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.