What this is for: Quick melting temperatures for a primer pair — base counts, GC%, Wallace and basic-formula Tm, approximate MW, and a suggested annealing temperature.
The formula: Wallace Tm = 2×(A+T) + 4×(G+C) °C; basic Tm = 64.9 + 41×(GC count − 16.4) ÷ length °C
How to use:
- Paste the forward primer sequence (5'→3', A/T/G/C) in the Forward Primer section.
- Paste the reverse primer sequence (5'→3') in the Reverse Primer section.
- For each primer, read the base counts, length, GC %, Wallace Tm, basic Tm and approx. MW.
- Read the suggested annealing temperature — the lower basic Tm minus 5 °C. For critical work, confirm with a nearest-neighbour calculator using your exact salt and primer concentrations.
Forward Primer
Paste the primer sequence (5'→3'). Base counts, GC% and both Tm estimates are computed from the sequence itself.
#A
#T
#G
#C
Length
GC %
Tm Wallace (°C)
Tm basic (°C)
Approx MW (g/mol)
Reverse Primer
#A
#T
#G
#C
Length
GC %
Tm Wallace (°C)
Tm basic (°C)
Approx MW (g/mol)
Suggested Annealing Temperature
Annealing ≈ lower primer Tm − 5 °C.
Suggested annealing temperature (°C) = lower basic Tm − 5
Prefer to work offline or keep a copy? The download link is right below.
Download the Oligo / Primer Tm Calculator workbook (.xlsx)
Download the Oligo / Primer Tm Calculator workbook (.xlsx)
Notes & limits (from the workbook): Paste each primer sequence (UPPERCASE A/T/G/C) — base counts, GC%, and two Tm estimates are computed from the sequence itself. Wallace rule (2/4 °C) suits primers < 14 nt; the basic salt-adjusted formula suits longer primers. Annealing ≈ lower primer Tm − 5 °C. For critical work use a nearest-neighbour calculator with your exact salt/primer concentrations.