How is primer Tm calculated?

This tool uses the SantaLucia (1998) nearest-neighbor method, which considers stacking interactions between adjacent base pairs. Salt correction uses Owczarzy (2008) for monovalent and divalent cations. This is more accurate than the simple Wallace rule (2×AT + 4×GC), which is only valid for very short oligonucleotides under 14 bases.

Why do different Tm calculators give different results?

Different calculators use different methods (Wallace rule, %GC method, nearest-neighbor), different salt correction formulas, and different default conditions (Na+, Mg2+, oligo concentration). Vendor-specific calculators like NEB also apply proprietary polymerase-specific corrections. This tool shows both nearest-neighbor and Wallace results so you can compare.

What is construct-aware primer analysis?

Cloning primers often have a 5' extension (Gibson overlap, restriction site) that doesn't anneal to the template during PCR. The annealing region Tm — not the full primer Tm — determines your PCR cycling temperature. This tool detects the boundary and reports separate Tms for each region.

Primer Tm Calculator

Nearest-neighbor Tm with 12+ polymerase presets, hairpin/dimer ΔG analysis, and construct-aware primer regions — no signup required.

Forward Primer 5′ → 3′

Polymerase

Oligo Concentration (nM)

Na+ (mM)

Mg2+ (mM)

dNTP (mM)

Primer Tm Calculator

Primer Input