This primer Tm calculator estimates the melting temperature of a PCR or sequencing primer from its sequence — by the Wallace rule for short primers and the standard basic formula for longer ones — and reports GC content and length alongside.
Two Formulas, Two Regimes
Wallace rule (short primers): Tm = 2 °C per A/T pair + 4 °C per G/C pair — G≡C's third hydrogen bond priced in. Basic formula (14+ nt): Tm = 64.9 + 41 × (nGC − 16.4) ÷ length. Both are first approximations: the physical gold standard is nearest-neighbor thermodynamics with salt corrections (what primer vendors' tools run), which weighs stacking between adjacent bases — sequence order, not just composition. Composition digests live at our GC Content Calculator. Part of our Biology Calculators collection.
Worked Examples (Verified)
- 20-mer at 50% GC (the default ATGC×5): Wallace: 2×10 + 4×10 = 60 °C (overestimates at this length — Wallace is a short-primer tool) · Basic: 64.9 + 41 × (10 − 16.4) ÷ 20 = 51.8 °C → suggested annealing ≈ 47 °C starting point
- A 12-mer, 6 GC: Wallace 2×6 + 4×6 = 36 °C — plausible; the basic formula returns 41.7 °C here (its long-primer assumptions bending) — the pair of readouts bracket reality, and the calculator flags which to trust
- GC reality checks: primers are designed to 40–60% GC with a G/C clamp at the 3′ end — composition drives Tm more than length does past ~18 nt
Design Notes the Formulas Assume
- Primer pairs should match Tm within ~2–5 °C of each other; the lower-Tm primer sets the annealing step.
- Avoid 3′ complementarity (primer-dimers), hairpins, and runs of 4+ identical bases — Tm math is innocent of all three failure modes; dedicated design tools check structure too.
- Mismatches drop effective Tm (~1 °C per % mismatch as a rough guide) — allele-specific PCR weaponizes exactly that.
- For qPCR and anything quantitative, vendor nearest-neighbor values at your reaction's actual salt/Mg²⁺ are worth the extra minute.
Related Tools
- GC Content Calculator — composition and the Wallace estimate in context
- Codon Calculator — what the amplified gene will say
- Biology Calculators — the full collection
Frequently Asked Questions
How do you calculate primer melting temperature?
Short primers (<14 nt): Wallace rule, 2 °C × (A+T) + 4 °C × (G+C). Longer primers: Tm = 64.9 + 41×(GC count − 16.4)/length — a 20-mer at 50% GC gives 51.8 °C by this formula. Nearest-neighbor methods refine both.
What annealing temperature should I use?
Start ~5 °C below the lower primer's Tm (with the estimates here, that is the number the results card prints) and optimize with a gradient run. Too high: no product. Too low: nonspecific bands join the party.
Why do GC pairs count double in the Wallace rule?
G≡C pairs share three hydrogen bonds versus A=T's two, so they hold the duplex together to higher temperatures — the 2/4 pricing is that chemistry rounded for mental math.
What GC content should a primer have?
40–60% is the standard design window, ideally with one or two G/C bases at the 3′ end (the "GC clamp") to anchor polymerase start — balanced against avoiding 3′ runs that invite mispriming.
How accurate are these formulas?
Within a few degrees for typical primers in standard buffers — good enough for teaching, screening and first-pass protocols. Exact values depend on salt, Mg²⁺, primer concentration and additives (DMSO lowers Tm ~0.5–0.75 °C per %), which is why nearest-neighbor vendor tools exist for final numbers.
Is this primer Tm calculator free?
Yes — free, instant, and your sequence stays in the browser.