PCR Primer Calculator

For primers of 13 bases or fewer, the calculator uses the Wallace rule, Tm = 2°C × (A+T count) + 4°C × (G+C count), a simple linear approximation that works reasonably well only at short lengths. For anything longer — including the 18–25 bp range typical of real PCR primers — it switches to the more accurate empirical formula Tm = 64.9 + 41 × (GC count − 16.4) ÷ length, which accounts for how Tm scales with both length and GC content rather than just summing fixed per-base contributions.

Annealing exactly at the calculated Tm would mean only half the primer-template duplexes are actually formed at any moment, since Tm is defined as the temperature where half have dissociated. Starting 5°C below Tm — the rule-of-thumb this calculator applies to your computed Tm — biases the reaction toward stable, specific binding while leaving some margin; real-world optimization often means running a temperature gradient around this starting point.

G-C base pairs form three hydrogen bonds each, compared to two for A-T pairs, making GC-rich regions of the duplex more thermally stable — which is exactly why the Tm formulas in this calculator weight the G+C count so heavily. The generally recommended range is 40–60% GC; primers far outside that range can have unpredictable melting behavior or be prone to secondary structure.

The calculator multiplies your primer length by 330 Da, the average mass of one single-stranded DNA nucleotide, to estimate the primer's total molecular weight. This is a practical number for converting between the mass of primer you receive from a synthesis order and the molar concentration you need for your PCR reaction.