Why does the Tm on my spec sheet differ from that generated by the IDT SciTools® programs (e.g., RealTime PCR, OligoAnalyzer® tools)?
The IDT RealTime PCR design tool (found under the SciTools®
> qPCR tab of the IDT website) calculates Tm
under real-time PCR conditions of 50 mM Na+, 3 mM Mg2+, and 0.8 mM dNTPs.
While the Tm
that appears on the spec sheet is calculated based on default conditions of 50 mM Na+, it does not take into account Mg2+ and dNTP concentrations. Hence you may observe that the Tm
calculated by the design tool is a few °C higher than that reported on the spec sheet.
For real-time PCR experiments, use the Tm
generated by the RealTime PCR design tool
or the OligoAnalyzer® tool
(also found under the SciTools®
> qPCR tab of the IDT website), using parameters of 50 mM Na+, 3 mM Mg2+, and 0.8 mM dNTPs.