A PCR primer's melting temperature (Tm) is one of the first numbers a molecular biologist checks when designing or troubleshooting a reaction — it drives the annealing temperature, and a poorly estimated Tm is a common reason PCR reactions fail to amplify or amplify the wrong product. This calculator implements the two textbook estimation methods, tells you which one fits your primer's length, and converts the result into a starting annealing temperature.

How the Primer Tm Calculator works

The calculator counts the A, T, G, and C bases in your pasted sequence and applies one of two classic formulas. The Wallace rule (Tm = 2(A+T) + 4(G+C)) is a simple weighted count that works well for short oligonucleotides, roughly 14 nucleotides or fewer. The GC% method (Tm = 64.9 + 41·(GC − 16.4)/N) instead scales with GC content relative to the total primer length, and is the more commonly recommended formula for the 15-30 nucleotide primers used in typical PCR. The calculator auto-selects whichever method fits your sequence's length, though you can always override it — the Method Compare tab shows both values side by side so you can see how far apart they are for your specific primer.

Choosing a method and reading the annealing temperature

If you're not sure which method to trust, the length-based recommendation is a reasonable default: use Wallace for very short primers and GC% for standard-length PCR primers. The suggested annealing temperature (Tm − 5°C) is a widely used starting point, not a guarantee — in practice, labs often run a gradient PCR across a few degrees around this estimate to find the temperature that gives the cleanest, most specific amplification for their particular primer pair, template, and polymerase.

Limits and edge cases

Both formulas here are sequence-composition estimates and do not account for salt/ion concentration, primer concentration, or the specific stacking energies between neighboring bases — all of which shift real Tm. Labs that need higher precision typically use a nearest-neighbor thermodynamic model, which accounts for the sequence context of each base pair rather than just the overall A/T/G/C counts; that method isn't implemented here. Non-ACGT characters (ambiguity codes, whitespace, stray text from a pasted file) are stripped out and counted separately rather than silently miscounted — check the Primer Stats tab if your result looks off, since an unexpected character count usually means something didn't paste cleanly.