rule, Tm = 2(A+T) + 4(G+C), to primers shorter than 14 nucleotides. At 14 bases or longer it uses the more accurate GC-based formula instead.
What does the salt-adjusted method add?
It corrects Tm for the sodium ion concentration of the reaction buffer using a log term, since higher salt stabilises the duplex and raises the melting temperature.
How is the annealing temperature chosen?
A common rule of thumb sets the PCR annealing temperature about 5 °C below the primer Tm. It is a starting point that you may optimise experimentally.
Are these values exact?
No. These are nearest-neighbour-free approximations useful for quick estimates. For critical work use a full nearest-neighbour thermodynamic model with your exact salt and primer concentrations.

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