Estimate the melting temperature (Tm) of short PCR primers and oligonucleotides (roughly ≤ 14 bp). Paste your primer below: the calculator shows the quick Wallace rule estimate alongside the more accurate nearest-neighbor value, with salt and concentration corrections.
A, C, G, T only. Spaces, numbers and line breaks are ignored. Calculation is instant.
The Wallace rule for short primers
For short oligonucleotides, a fast and surprisingly useful estimate is the Wallace rule:
Tm = 2 × (A + T) + 4 × (G + C)
Each A or T adds 2 °C and each G or C adds 4 °C. It assumes standard salt conditions and works best for primers up to about 14 bp, which is why it is the classic rule of thumb for short oligos. Above that length it becomes increasingly inaccurate — use the nearest-neighbor value instead.
When to use which method
- ≤ 14 bp: the Wallace rule is quick and adequate for ordering and quick checks.
- > 14 bp, or for reaction setup: use the nearest-neighbor estimate (also shown by the tool above), which accounts for sequence context, salt and primer concentration. See our full Tm calculator and guide for the details.
Worked example
For a 12-mer with 6 G/C and 6 A/T: Wallace Tm = 2×6 + 4×6 = 36 °C. The annealing temperature in a PCR would typically be set a few degrees below the lower primer Tm.
Tips for short primers
- Keep the two primers within a few °C of each other so one annealing temperature suits both.
- Very short primers have low specificity — for standard PCR, 18–24 nt primers are usual.
- Always enter the real salt and primer concentration for the nearest-neighbor estimate.
Frequently asked questions
What is the Wallace rule?
A quick Tm estimate for short oligos: 2 °C per A/T plus 4 °C per G/C. It is most reliable for primers up to about 14 bp.
Why is my nearest-neighbor Tm different from the Wallace value?
The nearest-neighbor model accounts for sequence context, salt and concentration, so it is more accurate; the Wallace rule is a simplified approximation.
What annealing temperature should I use?
A common starting point is a few degrees below the lower primer Tm, refined with a temperature gradient if needed.
Related guide: How to design PCR primers →
