How to use (3 steps)
- Select an example or enter primer sequences (Forward/Reverse).
- Adjust method and conditions (Na+, primer concentration) if needed.
- Tm and the annealing temperature guide (Ta) are shown.
Tm and Ta are guides. Final conditions depend on reagents, instrument, and template quality; optimize with gradient PCR if needed.
Inputs (primer sequences)
Results (Tm / Ta guide)
| Primer | Tm | Length | GC% |
|---|---|---|---|
| Forward | — | — | — |
| Reverse | — | — | — |
Method comparison (reference)
| Primer | Nearest-neighbor | Salt-adjusted | Wallace |
|---|
Nearest-neighbor is not applied when IUPAC bases (N/R/Y, etc.) are present.
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How it’s calculated
- Recommended (nearest-neighbor): sums dinucleotide thermodynamics (ΔH/ΔS) and applies Na+ correction and primer concentration.
- Basic (salt-adjusted): estimates Tm from GC%, length, and Na+.
- Very simple (Wallace): rough estimate from A/T and G/C counts (best for short oligos).
- Ta (guide): lower Tm minus offset (default 3°C), plus a suggested range (Ta ± 3°C).
If IUPAC bases (N/R/Y, etc.) are present, nearest-neighbor is not applied and a simple method is used.
FAQ
What is Tm (melting temperature)?
Tm is the temperature at which roughly half of a DNA duplex is denatured. It depends on length, GC%, and salt conditions.
How should I choose annealing temperature?
This tool shows Ta (lower Tm minus offset) as a guide. Optimize with gradient PCR when needed.
Why are there multiple formulas?
Different formulas make different assumptions. Nearest-neighbor is more detailed; simple formulas and Wallace are quick estimates.
Does salt concentration affect Tm?
Yes. Higher salt typically stabilizes duplexes and raises Tm.
What if Forward/Reverse Tm differ a lot?
A large ΔTm suggests a wider temperature range (gradient PCR) or redesigning primers.
Are my sequences sent anywhere?
No. All calculations run in your browser and no data is sent.
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