Oligo Melting Temperature Calculator
The melting temperature of a DNA oligonucleotide is the point where half the duplex has dissociated into single strands. It depends on length and base composition, since G-C pairs bond more strongly than A-T pairs. This calculator takes the counts of each base and returns two standard estimates: the Wallace rule for short primers and the GC-content formula for longer oligos, along with the GC percentage. The formulas are established published approximations; for demanding work, a nearest-neighbour thermodynamic model is more precise.
Melting temperature formulas
length L = A + T + G + C
Wallace Tm = 2*(A + T) + 4*(G + C)
GC-content Tm = 64.9 + 41*((G + C) - 16.4) / L
GC percent = (G + C) / L * 100
For a 20-mer with 5 of each base, the Wallace rule gives 2*10 + 4*10 = 60 degrees Celsius, and the GC-content formula gives 64.9 + 41*(10 - 16.4)/20, about 51.8 degrees Celsius.
Primer design facts
- Tm rises with both length and guanine-cytosine content.
- G-C pairs form three hydrogen bonds; A-T pairs form two.
- The Wallace rule suits short oligos under about 14 bases.
- The GC-content formula better fits longer oligos.
- Higher salt concentration stabilises the duplex and raises the true Tm.
Oligo melting temperature: frequently asked questions
What is the melting temperature of an oligo?
The melting temperature, Tm, is the temperature at which half of the DNA duplex has separated into single strands. It rises with length and with guanine-cytosine content, because G-C pairs form three hydrogen bonds compared to two for A-T pairs.
What is the Wallace rule?
The Wallace rule is a simple estimate for short oligos (roughly under 14 bases): Tm equals 2 times the number of A and T bases plus 4 times the number of G and C bases, in degrees Celsius. It assumes standard salt and is a quick rule of thumb for primer design.
What is the GC-content formula?
For longer oligos the GC-content formula is more appropriate: Tm equals 64.9 plus 41 times (number of G plus C minus 16.4) divided by the total length. It better captures the diminishing effect of each base as the oligo gets longer.
Which estimate should I use?
Use the Wallace rule for short primers under about 14 bases, and the GC-content formula for longer sequences. Both are approximations; for demanding applications, a nearest-neighbour thermodynamic calculation with explicit salt and concentration is more accurate.
Why does salt concentration matter?
Cations such as sodium and magnesium shield the negative charges on the DNA backbone, stabilising the duplex and raising Tm. The simple formulas here assume standard reaction salt; large deviations in salt or the presence of additives shift the true Tm.
Official sources
Reviewed by the CalculatorHub team, edited by James Graham, 16 June 2026. See our methodology.