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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.
Tags:
  • Tm
  • RealTime PCR
  • Oligoanalyzer