Plasmid Map
Tools

Primer Tm calculator

Estimate DNA melting temperature with the sequence, salt and strand concentrations in view.

01

Your values

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

nM

Sum of both complementary strands; equal amounts assumed.

mM
mM
mM

Calculations stay in this browser. No account or upload.

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
Calculation & assumptions

Tm = ΔH / (ΔS + R ln Ceff) − 273.15

  • DNA_NN3 parameters; entropy salt correction (method 5). Equal complementary strand amounts; self-complementarity is detected.
  • Tm is not a polymerase-specific annealing temperature. Mismatches, modified bases, secondary structures and additives 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 2–200 unambiguous DNA bases, 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 an estimated temperature, sequence length and GC percentage, along with the concentrations used.

Concentration settings and assumptions

The default total strand concentration is 50 nM. For a non-self-complementary sequence, this represents equal complementary strand amounts of 25 nM each. Self-complementarity is detected from the sequence and uses the corresponding symmetry and concentration treatment. This total-strand field should not be confused with the concentration of one PCR primer.

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.

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 Plasmid Map sequence editor. Its numerical calculations are checked against independently generated Biopython values, including self-complementary sequences and different salt settings. Agreement between implementations checks the arithmetic and chosen model; it does not establish experimental performance.

Tm is not an annealing-temperature recommendation

A PCR annealing temperature also depends on the polymerase, primer pair and reaction method. This page does not subtract an arbitrary fixed number from Tm or present that result as an optimized protocol. Follow the model and guidance applicable to your reagents.

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.