Primer Melting Temperature (Tm) Calculator
Estimate the melting temperature of a short PCR primer from its base composition using the Wallace (2+4) rule.
How it works
Tm = 2 × (A + T) + 4 × (G + C) [Wallace / basic rule, °C]
The Wallace rule is a quick estimate for short oligonucleotides (roughly 14–20 bases). It credits each A–T pair 2 °C and each G–C pair 4 °C because G–C pairs share three hydrogen bonds versus two for A–T, so GC-rich primers stay annealed at higher temperatures. It ignores salt and primer concentration, so treat it as a starting point rather than a nearest-neighbour thermodynamic value.
Worked example
A 20-base primer with 10 A/T bases and 10 G/C bases gives Tm = 2 × 10 + 4 × 10 = 20 + 40 = 60 °C, and its GC content is 10 ÷ 20 = 50%.
Sources: Wallace rule — Wikipedia
Frequently asked questions
When is the Wallace rule accurate?
It works best for primers of about 14 to 20 bases at standard salt concentrations. For longer oligos or precise work, use a nearest-neighbour model that accounts for stacking energies, salt and primer concentration.
Why do G and C count double?
Guanine and cytosine form three hydrogen bonds, while adenine and thymine form only two. The extra bond makes G–C pairs more stable, so each is weighted 4 °C versus 2 °C for A–T.
How does Tm relate to the annealing temperature?
Annealing temperature is usually set about 3–5 °C below the lower primer Tm so the primers bind specifically without falling off. See the annealing temperature calculator.
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This calculator is for educational purposes. Double-check important results before acting on them.