Primer Tm Calculator
Enter the counts of A, T, G, C in a primer to compute the Wallace-rule and salt-corrected (GC%) melting temperature Tm, for PCR primer design.
Input Data
Results
At a glance:Primer melting temperature (Tm) is the temperature at which half of the primer-template duplex dissociates (double strand becomes single strand), the key to setting PCR annealing temperature. For short primers (< 14 nt), the Wallace rule is common: Tm = 4×(G+C) + 2×(A+T). For longer primers, use the salt-corrected GC% method: Tm = 81.5 + 0.41×%GC − 675÷length (about 50 mM Na⁺). Annealing temperature is usually set about 5 °C below the lower primer Tm.
Formula
Wallace rule (short primer): Tm = 4 × (G + C) + 2 × (A + T).
Salt-corrected GC% method: Tm = 81.5 + 0.41 × %GC − 675 ÷ length.
Annealing reference: Ta ≈ lower primer Tm − 5 °C.
$$T_m = 4(G + C) + 2(A + T)$$$$T_m = 81.5 + 0.41 \times \%GC - \dfrac{675}{L}$$How to Use
- Count the A, T, G, C bases in the primer sequence and enter them.
- The tool shows both Wallace-rule and salt-corrected GC% Tm and primer length.
- For short primers (< 14 nt) use Wallace; for longer primers use the GC% method.
Common primer compositions and Tm (Wallace rule)
| Composition | Length (nt) | Wallace Tm (°C) |
|---|---|---|
| A3 T3 G3 C3 | 12 | 36 |
| A2 T2 G4 C4 | 12 | 40 |
| A5 T5 G5 C5 | 20 | 60 |
| A4 T4 G6 C6 | 20 | 64 |
The Wallace rule is simple but only for < 14 nt short primers; for longer primers it overestimates Tm, use the salt-corrected GC% method or the nearest-neighbor model.
Case Studies
Wallace estimate for a short primer
A 12 nt primer with A3, T3, G3, C3.
Wallace Tm = 4×(3+3) + 2×(3+3) = 24 + 12 = 36 °C.
Being short, annealing temperature can start around 31 °C and be fine-tuned by specificity.
GC% method for a longer primer
A 20 nt primer with A5, T5, G5, C5, 50% GC.
GC% Tm = 81.5 + 0.41×50 − 675÷20 = 81.5 + 20.5 − 33.75 ≈ 68.25 °C.
Annealing can be set around 63 °C, closer to the long-primer reality than the Wallace rule (here 60 °C).
FAQ
Which, Wallace rule or GC% method?
The Wallace rule (4×GC + 2×AT) suits only about 14 nt or shorter primers/oligos; beyond that it systematically overestimates Tm. For longer primers use the salt-corrected GC% method, or the more accurate nearest-neighbor thermodynamic model. This calculator shows both for comparison.
How to set annealing temperature from Tm?
A common rule is to take the lower Tm of the primer pair and subtract about 5 °C as the starting annealing temperature. In practice run a gradient PCR scanning a few degrees around Tm−5 to find the specific, high-yield temperature. GC-rich templates may need higher annealing or DMSO.
Why should the two primers have close Tm?
If the forward and reverse primer Tm differ too much (generally keep < 5 °C), their binding efficiencies at one annealing temperature become uneven, causing amplification bias or non-specific products. Adjust lengths and GC% during design to keep both Tm close.
Do salt and primer concentration affect Tm?
Yes. Higher monovalent cations (Na⁺, K⁺) and Mg²⁺ shield the phosphate backbone, stabilize the duplex, and raise Tm; primer concentration also matters slightly. The GC% method here includes a ~50 mM Na⁺ correction; precise Tm needs models that incorporate these variables.
How much does GC content affect Tm?
Greatly. G–C has three hydrogen bonds, A–T only two, so higher GC raises Tm. In the Wallace rule each G/C contributes 4 °C, each A/T only 2 °C; in the GC% method each 1% GC adds about 0.41 °C.
Related Tools
References
Content review: Calculatorism Science Team. Results are for reference only; please refer to the relevant authorities for the official figures.