Biology
Annealing Temperature Calculator
Estimate a PCR primer's melting temperature (Tm) and a sensible starting annealing temperature (Ta) from base composition.
Your inputs
Change any value to update the result instantly.
Suggested annealing temperature
59.5 °C
Primer melting temperature (Tm)
51.8 °C
Product melting temperature
84.1 °C
Primer length
20 nt
Primer GC content
50 %
A starting point for gradient PCR only — always confirm empirically. Primers shorter than 14 bases use the Wallace rule (2 × (A+T) + 4 × (G+C)).
What this calculator does
Estimates a PCR primer's melting temperature and a starting annealing temperature from base composition.
What your result means
The annealing temperature is a starting suggestion for gradient PCR optimisation, in degrees Celsius; it is not a guaranteed optimum.
Formula and working
Tm = 64.9 + 41 × (G + C − 16.4) ÷ (A + T + G + C); Ta = 0.3 × Tm(primer) + 0.7 × Tm(product) − 14.9
- 1
Collect the inputs
Primer A bases = 5; Primer T bases = 5; Primer G bases = 5; Primer C bases = 5; Product length = 500 bp; Product G + C content = 50 %
- 2
Apply the formula
Tm = 64.9 + 41 × (G + C − 16.4) ÷ (A + T + G + C); Ta = 0.3 × Tm(primer) + 0.7 × Tm(product) − 14.9
- 3
Result
Suggested annealing temperature = 59.5 °C
Frequently asked questions
Symbols and assumptions
- Tm
- Melting temperature of primer or product
- Ta
- Suggested annealing temperature
- GC%
- Fraction of guanine and cytosine bases
- • Primers of 14+ bases use the salt-free Marmur-Doty style Tm estimate; shorter primers use the Wallace rule.
- • Standard salt and primer concentrations are assumed.
Sources and review
Reviewed 2026-09-22 · Next review 2027-09-22
- • Nearest-neighbour Tm with exact salt correction (as in vendor tools) will differ; always optimise empirically with gradient PCR.
Important limitation
This is an educational estimate based only on the values and formula shown. Verify current rates, rules, units, and professional standards before relying on the result. It is not personalized financial or investment advice.
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