Primer Melting Temperature (Tm) Calculator
By Santaji GadepatilinUpdated October 27, 2026
A primer's melting temperature (Tm) is where half of it is bound to its target DNA. Setting the PCR annealing temperature correctly depends on knowing Tm. This free primer melting temperature calculator solves for whichever value you leave blank and shows the formula and steps.
How to use the primer melting temperature calculator
Count the A, T, G and C bases in your primer and enter them. The melting temperature, a suggested annealing temperature and GC content are calculated.
Formula (Wallace rule)
Tm = 2(A + T) + 4(G + C)
Example
A 20-mer with 5 of each base:
- Tm = 2 × 10 + 4 × 10 = 60 °C
- Annealing ≈ 55 °C
- GC = 50%
For longer primers or exact work, nearest-neighbour methods that account for salt concentration are more accurate.
Primer design guidelines
| Property | Recommended |
|---|---|
| Length | 18–25 nucleotides |
| GC content | 40–60% |
| Tm | 52–65 °C |
| Tm difference between primers | Within 2–5 °C |
| 3′ end | End in G or C (GC clamp), avoid more than 3 G/C in the last 5 bases |
| Repeats | Avoid runs of 4+ identical bases |
Other Tm methods
| Method | Best for |
|---|---|
| Wallace rule (this calculator) | Primers under about 14–20 nt, quick estimates |
| Basic GC formula: 64.9 + 41(GC − 16.4)/N | Longer oligos |
| Nearest-neighbour thermodynamics | Most accurate; accounts for salt and primer concentration |
The Wallace rule tends to overestimate Tm for longer primers. Many polymerase suppliers provide nearest-neighbour Tm calculators tuned to their buffers.
Annealing temperature
Start about 3–5 °C below the lower primer Tm. If you see non-specific bands, raise it; if there is no product, lower it or run a gradient PCR.
Primer problems to avoid
- Self-dimers and hairpins: primers binding to themselves.
- Primer-dimers: forward and reverse primers binding each other at the 3′ ends.
- Off-target binding: check specificity against the genome with a sequence search tool.
Frequently asked questions
What is the Wallace rule?
Tm = 2 °C × (A + T) + 4 °C × (G + C). It is a quick estimate for short primers (about 14–20 bases).
What annealing temperature should I use?
Start about 3–5 °C below the lower primer Tm, then optimise with a gradient PCR.
What is an ideal primer?
Typically 18–25 bases, 40–60% GC, Tm of 55–65 °C, and primer pairs within about 5 °C of each other.
What is the Wallace rule?
Tm = 2 × (A + T) + 4 × (G + C) °C, a quick estimate for short primers.
How far below Tm should I anneal?
Usually 3–5 °C below the lower of the two primer Tms.