Primer Tm Calculator

This tool uses the SantaLucia (1998) nearest-neighbor method, which considers stacking interactions between adjacent base pairs (dinucleotide steps). Salt correction uses Owczarzy (2008) for both monovalent (Na⁺, K⁺) and divalent (Mg²⁺) cation effects. This is significantly more accurate than the simple Wallace rule (2×A/T + 4×G/C), which is only valid for very short oligonucleotides under 14 bases and ignores salt concentration entirely.

Tm (melting temperature) is a thermodynamic property of the primer–template duplex — the temperature at which 50% of duplexes dissociate in solution. It is calculated from the primer sequence and buffer conditions, independent of your thermocycler. Ta (annealing temperature) is the PCR cycling parameter you actually set. For standard Taq polymerases, Ta = Tm − 5°C; for high-fidelity polymerases (Q5, Phusion, KAPA HiFi), Ta = min(Tm) + 1–3°C. The formula depends on the enzyme’s buffer system. This calculator displays both Tm and the polymerase-specific Ta for each of the 13 vendor presets.

For most PCR cloning applications, aim for a primer Tm of 58–65°C using the nearest-neighbor method, with a ΔTm of less than 2°C between the forward and reverse primers. Primers below 55°C risk non-specific amplification; above 65°C can prevent complete denaturation of GC-rich amplicons. For primers with 5′ tails (Gibson Assembly overlaps, restriction sites), use the annealing region Tm, not the full-length Tm, to set your cycling temperature — a 45-nt primer with a 25-nt overlap and 20-nt annealing region should be cycled at the annealing region Tm (~58–62°C), not the full-primer Tm (~72°C).

Different calculators use different thermodynamic models (Wallace rule, %GC method, nearest-neighbor), different salt correction formulas, and different default reaction conditions (Na⁺ concentration, Mg²⁺ concentration, oligo concentration). Vendor-specific calculators like the NEB Tm Calculator also apply proprietary polymerase-specific corrections calibrated to their buffers. As a result, the same primer sequence can yield Tm values that differ by 2–8°C across tools. This tool shows both nearest-neighbor and Wallace results for direct comparison and lets you select your specific polymerase to get the appropriate annealing temperature.

Cloning primers often contain two functional regions: a 5′ extension (Gibson Assembly overlap, restriction enzyme site, attB gateway sequence) that does not anneal to the template during PCR, and a 3′ annealing region that base-pairs with the template. Only the annealing region Tm determines your PCR cycling temperature. This tool automatically detects the boundary for primers over 30 nt and reports separate Tm values for each region, labeled “Use this for PCR cycling” on the annealing Tm. The boundary position is adjustable via slider if you know the exact split.

Both use the SantaLucia nearest-neighbor thermodynamics. The NEB Tm Calculator is optimized for NEB polymerases (Q5, Phusion, Taq, OneTaq) and does not support other vendors. This tool supports NEB presets plus polymerases from Thermo Fisher (Platinum SuperFi II, DreamTaq), Takara (PrimeSTAR GXL, PrimeSTAR Max, Ex Taq), Bio-Rad (iProof), Roche (KAPA HiFi), and PCR Biosystems (VeriFi) — 13 presets total. It additionally provides hairpin and dimer ΔG analysis and construct-aware primer region detection, which the NEB calculator does not provide.

Yes, for Tm calculation and secondary structure analysis. IDT OligoAnalyzer requires account creation and covers additional features (heteroduplex Tm, siRNA design, modifications). This tool focuses on primer Tm with multi-vendor polymerase support, hairpin and dimer ΔG analysis using the same threshold conventions as OligoAnalyzer (green > −3, yellow −3 to −9, red < −9 kcal/mol), and construct-aware 5′ extension detection — all without signup or account creation.