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Primer Tm Calculator — Melting Temperature, GC% & Primer Pair Check
Estimate DNA primer melting temperature from sequence, compare forward and reverse primers, inspect GC content and ΔTm, and see the calculation method used.
Last updated: 2026-09-17
Results
Forward Tm 53.83 °C
Forward GC 55%
Forward length 20 nt
Formula, example and assumptions
Short oligos: Tm≈2(A+T)+4(G+C). Longer oligos: Tm≈64.9+41·(GC−16.4)/N.
A 20-base primer with balanced GC content typically lands near the temperature range used as a starting point for routine PCR optimization.
- This is an educational planning estimate; buffer chemistry and polymerase systems can shift experimental Tm.
- For sequence-critical work, compare against a nearest-neighbor calculator using your actual ionic and oligo concentrations.
Frequently asked questions
Why do primer Tm calculators disagree?
They may use different thermodynamic models, salt corrections, oligo concentrations and buffer assumptions. Compare the method and conditions, not only the final number.
Should a 5-prime cloning tail be included?
For first-cycle priming, evaluate the template-binding region separately because a non-annealing tail does not initially pair with the template.