DNA Melting Temperature Calculator
Estimate the melting temperature of a DNA duplex or primer.
About DNA Melting Temperature Calculator
GC pairs form three hydrogen bonds against two for AT, so higher GC content raises melting temperature. Salt stabilises the duplex by screening phosphate charge, which is why the correction is needed. Primer pairs should have Tm values within about 2 °C of each other, or one will anneal preferentially and bias the amplification.
Formula
DNA Melting Temperature
Tm = 64.9 + 41 × (GC − 16.4) ÷ length, with a salt correction of 16.6 log₁₀[Na⁺]
- Tm
- Temperature at which half the duplex has separated
- annealing temperature
- Usually about 5 °C below Tm
Worked example
With the default values below, DNA Melting Temperature Calculator returns Melting Temperature of 37.383. Change any input above and every figure here is recalculated in your browser.
Inputs used
- Primer or duplex lengthminimum 1 bases
- 20 bases
- GC contentaccepts 0–100 %
- 50 %
- Monovalent salt concentrationminimum 1 mM
- 50 mM
- Primer concentrationminimum 1 nM
- 500 nM
Results
- Melting Temperature
- 37.383 C
- Suggested Annealing Temperature
- 32.383 C
- Simple Estimate Ignoring Salt
- 51.780 C
- Number of GC Bases
- 10.0
- Verdict
- Well designed primer parameters
Step by step
- 1Values used
- 2DNA Melting Temperature
- 3Melting Temperature= 37.383 C
- 4Suggested Annealing Temperature= 32.383 C
- 5Simple Estimate Ignoring Salt= 51.780 C
- 6Number of GC Bases= 10.0
- 7Verdict= Well designed primer parameters
Assumptions and limits
- The result is derived only from the 4 inputs listed above. Anything not among them is not part of the calculation.
- In particular, this calculator does not take inflation, tax, fees or charges or compounding frequency into account.
- Figures are rounded for display to 3 decimal places, so totals built from several rounded parts can differ slightly from the unrounded arithmetic.
How these calculators are built and tested is described on the Methodology page. If a figure looks wrong, please report it.
Frequently Asked Questions
How is DNA Melting Temperature calculated?
Tm = 64.9 + 41 × (GC − 16.4) ÷ length, with a salt correction of 16.6 log₁₀[Na⁺] GC pairs form three hydrogen bonds against two for AT, so higher GC content raises melting temperature. Salt stabilises the duplex by screening phosphate charge, which is why the correction is needed.
Why does DNA Melting Temperature matter?
Primer pairs should have Tm values within about 2 °C of each other, or one will anneal preferentially and bias the amplification.
What values do I need to enter?
This calculator takes 4 inputs: Primer or duplex length, GC content, Monovalent salt concentration, Primer concentration. The pre-filled defaults are a realistic starting point — replace them with figures from your own environment for a result you can act on.