PCR Primer Designer
Primer design with SantaLucia 1998 nearest-neighbour Tm, GC clamp checking, hairpin and dimer screening.
How it works
Melting temperature is calculated with nearest-neighbour thermodynamics, summing enthalpy and entropy contributions for each adjacent base pair and applying a salt correction for the reaction’s monovalent cation concentration. Candidates are screened for GC clamps, self-complementarity that would form hairpins, and cross-complementarity that would form primer dimers.
Frequently asked questions
What is a good melting temperature for PCR primers?
Most primers work well between 55 and 65 °C, with the annealing temperature set roughly 3–5 °C below the lower of the two primer Tm values. What matters more than the absolute value is that the pair is matched: primers differing by more than about 5 °C in Tm will not anneal efficiently at the same temperature, which reduces yield and specificity.
Why do nearest-neighbour and basic Tm calculations disagree?
The basic rule of 2 °C per A/T and 4 °C per G/C counts bases in isolation and ignores that duplex stability depends on which bases are adjacent to which. Nearest-neighbour models sum measured thermodynamic parameters for each dinucleotide step and correct for salt concentration and strand concentration, which is why they agree far better with experiment — often differing from the basic rule by 5 °C or more.
Method reference
SantaLucia J Jr (1998) PNAS 95(4):1460-1465.