Primer Melting Temperature
Calculator
Results
- Melting temperature Tm (°C)
- 62
- Primer length (nucleotides)
- 20
- GC content (%)
- 55
- Suggested annealing temperature (°C)
- 57
Biology and lab results
| Melting temperature Tm (°C) | 62 |
| Primer length (nucleotides) | 20 |
| GC content (%) | 55 |
| Suggested annealing temperature (°C) | 57 |
formula-map diagram
- Melting temperature Tm (°C)
- 62
- Primer length (nucleotides)
- 20
- GC content (%)
- 55
- Suggested annealing temperature (°C)
- 57
Formula breakdown
Formula
Tm = 2 × (A + T) + 4 × (G + C) (Wallace rule, short primers)= 62
Note
Simplified model: these results use standard textbook laboratory relationships and average constants (A260 = 1 for 50 µg/mL dsDNA, 617.96 g/mol per base pair, ~110 Da per amino acid, ideal exponential growth). Real samples vary with purity, contaminants, buffer, temperature and instrument calibration. Always confirm against your own standards and protocol; do not use for diagnostic or safety-critical work.
More in Biology and lab
See all →Frequently asked questions
What does the Wallace rule calculate?+
The Wallace rule gives a quick estimate of primer melting temperature (Tm) using the formula Tm = 4°C x (G+C count) + 2°C x (A+T count), based on the idea that G-C pairs (three hydrogen bonds) contribute more thermal stability than A-T pairs (two hydrogen bonds).
How accurate is the Wallace rule compared to other Tm methods?+
It is a fast approximation best suited to short primers, roughly 14 to 20 bases; for longer primers or higher accuracy needs, nearest-neighbor thermodynamic methods account for the specific sequence context and salt concentration and are considered more reliable.
Why do two primers with the same GC content give the same Tm here?+
The Wallace rule only counts how many G/C and A/T bases are present, not their order along the sequence, so any two primers of the same length and same base composition will always return an identical Tm under this method even if nearest-neighbor methods would distinguish them.
Does salt concentration affect the result?+
The basic Wallace rule does not include a salt correction term, unlike nearest-neighbor calculations; it implicitly assumes standard PCR salt conditions, so it will be less accurate for reactions with unusual monovalent or divalent cation concentrations.
What Tm difference between a primer pair is considered acceptable for PCR?+
Most protocols recommend keeping the Tm of the forward and reverse primers within about 1-2°C of each other so that both anneal efficiently at the same annealing temperature; a larger mismatch can bias amplification toward the primer with the higher Tm.