Plasmid Map
Tools

Primer Tm calculator

Estimate Tm with your sequence and salts.

01

Your values

Matches Primer3 calc_tm defaults. For PCR annealing guidance, choose the PCR primer model.

2–200 A, C, G and T bases. Whitespace and lowercase are accepted.

Add the second primer to compare Tm values and get a pair's annealing starting point.

nM

Sum of both complementary strands; equal amounts assumed.

mM
mM
mM
02

Example result

Estimated melting temperature

56.3°C

20 DNA bases, 45% GC. Calculated for a perfectly complementary duplex with the settings shown.

Sodium-equivalent salt
163.84 mM
Total strand concentration
50 nM
Annealing starting point
Choose the PCR primer model
Calculation & assumptions

Tm = ΔH / (ΔS + R ln Ceff) − 273.15

  • Primer3 / Primer-BLAST convention: two equal complementary strands, effective concentration total ÷ 4 (total when self-complementary). This matches Primer3 calc_tm with its defaults. Vendor PCR calculators assume the primer is in excess and usually read about 4–6 °C higher.
  • DNA_NN3 parameters with the entropy salt correction (method 5). Mismatches, modified bases, secondary structures and additives such as DMSO are not modeled.

What this Tm calculator estimates

Melting temperature, or Tm, describes the transition of a DNA duplex under specified conditions. It depends on the sequence and solution conditions, so GC percentage alone does not determine it. The calculator uses adjacent base-pair contributions in a nearest-neighbor model for a perfectly complementary DNA duplex.

Enter one primer, or both primers of a pair: 2–200 unambiguous DNA bases each, written 5′ to 3′. Lowercase and whitespace are accepted. RNA letters, ambiguity codes, FASTA headers and modified-base notation are rejected instead of silently converted. The result reports each estimated temperature, sequence length and GC percentage, the concentrations used and, for a pair, the Tm difference.

Two concentration models

The default is the Primer3 / Primer-BLAST convention: a total strand concentration of 50 nM, meaning equal complementary strand amounts of 25 nM each. Self-complementarity is detected from the sequence and uses the corresponding symmetry and concentration treatment. With the default salts, this gives the same Tm as Primer3's calculation, so it matches the values Primer-BLAST reports.

In a PCR, each primer is far more concentrated than the template it binds. PCR primer in excess over template uses the concentration of one primer (500 nM by default) as the effective concentration, the treatment Biopython documents for PCR. This is the assumption vendor PCR calculators make, so their Tm values are usually 4–6 °C higher than the Primer3 default. When we compared six primers with Thermo Fisher's calculator at 0.5 µM on September 29, 2026, this model came within 0.8 °C of its values.

The other defaults are 50 mM sodium, 1.5 mM magnesium and 0.6 mM total dNTPs. These are example calculation conditions, not a recommendation for a particular polymerase or experiment. Change them to describe the model you intend to use. The dilution calculator can help with a separate stock-to-working-concentration calculation. For a dry primer, its resuspension worksheet starts with the delivered quantity in nmol.

Model and numerical checks

The implementation uses the DNA_NN3 parameter set, identified in Biopython's melting-temperature documentation as Allawi and SantaLucia (1997). The entropy salt adjustment follows method 5, attributed there to SantaLucia (1998). Sodium-equivalent salt accounts for magnesium above the total dNTP concentration; it does not model each buffer component separately.

The engine is shared with the sequence editor. Its numerical calculations are checked against independently generated Biopython values, including self-complementary sequences and different salt settings, for both concentration models, and the default is checked against Primer3's own calculation. Agreement between implementations checks the arithmetic and chosen model; it does not establish experimental performance.

Annealing temperature starting points

Annealing temperature is set below the primers' Tm and then optimized. Choose PCR primer in excess over template to see a starting point for a temperature gradient, taken from the named vendor's published rule and applied to the lower Tm shown:

Guidance Rule Source
Taq-type polymerase 3–5 °C below the lower primer Tm NEB application note, Universal Annealing Temperature in PCR (06/26)
DreamTaq 5 °C below the primer Tm Thermo Fisher DreamTaq DNA Polymerase manual, MAN0012036
Q5 or Phusion No offset applied here Thermo Fisher Phusion manual, MAN0012393

The DreamTaq manual pairs its rule with a simpler Tm estimate, 4(G + C) + 2(A + T), for primers under 25 bases; this page applies the offset to the nearest-neighbor Tm instead, and says so in the result. NEB and Thermo Fisher tie their high-fidelity rules to their own calculators, which account for their buffers, so use NEB's calculator for Q5 and Thermo Fisher's for Phusion. With two primers, the result also reports their Tm difference; the DreamTaq manual advises keeping it within 5 °C.

A starting point is not an optimized protocol. Confirm it with a temperature gradient for your template, polymerase and buffer. The estimate does not include mismatches, modified bases, hairpins, primer dimers, DMSO or other additives. It does not search a template for binding sites or test specificity. Use the editor to inspect your sequence and annotations; use the reverse complement tool when you need to orient the opposite DNA strand.

For sequence-based molecular weight and mass or concentration conversions, use the oligo calculator. Its unmodified hydroxyl-end model is separate from the Tm calculation.