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.
Use this page to compare primer pairs before wet-lab testing
Primer Tm checks are most useful when you want one place to compare forward and reverse primers, salt assumptions, and a first annealing-temperature range before gradient PCR. Use qPCR ΔΔCt Calculator after you have assay data, switch to Reagent Table when you are planning mix volumes, and open A260 Calculator when concentration or purity is the immediate question instead of annealing behavior.
Good workflow
- Paste the exact primer sequences you intend to order or test.
- Keep salt and primer concentration assumptions aligned with the PCR protocol you will actually run.
- Treat the suggested Ta as a starting point, then confirm with gradient PCR or redesign when the pair remains far apart.
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 (Ta)?
This tool shows Ta from the lower primer Tm minus an offset as a starting guide. Final PCR conditions should be optimized with gradient PCR when needed.
Why are there multiple Tm formulas?
Different formulas make different assumptions. Nearest-neighbor uses sequence context and salt correction, while simpler formulas and Wallace are faster rough estimates.
Does salt concentration affect Tm?
Yes. Higher salt typically stabilizes duplexes and raises Tm.
What if Forward and Reverse primer Tm differ a lot?
A large delta Tm usually means you should test a wider annealing range with gradient PCR or redesign one primer so the pair is closer.
Are my sequences sent anywhere?
No. All calculations run in your browser and no data is sent.
Related tools
- A260 Calculator (DNA/RNA Concentration & Purity) | CalcBEConfirm template concentration and purity before blaming primer design for weak amplification.
- Beta diversity calculator (Jaccard / Bray–Curtis) | CalcBEMove here only after primer validation if your downstream work compares sample composition or ecology data.
- Cell seeding calculator | cells/well → required volume | CalcBEUse seeding plans upstream when primer tests are tied to consistent plated cell numbers.
- Centrifuge converter (rpm ↔ RCF ×g) | by rotor radius | CalcBEConvert rotor settings separately when sample prep requires pelleting before PCR or sequencing.
- Diversity index calculator (Shannon & Simpson) | CalcBEUse diversity summaries later when the workflow moves beyond primer screening into sample-level comparison.
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