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Melting temperature calculator

Primer Tm under every model at once, with the salt and concentration you actually used.

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The unified parameter set most primer suppliers and design tools use. Match this one when your number has to agree with an order form.

nM

Of the primer strand. 200–500 nM is typical in PCR.

Buffer

mM
mM
mM
mM

Tris counts as half its concentration. dNTPs chelate magnesium, so they are subtracted before it is converted — which is why adding nucleotides lowers the melting temperature.

Tm

68.42°C

Length

28nt

GC

50%

ΔG 37 °C

-36kcal/mol

Every model, same sequence

  • Nearest neighbour — Allawi & SantaLucia 199768.42 °C
  • Salt-adjusted GC67.15 °C
  • Wallace rule84 °C

A spread of 16.9 °C across the three. This is why a melting temperature quoted without its model, salt and concentration cannot be reproduced.

ΔH°

-222.9kcal/mol

ΔS°

-602.5cal/mol·K

3′ GC clamp

2of 5

Secondary structure

Self-dimer · 4 bp · -2.32 kcal/mol · reaches the 3′ end

5'-CGTTCCAAAGATGTGGGCATGAGCTTAC-3'
                        ||||
3'-                  CATTCGAGTACGGGTGTAGAAACCTTGC-5'

Hairpin · 3 bp · -1.29 kcal/mol

5'-CGTTCCA
       |||  loop 6 nt
3'-CATTCGAGTACGGGT
FormulaTm = ΔH° / (ΔS° + R·ln(C_T/4)), ΔS° corrected by 0.368·(N−1)·ln[Na⁺]
ModelNearest neighbour — Allawi & SantaLucia 1997

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Melting temperature is not annealing temperature. A common starting point is 3–5 °C below the lower primer Tm, but the optimum is found by gradient. The secondary structure check finds contiguous pairing, not a minimum free energy fold — it catches the ordinary mistakes and does not replace a folding program.

About this tool

Calculate oligonucleotide melting temperature by nearest-neighbour thermodynamics, the salt-adjusted GC formula and the Wallace rule — all three at once, because they disagree by more than ten degrees and a Tm without its model is not reproducible. Takes the sodium, potassium, Tris, magnesium and dNTP concentrations from your actual buffer, and checks the primer for self-dimers, hairpins, runs and a workable 3′ end.

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Science last reviewed .