What Tm means
The melting temperature (Tm) is the temperature at which half of the primer is bound to its target and half is free. The annealing step of a PCR is set a little below it, so the primers bind firmly but only to the right place.
Three ways to calculate it
- The Wallace rule, for primers under about 14 bases: Tm = 2 × (A + T) + 4 × (G + C). Quick, but it ignores the order of the bases and the buffer.
- The GC formula, for longer primers: Tm = 64.9 + 41 × (G + C − 16.4) ÷ length. A 20-mer with 10 G or C gives 64.9 + 41 × (10 − 16.4) ÷ 20 = 51.8 °C.
- Nearest-neighbor thermodynamics, the most accurate: it adds up the stacking energy of each pair of neighboring bases, then corrects for the primer concentration and the salt in the reaction.
Why the buffer matters
Cations shield the negative charge of the DNA backbone, so more salt means a higher Tm. Magnesium has a much stronger effect than sodium, and dNTPs bind some of it. The same primer can differ by 5 °C or more between calculators that assume different buffers, so set the sodium, magnesium and dNTP concentrations of your own mix.
Choosing the annealing temperature
Start about 5 °C below the lower Tm of the pair. If you see no product, lower it; if you see extra bands, raise it. A gradient PCR tests a range in one run. Some high-fidelity polymerases come with their maker's own rules for their buffer, which can give a different starting point.
Primer design rules of thumb
- 18 to 25 bases long, with 40% to 60% GC.
- The two primers' Tm within about 5 °C of each other.
- A G or C at the 3' end, but no more than 3 G or C in the last 5 bases.
- No runs of 4 or more of one base, and no stretches that pair with themselves or with the other primer.
Calculators
The Primer Tm Calculator uses nearest-neighbor thermodynamics with your salt, magnesium and primer concentrations, and suggests an annealing temperature. PCR Primer Stats compares all three methods and warns about hairpins, self-dimers and runs.