Melting temperature of a PCR product (not the primer)

Two very different numbers both get called “Tm”. The primer Tm is where a short oligo melts off its target — around 55–65 °C. The product Tm is where the whole double-stranded amplicon comes apart — often near 80 °C. They are not interchangeable.

Why the product melts higher

Melting temperature rises with length: a longer duplex has more base pairs holding it together, so it takes more heat to separate. A 20 nt primer and the 500 bp product it helps make differ by hundreds of base pairs, which is why the product melts tens of degrees higher than the primers that built it.

The salt-adjusted formula

For products longer than ~50 bp, the classic estimate is:

Tm = 81.5 + 16.6 · log₁₀[Na⁺] + 0.41 · (%GC) − 675 ÷ length

For a 500 bp product that is 50% GC at 50 mM Na⁺ (0.05 M): 81.5 − 21.6 + 20.5 − 1.35 ≈ 79.1 °C. GC content pushes Tm up (0.41 °C per %GC); higher salt stabilises the duplex and raises Tm; length’s contribution flattens out once the product is long.

Where you use it

Product Tm matters for qPCR melt-curve analysis, where a single sharp melt peak at the expected temperature confirms one specific product, and a low, broad peak flags primer-dimers — which melt much lower because they are tiny. It also underpins high-resolution melt (HRM) genotyping, where small sequence differences shift the melt.

Don’t use it for primers

This formula is calibrated for long duplexes. For designing primers, use the Wallace rule (very short oligos) or the nearest-neighbor model (real design), which account for sequence context and primer concentration that the amplicon formula ignores.