Primer Melting Temperature Calculator

Enter DNA in the 5′→3′ direction, one primer per line. Names are optional: Fwd: ATGC…. Spaces and lower case are accepted; blank lines and lines beginning with # are ignored. An invalid line stops the calculation so no primer disappears unnoticed. Up to {max} primers, 2–{len} bases each.

Temperature model

Tm uses the perfect DNA/DNA nearest-neighbor parameters in SantaLucia (1998), Table 2, terminal corrections and the length-dependent sodium entropy correction (Eq. 8). Adjacent bases matter, so sequences with the same length and GC content need not have the same Tm. No terminal phosphates are assumed.

Concentrations

The concentration field is the primer strand concentration in nM. In PCR mode, the target concentration is neglected and the effective concentration is the primer concentration. In DNA duplex mode, each distinct complementary strand has the entered concentration, giving an effective concentration of half that value. A self-complementary sequence instead uses the entered concentration of that single species and the symmetry entropy correction.

Limits

This is a model of a perfectly matched duplex, not a PCR annealing-temperature recommendation. Mg²⁺, K⁺, Tris, dNTPs, DMSO, mismatches, hairpins and dimers are not modelled. Sodium is limited to 10–1000 mM; the salt correction was fitted at 10–300 mM. Below 8 or above 60 bases, or above 300 mM, an extrapolation warning appears. Long DNA can melt in several transitions.

The optional Wallace value, 2(A+T)+4(G+C), is only a rule of thumb and ignores salt and concentration; its usual range is 14–20 bases. CSV exports both estimates, the sequence and the model settings as unrounded, language-independent values.