PCR Primer Tm Calculator
Primer melting temperature sets the annealing temperature for a PCR reaction, and getting it right is the difference between clean amplification and no product. Enter the count of each base in your primer and this calculator returns the Wallace 2+4 estimate, the GC-content estimate, the length, and the GC percentage. Both formulas are standard teaching approximations: they assume standard salt conditions and ignore nearest-neighbour effects, so use them as a starting point and confirm with a temperature gradient in the lab.
Primer Tm formulas
Length L = A + T + G + C
GC count = G + C
Wallace Tm = 2 * (A + T) + 4 * (G + C)
GC-content Tm = 64.9 + 41 * (GC count - 16.4) / L
GC content (%) = 100 * GC count / L
The Wallace rule suits short oligos (about 14 to 20 bases). The GC-content formula is better for longer primers. Neither corrects for salt or oligo concentration, so treat both as approximations.
Primer design notes
- Aim for the two primers in a pair to have similar Tm, within a couple of degrees of each other.
- Target 40 to 60 percent GC content for stable, specific binding.
- Set the annealing temperature roughly 3 to 5 degrees Celsius below the lower primer Tm.
- Avoid long single-base runs and strong 3-prime self-complementarity.
- For demanding applications, confirm with a nearest-neighbour model using your actual buffer conditions.
PCR primer Tm: frequently asked questions
What is primer melting temperature (Tm)?
The melting temperature is the temperature at which half of a primer-template duplex has dissociated into single strands. It governs the annealing temperature you use in PCR. Higher GC content and longer primers raise Tm. This calculator reports two widely taught estimators: the Wallace rule for short oligos and a GC-content formula for longer ones.
What is the Wallace rule?
The Wallace rule, or 2+4 rule, estimates Tm = 2 times (number of A and T bases) plus 4 times (number of G and C bases). It is a quick approximation valid for short oligonucleotides, roughly 14 to 20 bases, at standard salt conditions. It ignores nearest-neighbour stacking and salt, so it is a rule of thumb rather than a precise thermodynamic value.
When should I use the GC-content formula instead?
For primers longer than about 13 bases the GC formula Tm = 64.9 + 41 times (GC count minus 16.4) divided by primer length gives a better estimate than Wallace. Both are approximations; for high-stakes design use a nearest-neighbour thermodynamic model with your actual salt and primer concentrations.
What annealing temperature should I run?
A common starting point is an annealing temperature about 3 to 5 degrees Celsius below the lower primer Tm of the pair. Optimal annealing depends on the enzyme, buffer, and template, so a temperature gradient is the reliable way to find the best setting for a new primer pair.
Do these formulas account for salt and primer concentration?
No. The Wallace and basic GC formulas assume standard conditions and do not correct for monovalent salt, magnesium, or oligo concentration, all of which shift the true Tm. For accurate design, use a nearest-neighbour model that takes those parameters as inputs. Treat these values as quick estimates.
Official sources
- National Center for Biotechnology Information: NCBI primer design and PCR literature.
- National Human Genome Research Institute: NHGRI molecular biology education.
Reviewed by the CalculatorHub team, edited by James Graham, 17 June 2026. See our methodology.