Estimate the melting temperature of a DNA duplex or primer.
GC pairs form three hydrogen bonds against two for AT, so higher GC content raises melting temperature. Salt stabilises the duplex by screening phosphate charge, which is why the correction is needed. Primer pairs should have Tm values within about 2 °C of each other, or one will anneal preferentially and bias the amplification.
DNA Melting Temperature
Tm = 64.9 + 41 × (GC − 16.4) ÷ length, with a salt correction of 16.6 log₁₀[Na⁺]
Tm = 64.9 + 41 × (GC − 16.4) ÷ length, with a salt correction of 16.6 log₁₀[Na⁺] GC pairs form three hydrogen bonds against two for AT, so higher GC content raises melting temperature. Salt stabilises the duplex by screening phosphate charge, which is why the correction is needed.
Primer pairs should have Tm values within about 2 °C of each other, or one will anneal preferentially and bias the amplification.
This calculator takes 4 inputs: Primer or duplex length, GC content, 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.