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Primer Tm Calculator (Four Methods)

Calculate a primer's melting temperature by nearest-neighbour thermodynamics, the salt-adjusted GC formula, the basic GC formula or the Wallace rule, and compare them.

Formula

Tm=ΔH∘ΔS∘+Rln⁡(C/4)−273.15T_{\mathrm{m}} = \dfrac{\Delta H^\circ}{\Delta S^\circ + R\ln(C/4)} - 273.15
ΔSsalt∘=ΔS∘+0.368 (N−1)ln⁡[Na+]\Delta S^\circ_{\text{salt}} = \Delta S^\circ + 0.368\,(N-1)\ln[\mathrm{Na}^+]
Tm=81.5+16.6log⁡10[Na+]+0.41 (%GC)−675NT_{\mathrm{m}} = 81.5 + 16.6\log_{10}[\mathrm{Na}^+] + 0.41\,(\%\mathrm{GC}) - \dfrac{675}{N}
Tm=64.9+41 (G+C−16.4)NT_{\mathrm{m}} = 64.9 + \dfrac{41\,(G + C - 16.4)}{N}
Tm=2 (A+T)+4 (G+C)T_{\mathrm{m}} = 2\,(A + T) + 4\,(G + C)
ΔH∘, ΔS∘\Delta H^\circ,\ \Delta S^\circ
enthalpy and entropy of duplex formation, summed over each pair of neighbouring bases
RR
gas constant, 1.987 cal/(mol·K)
CC
primer concentration, in mol/L
NN
primer length, in nucleotides
%GC\%\mathrm{GC}
percentage of G and C among the bases

How it works

The melting temperature is the temperature at which half of the primer is bound to its complement. The nearest-neighbour model treats the stability of a duplex as the sum of contributions from each pair of adjacent bases, plus terms for starting the helix, using the unified parameters of SantaLucia (1998). Those parameters apply at 1 M sodium, so the entropy is corrected for the lower salt of a PCR.

There is no single correct Tm formula, and the methods can disagree by ten degrees or more for the same primer. The two GC formulas are empirical fits that were developed for long DNA; the Wallace rule is a rule of thumb for very short oligos at high salt. The nearest-neighbour method is the usual basis for primer design because it uses the actual sequence, the primer concentration and the salt. All four are shown so the difference is visible. Use one method consistently for both primers of a pair.

Worked example

The M13 forward (−20) primer GTAAAACGACGGCCAGT at 500 nM in 50 mM monovalent salt.

  1. Summed parameters: ΔH° = −133.6 kcal/mol; salt-corrected ΔS° = −378.3 cal/(mol·K).
  2. Tm = −133,600 / (−378.3 + 1.987 × ln(5 × 10⁻⁷ / 4)) − 273.15 = 52.8 °C.

Nearest-neighbour Tm is 52.8 °C. For the same primer the salt-adjusted formula gives 41.9 °C, the basic formula 47.1 °C and the Wallace rule 52.0 °C.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The primer pairs perfectly with its target, with no mismatches.
  • Only monovalent cations are modelled. Magnesium and dNTPs, which shift Tm in a real PCR, are not included.
  • The primer is in large excess over the template.

Common mistakes

  • Treating Tm as the annealing temperature. Annealing is usually set a few degrees below the lower Tm of the pair and then optimised.
  • Comparing values from different calculators. Different parameter sets and salt corrections routinely differ by several degrees, and so do the four methods here.
  • Including a 5′ tail or restriction site that does not anneal in the first cycles.

Related tools

Oligo and Primer AnalyzerLength, GC, Tm, weight and a design-guideline check for a primerOpen calculatorPrimer Pair AnalyzerTm match, GC, dimers and product size for a forward-reverse pairOpen calculatorPCR Annealing Temperature CalculatorOptimal annealing temperature from primer Tm and productOpen calculatorGC Content CalculatorGC percentage and base composition of a sequenceOpen calculatorPCR Master Mix CalculatorComponent volumes for any number of reactionsOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.