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🌡️ Primer Tm Calculator

Calculate primer melting temperature (Tm) from a DNA sequence, automatically choosing the salt-adjusted or Wallace rule formula based on primer length.

Primer Sequence 5'→3', letters A/C/G/T only

Na⁺ Concentration mM, default 50

Melting Temperature (Tm)

— °C

💡 Note: For primers ≥14 nt, this uses the salt-adjusted formula (more accurate for standard PCR primers). For very short primers (<14 nt), the simpler Wallace rule is used instead, since the salt-adjusted formula becomes unreliable at short lengths. Both are estimates — for critical applications, cross-check with your primer synthesis vendor's calculated Tm.

📖 Example: 21-nt primer, 12 G/C out of 21 bases (57.1% GC), 50 mM Na⁺: Tm = 81.5 + 16.6×log₁₀(0.05) + 0.41×57.1 − 600/21 ≈ 54.8°C.

Frequently asked questions

Why do different Tm calculators give different results for the same primer?

Several formulas exist (Wallace rule, salt-adjusted %GC method, and nearest-neighbor thermodynamic methods), each with different assumptions and levels of sophistication. Nearest-neighbor methods, used by most commercial primer design tools, account for the specific sequence of neighboring bases and are generally more accurate than the simpler formulas used here, which is why results can differ by a few degrees between calculators.

Which formula does this calculator use?

For primers 14 nucleotides or longer, this uses the salt-adjusted formula (Tm = 81.5 + 16.6×log10[Na+] + 0.41×%GC − 600/N), a widely used approximation for standard PCR primers. For shorter primers, it falls back to the simpler Wallace rule (Tm = 2(A+T) + 4(G+C)), since the salt-adjusted formula becomes unreliable below about 14 nucleotides.

Does primer concentration affect Tm?

Yes, in principle — higher primer concentrations slightly increase Tm. This calculator's salt-adjusted formula only accounts for monovalent cation (Na+) concentration, not primer concentration, which is a simplification. Nearest-neighbor thermodynamic calculators typically incorporate primer concentration for a more complete estimate.

Accuracy & how this is derived

Derivation: Two standard approximations depending on primer length: the salt-adjusted formula (Tm = 81.5 + 16.6*log10[Na+] + 0.41*%GC - 600/N) for primers 14nt and longer, and the Wallace rule (Tm = 2(A+T) + 4(G+C)) for shorter primers.

Validated against: Salt-adjusted formula from Marmur & Doty (1962) with subsequent salt correction; Wallace rule from Wallace et al. (1979) — both widely used approximations in molecular biology primer design, though nearest-neighbor thermodynamic methods are more accurate for critical applications.

⚠️ For educational and research support only — verify critical results independently before use in regulated, clinical, or publication-bound work.

✔ Last updated: July 2026 · Report an error

Estimating primer melting temperature for PCR design

Primer melting temperature estimates the temperature at which half of a primer-template duplex has dissociated into single strands, a key parameter for choosing appropriate PCR annealing temperatures. The salt-adjusted formula used for longer primers accounts for both GC content, which stabilizes the duplex through additional hydrogen bonding, and monovalent cation concentration, which screens the negative charge of the DNA backbone and also stabilizes the duplex, while very short primers rely on the simpler Wallace rule since the salt-adjusted approximation loses accuracy at short lengths.