What is the Melting Temperature Calculator?
Estimate DNA primer melting temperature (Tm) in °C using Wallace, salt-adjusted, or long-oligo formulas. Set sodium ion concentration for salt-adjusted Tm, compare all methods side by side, and see GC content and base counts. Free, private, and instant in your browser.
How to use the Melting Temperature Calculator
- Paste a DNA primer sequence (A, T, G, C only).
- Choose Auto or a specific Tm formula.
- Enter [Na⁺] in mM (typically ~50 for PCR buffers).
- Read the primary Tm and compare estimates from all methods.
- Copy the summary for PCR setup notes.
Common use cases
- Setting PCR annealing temperature for a new primer pair
- Comparing Wallace vs salt-adjusted Tm for short oligos
- Estimating Tm at different salt concentrations
Frequently asked questions
- What is melting temperature (Tm)?
- Tm is the temperature at which half of the DNA primer duplexes are denatured into single strands. PCR annealing temperature is often set a few degrees below Tm.
- Which formula should I use?
- Auto uses Wallace (2×AT + 4×GC) for primers ≤14 bp and salt-adjusted for longer oligos. Salt-adjusted accounts for GC% and sodium concentration.
- What sodium concentration should I enter?
- Use the approximate monovalent ion concentration of your PCR buffer. Many standard buffers are near 50 mM Na⁺ when KCl and Tris contributions are combined.
- How accurate are these Tm values?
- These are common empirical estimates. Nearest-neighbor thermodynamic models (e.g. Primer3) can be more accurate for demanding assays — validate critical primers experimentally.
- Does this work for RNA?
- This tool is for DNA primers. RNA oligos use different thermodynamic parameters.
