What is melting temperature (Tm)?

Tm is the temperature at which 50% of the DNA primer–template duplexes are dissociated into single strands. Higher GC content raises Tm because G:C base pairs form three hydrogen bonds, while A:T pairs form only two.

What is the Wallace rule?

The Wallace rule estimates Tm as 2°C × (A+T) + 4°C × (G+C). It is fast but best suited for primers of 14–20 bp under standard salt conditions. It is less accurate for short or long primers and non-standard salt concentrations.

What is the nearest-neighbor method?

The nearest-neighbor (SantaLucia 1998) method uses experimentally measured enthalpy (ΔH) and entropy (ΔS) values for each adjacent dinucleotide pair. It is more accurate than the Wallace rule, especially for primers under 20 bp, and accounts for sequence context rather than just base composition.

What salt concentration should I use?

Standard PCR uses 50 mM KCl or ~50 mM Na+. The nearest-neighbor formula includes a salt correction term (16.6 × log10[Na+]) so changing the value here shifts the calculated Tm accordingly. For typical PCR buffers, 50 mM is a good default.

What primer concentration should I use?

Typical PCR primers are used at 200–500 nM. The nearest-neighbor formula includes a concentration term (−10.8 × ln(CT/4)) where CT is total primer concentration. Lowering concentration decreases Tm slightly.

Why do the two methods give different results?

The Wallace rule only counts G+C and A+T bases, ignoring the order of bases. The nearest-neighbor method accounts for stacking interactions between adjacent base pairs, which depend on sequence context, giving a more accurate result especially for short primers or sequences with unusual base runs.

Tm Primer Calculator

Name: Tm Primer Calculator
Availability: InStock
Author: Nham Vu

Enter a DNA primer sequence to calculate its melting temperature using both the Wallace rule and nearest-neighbor thermodynamics.

Enter Primer Sequence

0 bp

Reaction Parameters

Salt Concentration (Na⁺ mM)

Typical PCR: 50 mM

Primer Concentration (nM)

Typical PCR: 200–500 nM

Enter a primer sequence on the left to calculate Tm.

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Summary

Enter a DNA primer sequence to calculate its melting temperature using both the Wallace rule and nearest-neighbor thermodynamics.

How it works

Enter your DNA primer sequence (5'→3') in the input box — uppercase or lowercase letters are accepted.
Adjust the salt concentration (Na+ mM) and primer concentration (nM) if needed, or leave defaults for a standard PCR estimate.
The tool instantly displays Tm calculated by both the Wallace rule and the nearest-neighbor method.
Review the GC content, sequence length, and base composition breakdown.
Use the nearest-neighbor Tm for short oligonucleotides (under 20 bp) and primers in non-standard salt conditions.

Use cases

Estimate annealing temperature for PCR primer pairs before running a reaction.
Compare Wallace rule vs. nearest-neighbor Tm for short primers.
Adjust Tm predictions for different salt concentrations in buffer optimization.
Verify primer quality metrics (GC content, length) during primer design.
Screen primers for hairpin-prone GC-rich regions using the base composition view.
Quickly validate primer Tm without installing desktop software.
Teach students the principles of DNA hybridization thermodynamics.
Cross-check Tm values from primer synthesis reports.

Frequently Asked Questions

Last updated: 2026-07-01 · Reviewed by Nham Vu