Oligo Tm Calculator
Calculate the melting temperature of a DNA oligo or PCR primer. The headline value uses the nearest-neighbor method (SantaLucia 1998) with salt correction; the basic Wallace rule and GC% estimate are shown for comparison.
Enter a DNA sequence (A, C, G, T only) to calculate its Tm.
How it works
The melting temperature (Tm) is the temperature at which half of a DNA duplex is dissociated into single strands. The most accurate estimate for oligos uses nearest-neighbor thermodynamics: each adjacent base-pair step contributes a known enthalpy (ΔH) and entropy (ΔS), and the Tm follows from Tm = ΔH / (ΔS + R·ln(C_T/4)) with a correction for salt concentration. This calculator uses the unified SantaLucia 1998 parameters.
The monovalent cation concentration (Na⁺) and the oligo strand concentration both affect the Tm, so enter the values for your reaction. For quick checks, the Wallace rule — 2 °C per A/T and 4 °C per G/C — works for short oligos, and a GC%-based formula is shown for longer sequences. The sequence must contain only A, C, G and T (U is read as T).
Examples
- GTAAAACGACGGCCAGT (M13 forward primer) melts around 50 °C at 50 mM Na⁺.
- Wallace rule: for a 17-mer with 8 A/T and 9 G/C, Tm = 2×8 + 4×9 = 52 °C.
- Raising the salt concentration or the oligo concentration increases the Tm.
Frequently asked questions
- Which method is most accurate?
- The nearest-neighbor method is the most accurate for oligonucleotides because it accounts for the identity of neighbouring bases, not just the overall GC content. It is the headline value here.
- Why do salt and oligo concentration matter?
- Cations stabilise the DNA duplex, raising the Tm, and a higher strand concentration shifts the equilibrium toward the duplex. Both appear in the nearest-neighbor equation, so entering your reaction's values gives a realistic Tm.
- When is the Wallace rule good enough?
- The Wallace rule (2 °C per A/T, 4 °C per G/C) is a reasonable quick estimate for oligos shorter than about 14 bases, but it ignores salt, concentration and sequence context, so it drifts for longer primers.
- Does it handle RNA or ambiguous bases?
- Uracil is read as thymine, but the nearest-neighbor parameters are for DNA. Ambiguous IUPAC codes are not supported — the sequence must be A, C, G and T (or U).