Primer Tm Calculator – Melting Temperature & GC%
Estimate primer Tm, GC% and molecular weight from a sequence.
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This primer Tm calculator takes a single PCR primer or oligo and reports its melting temperature three ways: nearest-neighbor (SantaLucia 1998), the Wallace rule, and the salt-adjusted formula, with the length-appropriate value highlighted — nearest-neighbor for anything 14 nt or longer, Wallace below that. It also shows length, GC content, and single- and double-stranded oligo molecular weight, a quick primer design sanity check before you order oligos or set up a PCR.
0 nt
| Tm — Wallace rule (2AT+4GC) | — |
| Tm — salt-adjusted (Schildkraut-Lifson, 100 mM Na⁺) | — |
| Tm — nearest-neighbor (SantaLucia) | — |
| Single-stranded MW (g/mol) | — |
| Double-stranded MW (g/mol) | — |
The headline Tm is the nearest-neighbour (SantaLucia 1998) value from 14 nt up — it is the only figure here that responds to the oligo, Na⁺, Mg²⁺ and dNTP conditions. Below 14 nt the Wallace rule (2AT+4GC) is quoted instead, since the nearest-neighbour model is not parameterised for very short duplexes. The salt-adjusted row is a composition-only Schildkraut-Lifson estimate pinned to 100 mM Na⁺ and does not see your buffer, so treat it as a reference point rather than a prediction. For primer dimers, hairpins, custom salt/Mg²⁺ conditions and batch analysis, use the Oligo Analyzer.
Working on a whole construct rather than a single Tm check? Open SeqStudio — the full editor, with live feature annotation, plasmid maps, restriction and primer panels, undo/redo, multi-document tabs and GenBank / SnapGene import and export.
How to use the Primer Tm tool
- 1Paste one primer or oligo into the "Primer / oligo sequence" box. The nucleotide count under the box updates as you type.
- 2Read the four summary tiles: Length, GC content, the recommended Tm (labelled with the formula it came from — "Wallace (2AT+4GC)" under 14 nt, "Nearest-neighbour (SantaLucia 1998)" at 14 nt and above), and ssDNA MW.
- 3Compare all three values in the results table: Tm by the Wallace rule, Tm salt-adjusted, and Tm nearest-neighbor (SantaLucia), plus single-stranded and double-stranded molecular weight.
- 4Aim for closely matched Tm values across a forward/reverse pair, and click Share to copy a link that reloads the same sequence.
Frequently asked questions
Which Tm formula should I use?
The Wallace rule (2x(A+T) + 4x(G+C)) is a quick estimate for primers under 14 nt, and it is what the headline Tm tile reports at those lengths. At 14 nt and above the tile switches to the nearest-neighbor (SantaLucia 1998) value, which is the most accurate of the three because it accounts for which bases sit next to each other rather than only counting them. The salt-adjusted formula 64.9 + 41x(GC-16.4)/N is listed in the table for reference — it beats Wallace on a long primer but still ignores stacking, and it assumes 100 mM Na+. To recompute Tm under your own oligo, Na+, Mg2+ and dNTP concentrations, use the Oligo Analyzer.
How is molecular weight calculated?
From standard anhydrous nucleotide monophosphate masses with an end correction for a 5'-OH oligo. Both single-stranded and double-stranded values are given.
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