Primer Melting Temperature (Tm) Calculator
Written by Thierno Sadou Diallo, formula verified per our methodology • Last checked on 9/3/2026
The Wallace rule quickly estimates a short primer's Tm with 2°C × (A+T) + 4°C × (G+C). For a 20-base primer with 50% GC content (10 A/T and 10 G/C), the Wallace Tm is 60°C, versus about 51.8°C with the Bolton-McCarthy empirical formula, which is better suited to primers of this length.
Explanation
A PCR primer's melting temperature (Tm) is the temperature at which half of the double-stranded DNA molecules formed by the primer and its complementary sequence are denatured (separated into single strands). Knowing the Tm is essential for choosing a suitable annealing temperature during PCR: too high an annealing temperature prevents the primer from binding, too low favors nonspecific binding. Two classic formulas exist, with different ranges of validity. The Wallace rule (Tm = 2×(A+T) + 4×(G+C)) is the simplest, but stays reliable mainly for short primers, under about 20 bases. For more standard-length primers (18 to 25 bases, today's typical range for PCR), the Bolton-McCarthy empirical formula (Tm = 64.9 + 41 × (G+C − 16.4) / total length) — the one used by default in the reference software Primer3 for sequences longer than 13 bases — generally gives a more realistic estimate. This calculator shows both, for comparison. Neither replaces a full primer-design tool (such as Primer3), which also factors in salt and primer concentration, but they're enough for a quick first estimate. For the amount of DNA obtained after amplification, see our PCR amplification calculator; for the concentration of a DNA sample before PCR, see our nucleic acid concentration calculator.
Example: a 20-base primer with 50% GC content
Inputs
A+T bases: 10. G+C bases: 10 (20 bases total, 50% GC).
Calculation
Wallace rule: 2 × 10 + 4 × 10 = 20 + 40 = 60°C. Empirical formula: 64.9 + 41 × (10 − 16.4) ÷ 20 = 64.9 + 41 × (−6.4) ÷ 20 = 64.9 − 13.12 = 51.78°C.
Result
The estimated Tm is 60°C (Wallace rule) or 51.78°C (empirical formula).
Frequently asked questions
Why do the two formulas give different results?
Because they rely on different approximations, with distinct ranges of validity. The very simple Wallace rule was designed for short probes or primers (under 20 bases); beyond that, it tends to overestimate the Tm. The Bolton-McCarthy empirical formula accounts for the sequence's total length, making it more reliable for the 18-to-25-base primers commonly used today.
What annealing temperature should I choose from the Tm?
A common rule of thumb is to set the annealing temperature about 3 to 5°C below the calculated Tm (the lower of the two Tm values if a primer pair has different values), then optimize it experimentally if needed. This calculator gives a starting estimate, not a final value.
Do these formulas account for salt concentration?
No, both of these simplified formulas assume standard conditions (the Bolton-McCarthy formula is calibrated for about 50 mM sodium). For an estimate that precisely adjusts the Tm to your reaction's actual salt and primer concentration, a dedicated primer-design tool like Primer3 remains more accurate.