Tm Calculator Primer
Enter a DNA primer sequence to instantly calculate its melting temperature using the Wallace rule and nearest-neighbor method.
Use the Tm Calculator Primer
Primer Sequence
Examples
Enter a primer sequence and click Calculate Tm
Wallace Rule Tm
—
°C
2(A+T) + 4(G+C)
Nearest-Neighbor Tm
—
°C
Thermodynamic model
Suggested Annealing Temp
—
Tm(NN) − 5 °C (starting point; optimize by gradient PCR)
Sequence Statistics
Length
—
nt
GC Content
—
%
A / T / G / C
—
counts
Nearest-Neighbor Thermodynamics
ΔH
—
kcal/mol
ΔS
—
cal/mol·K
Parameters from SantaLucia 1998 unified NN model. Tm = ΔH / (ΔS + R·ln(CT/4)) − 273.15, where CT is total strand concentration.
Cleaned Sequence (5′→3′)
Summary
The Tm Calculator estimates the melting temperature of a DNA primer — the temperature at which half the primer dissociates from its complement. Two methods are provided: the quick Wallace rule (works well for primers 14–20 nt) and the more accurate nearest-neighbor thermodynamic model that accounts for stacking interactions between adjacent bases. Both are widely used in PCR primer design.
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.