Why does the Tm on my spec sheet differ from that designed by the RealTime PCR design tool?
The RealTime PCR design Tool calculates Tm under real time PCR conditions of 32mM Na, 3 mMMg and 0.8mM dNTPs. However the Tms that appear on the spec sheet are under default conditions that are 50mM Na and does not take into account Mg and dNTP conc. 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 experimental purposes for RealTime PCR, use the Tm as designed by the RealTime PCR design tool or the Tm given when the sequence is entered in our OligoAnlayzer tool under the conditions of 32mM Na, 3mM Mg and 0.2 mM dNTPs.