PCR Annealing Temperature

Use this PCR annealing temperature calculator to estimate primer melting temperature (Tm) and a starting annealing temperature (Ta). The calculation uses the primer sequence, GC content, and an approximate monovalent sodium (Na+) concentration.

Two simplified Tm estimates are provided. The Wallace method estimates Tm from the number of A, T, G, and C bases. The salt-adjusted estimate also accounts for the sodium concentration in the reaction. For primers up to 13 nucleotides, the calculator uses the Wallace estimate to determine the suggested Ta; for longer primers, it uses the salt-adjusted estimate. The suggested annealing temperature is set 5 °C below the selected Tm estimate.

These formulas are empirical approximations and should be used as starting points rather than exact thermodynamic predictions. Actual PCR performance also depends on primer concentration, Mg2+, dNTPs, buffer composition, template concentration, mismatches, primer-dimer formation, secondary structure, and other experimental conditions. Use the result as a starting point for PCR optimization, not as a guaranteed optimal temperature.

Primer sequence: Enter a DNA or RNA primer sequence in the 5′ → 3′ direction. Spaces and other non-letter characters are removed before validation. The calculator accepts A, C, G, T, and U. For the Wallace calculation, U is treated as T; GC content counts only G and C.

Salt (mM Na+): Enter the approximate monovalent sodium concentration in the reaction mixture, in millimolar (mM). The calculator converts the value to molar concentration before applying the salt-adjusted formula. 50 mM is provided as the default starting value.

Length: Number of nucleotides (nt) in the cleaned primer sequence.

GC (%): Percentage of G and C nucleotides in the primer sequence.

Tm Wallace (°C): Simplified melting-temperature estimate calculated as 2 × (A + T) + 4 × (G + C), with U counted as T.

Salt-adjusted Tm (°C): Simplified melting-temperature estimate that incorporates the entered monovalent sodium concentration.

Suggested Ta (°C): Starting PCR annealing temperature, calculated as 5 °C below the Tm estimate selected by the calculator.