Primer Melting Temperature Calculator

The temperature at which half the primer is bound to its template and half is free. Annealing temperature is conventionally set a few degrees below it.

Tm = 2(A+T) + 4(G+C) in degrees Celsius. It is quick and holds reasonably below about 14 bases, but it ignores salt concentration entirely and drifts badly on longer sequences.

A G–C pair is held by three hydrogen bonds against two for A–T, and GC-rich stacking is more favourable. More energy is needed to separate the strands.

Considerably. Sodium ions screen the negative charge on the phosphate backbone, so the strands repel each other less and the duplex is more stable. Raising sodium raises Tm, which the Wallace rule and the basic GC formula both ignore.

Usually about 5 °C below the lower of the two primer Tm values. Too high and the primers do not bind; too low and they bind to unintended sites, giving spurious products.

None of these three. Nearest-neighbour thermodynamics, which accounts for each adjacent base pair rather than just base composition, is what a real design tool uses — these approximations are for sanity checks and quick comparisons.

It sums thermodynamic parameters for each adjacent base pair in the sequence rather than just counting bases, then computes Tm from the resulting ΔH and ΔS along with primer and salt concentrations. It is what commercial design tools use and what all three formulas here approximate.

Yes — duplex formation is a bimolecular reaction, so higher primer concentration favours the bound state and raises Tm. None of the three formulas here includes it; nearest-neighbour calculations do.

More, per mole. Mg²⁺ carries twice the charge and screens the phosphate backbone far more efficiently, so PCR buffers at 1.5 to 2 mM Mg²⁺ contribute meaningfully to duplex stability despite the low concentration.

18 to 25 bases, 40 to 60% GC, a G or C at the 3′ end to anchor extension, no runs of four or more identical bases, no self-complementarity that would form hairpins, and no complementarity between the pair that would give primer dimers.

Because both anneal at the same temperature in the same tube. A pair differing by more than about 5 °C cannot both work well — one binds poorly or the other binds non-specifically — so redesigning to match them beats adjusting the cycler.

No. RNA duplexes are generally more stable than DNA of the same sequence, and RNA–DNA hybrids are different again. Each needs its own set of nearest-neighbour parameters, so a DNA formula applied to RNA gives a systematically wrong answer.