Paste a forward and/or reverse primer sequence to get its melting temperature, GC content, and self-/hetero-dimer risk, the same nearest-neighbor calculation Ampliova uses internally 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
Defaults are a typical SYBR qPCR master mix. Mg²⁺ dominates the ionic strength here, far more than the 50 mM Na⁺, so leaving it out under-predicts Tm by several degrees. dNTPs chelate Mg²⁺ roughly 1:1, so only the excess counts — currently 2.2 mM free Mg²⁺. The two salt-correction models disagree by ~2 °C at qPCR salt: Owczarzy is the more accurate fit and is what IDT OligoAnalyzer reports, SantaLucia 1998 is what Primer-BLAST reports.
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.
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