Estimate primer melting temperature and recommend an annealing temperature.
Melting temperature rises with GC content because guanine and cytosine form three hydrogen bonds rather than two. Salt concentration stabilises the duplex, which is why the buffer composition shifts Tm measurably. Primer pairs should have Tm values within about two degrees of each other, because a single annealing temperature has to work for both.
Primer Tm
Tm = 64.9 + 41 × (GC − 16.4) ÷ length, with salt and concentration corrections
Tm = 64.9 + 41 × (GC − 16.4) ÷ length, with salt and concentration corrections Melting temperature rises with GC content because guanine and cytosine form three hydrogen bonds rather than two. Salt concentration stabilises the duplex, which is why the buffer composition shifts Tm measurably.
Primer pairs should have Tm values within about two degrees of each other, because a single annealing temperature has to work for both.
This calculator takes 4 inputs: Primer length, Number of G and C bases, Monovalent salt concentration, Primer concentration. The pre-filled defaults are a realistic starting point — replace them with figures from your own environment for a result you can act on.