Which Tm formula should I trust more?

The nearest-neighbor model is generally more accurate, especially for primers longer than 20 nt or those with unusual sequence composition. The Wallace rule is a quick estimate best suited for 14–20 nt primers.

What primer concentration should I use?

Most PCR reactions use 200–500 nM per primer. The default here is 250 nM. The nearest-neighbor Tm shifts by roughly 1–2 °C across this range, so the default is a good starting point.

Why does GC content affect Tm so strongly?

G-C base pairs form three hydrogen bonds, while A-T pairs form only two. More G and C bases means more energy is needed to separate the strands, raising Tm.

What annealing temperature should I use in PCR?

A common starting point is 5 °C below the lower of the two primer Tm values. Gradient PCR or incremental optimization from that point usually yields clean amplification.

Does this tool accept RNA or degenerate bases?

Only standard DNA bases (A, T, G, C) are accepted. Non-standard characters are stripped before calculation, which may affect accuracy if your primer contains degenerate positions.

Tm Calculator Primer

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

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

Primer Sequence

DNA sequence (5′→3′)

Primer conc. (nM)

Salt conc. (mM)

Examples

Enter a primer sequence and click Calculate Tm

Summary

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

How it works

Enter your DNA primer sequence (A, T, G, C) in the input box.
The Wallace rule Tm is computed as 2°C × (A+T) + 4°C × (G+C).
The nearest-neighbor Tm uses published ΔH and ΔS values for each dinucleotide pair, then applies the thermodynamic formula: Tm = ΔH / (ΔS + R·ln(C/4)) − 273.15.
GC content and basic sequence statistics are shown alongside the Tm values.
Adjust the primer concentration (default 250 nM) and salt concentration (default 50 mM) to refine the nearest-neighbor result.

Use cases

Design PCR primers with matching Tm values for both forward and reverse strands.
Choose annealing temperature for a new PCR protocol.
Compare the two Tm models to gauge uncertainty for short or long primers.
Verify that a primer falls within the 55–65 °C range recommended for most PCRs.
Estimate Tm when designing qPCR or RT-PCR assays.
Cross-check primer Tm before ordering oligonucleotide synthesis.

Frequently Asked Questions

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