Primer Tm & dimer calculator
Paste a forward primer, and a reverse one if you have it, to see its melting temperature, GC content, and self- and hetero-dimer risk. It's the same nearest-neighbor calculation Ampliova runs behind the scenes for its primer database QC.
5′→3′ · 15–35 bases · A, C, T, G only
5′→3′ · 15–35 bases · A, C, T, G only
Reaction conditions
The defaults match a typical SYBR qPCR master mix. At these concentrations Mg²⁺ matters much more than the 50 mM Na⁺, and leaving it out gives a Tm several degrees too low. dNTPs bind Mg²⁺ about one to one, so only what is left over counts (2.2 mM free with these settings). The two salt corrections differ by about 2 °C under qPCR conditions. Owczarzy fits measured data better and is what IDT OligoAnalyzer uses. SantaLucia 1998 is what Primer-BLAST uses.
Tm is computed with the SantaLucia (1998) nearest-neighbor thermodynamic model. Salt is corrected either with Owczarzy (2004, monovalent) and Owczarzy (2008, Mg²⁺ with monovalent competition), the default and the model behind IDT OligoAnalyzer, or with the SantaLucia (1998) correction and Primer3's 120·√([Mg²⁺]−[dNTP]) monovalent equivalent, which is what NCBI Primer-BLAST reports. Expect the two to differ by roughly 2 °C at qPCR salt concentrations. Dimer ΔG slides the primer against the reverse-complement of the other primer (or itself, for self-dimers) to find the most stable contiguous match, reported both as the best match anywhere in the primer and restricted to matches anchored at the primer's own 3′ terminal base, the case the polymerase can actually extend into a real dimer product. This is a fast screening estimate: it does not model bulges, internal loops, or hairpins the way Primer3 does.
Two things make this number differ from the one NCBI Primer-BLAST gives you, and neither is an error on either side. First, the conditions: Primer-BLAST defaults to 50 nM primer with 1.5 mM Mg²⁺ and 0.6 mM dNTP, while the defaults here are a typical qPCR master mix at 250 nM primer and 3 mM Mg²⁺. Second, the salt-correction model, worth about 2 °C. The Primer-BLAST value for its own default parameters is always shown next to each sequence above, so you can line the two up directly. Drag the sliders to whatever your protocol actually specifies, or hit "Primer-BLAST defaults" to reproduce its number exactly.
Primer QC is one step. Ampliova handles the full efficiency-corrected qPCR analysis too, from raw curves to fold-change. Try it free