Oligonucleotide Tm Calculator
Estimate the melting temperature of a DNA oligo using two complementary formulas. The Wallace rule (4·(G+C) + 2·(A+T)) is fast and accurate for short primers (<18 nt); the nearest-neighbour model uses a published parameter set and is preferred for longer oligos. Both honour a non-default sodium concentration.
Calculator
Enter a sequence and calculate to see the melting temperature
Melting temperature (Tm)
≤ 13 nt: Tm = 2(A+T) + 4(G+C) · 14–70 nt: Tm = 64.9 + 41 × (G+C − 16.4) / length
Short oligos use the Wallace rule (each A/T pair contributes 2 °C, each G/C pair 4 °C). Longer oligos use the basic GC formula. Both assume standard low-salt conditions — see the limitations below the calculator.
About this calculation
What this is
Tm is the temperature at which half the oligo is single-stranded and half is base-paired with its perfect complement. Below Tm the duplex is favoured; above it, the strands separate. PCR annealing temperatures are set a few degrees below Tm.
Why it is used
A working annealing temperature is the difference between a clean band and a smear. The Wallace rule is a quick estimate for short primers; the nearest-neighbour model accounts for sequence context and is the better choice for oligos longer than ~18 nt.
Why this calculator exists
Both formulas are short, but the salt correction and the choice between methods are easy to slip. The calculator shows the Wallace value, the nearest-neighbour value, and the salt-adjusted versions side by side, so the right number is obvious before you run the PCR.
Key assumptions
- The oligo is DNA (RNA needs a different parameter set, not provided here).
- The sodium concentration is the monovalent cation concentration in your reaction.
- The oligo has no modified bases or labelled dyes with their own Tm shift.
Limitations
- Tm estimates ignore DMSO, betaine, or other PCR additives that change duplex stability.
- The nearest-neighbour parameters are an average across conditions; mismatch prediction requires dedicated software.
- Hairpins and self-dimers can dominate real annealing behaviour for some sequences — use a dedicated tool for those.
What this calculates
- Melting temperature (Tm) °C
- Estimated temperature at which half of the duplexes are dissociated.
- Length nt
- Number of nucleotides in the sequence.
- GC content %
- Percentage of G and C bases in the sequence.
Frequently asked questions
What is the melting temperature (Tm) of an oligonucleotide?
Tm is the temperature at which half of the DNA duplexes have separated into single strands. It depends on length and base composition, with G·C pairs contributing more stability than A·T pairs. This calculator estimates Tm in °C from a 5–70 nt sequence and reports the length and GC content alongside it.
How do I use the oligo Tm calculator?
Enter a single-stranded DNA sequence of 5 to 70 nucleotides using only the bases A, C, G, and T; the check is case-insensitive. The calculator returns the melting temperature, the length, and the GC percentage, and it selects the formula for you based on how long the oligo is. Ambiguity codes and sequences longer than 70 nt are rejected rather than estimated.
Which formula does this calculator use, and why does it switch at 13 nt?
Oligos of 13 nt or fewer use the Wallace rule: 2 °C per A/T plus 4 °C per G/C, which is the 2+4 rule of thumb for short primers. Longer oligos up to 70 nt use the basic GC formula, 64.9 + 41 × (G+C − 16.4) / length, which accounts for length and GC content. The simple Wallace count loses accuracy as oligos grow, so the switch is made automatically at the boundary.
What is the difference between melting temperature and annealing temperature?
Tm is a property of the oligo duplex: the temperature where half of it is dissociated. The annealing temperature is the temperature you actually set on the thermocycler for primer binding during PCR, and it is typically chosen a few degrees below the primer Tm. This calculator gives you the Tm value only; it does not compute an annealing temperature for a given protocol.
Why is my measured Tm higher than the value from this calculator?
The Wallace and basic GC formulas are empirical estimates made under standard assay conditions of roughly 50 ng/µL oligo, low monovalent cation, and pH around 7. Under typical PCR salt conditions, such as 50 mM K⁺, the true Tm sits several degrees higher than these formulas report. They are also not nearest-neighbour thermodynamics and model no secondary structure, so for critical designs validate with a nearest-neighbour tool or a gradient PCR.
For research and educational use only. Not for clinical or diagnostic decisions. Always verify calculations independently before use in critical applications.