Why is the Tm provided on your oligonucleotide specification sheets different from that given by your oligonucleotide design tools (RealTime PCR, OligoAnalyzer)?

The IDT RealTime PCR design tool (found under the SciTools>PCR tab of the IDT website) calculates Tm under real-time PCR conditions of 32 mM Na1+, 3 mM Mg2+, and 0.8 mM dNTPs. However the Tm that appears on the spec sheet is calculated based on default conditions of 50 mM Na1+, and does not take into account Mg2+ and dNTP concentrations. Hence you may observe that the Tm calculated by the design tool is 5-8°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 OligoAnlayzer tool (also found under the SciTools>PCR tab of the IDT website), using parameters of 32 mM Na1+, 3mM Mg2+, and 0.2 mM dNTPs.

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