What is primer melting temperature?

The melting temperature, or Tm, is the temperature at which half of the primer-template duplexes have separated into single strands. It guides the annealing temperature of a PCR, since primers must bind their target but not stick non-specifically.

Which method is most accurate?

Nearest-neighbour thermodynamics is the most accurate because it accounts for how each adjacent base pair stacks, plus salt and primer concentration. The basic 2(A+T)+4(G+C) rule is only a rough guide for very short oligos under about 14 bases.

What annealing temperature should I use?

A common starting point is 3 to 5 degrees below the lower Tm of your two primers. If you see non-specific bands, raise the annealing temperature; if the product is weak or absent, lower it and optimise from there.

Why does salt concentration matter?

Monovalent cations such as sodium and potassium shield the negatively charged DNA backbone, stabilising the duplex and raising the Tm. Higher salt gives a higher Tm, which is why the nearest-neighbour calculation includes a salt correction term.

Should my two primers have matched Tm values?

Yes. Forward and reverse primers should have Tm values within about 5 degrees of each other so they anneal efficiently at the same temperature. A large mismatch means one primer binds poorly at the chosen annealing temperature, hurting yield and specificity.

Primer Tm (Melting Temperature) Calculator

A primer’s melting temperature sets the annealing temperature of your PCR and strongly affects specificity. This calculator reports the nearest-neighbour Tm — the most accurate method — alongside the classic basic and salt-adjusted estimates, straight from the primer sequence.

How it works

Three methods are computed. The basic Wallace rule is fast but ignores salt and sequence context:

basic Tm    = 2(A+T) + 4(G+C)
salt-adj Tm = 64.9 + 41 × (G+C − 16.4) / N

The nearest-neighbour method sums experimentally measured enthalpy (ΔH) and entropy (ΔS) for every adjacent base-pair step (SantaLucia 1998 parameters), adds initiation terms, then solves for temperature with a salt correction:

Tm(K) = ΔH×1000 / ( ΔS + R × ln(Ct/4) )
Tm(°C) = Tm(K) − 273.15 + 16.6 × log10([Na+])

where R is the gas constant, Ct the primer concentration, and [Na+] the monovalent salt concentration in molar.

Tips and example

For the M13 forward primer GTAAAACGACGGCCAGT at 50 mM salt and 250 nM primer, the nearest-neighbour Tm lands near the low-to-mid 50s °C, while the basic rule over-estimates it because that rule is calibrated only for short oligos. Use the nearest-neighbour value for any primer of 18 bases or more, and keep your forward and reverse primer Tm values within about 5 °C of each other.