Similar Tm values are one important requirement for uniform PCR reactions. Primer Tm is calculated using the nearest neighbor method with the latest thermodynamic parameters. The formula for Tm calculation is described in details in Section 5.1.6. 250 nM primer and 0.15 M Naeq+ concentrations are used as the default parameters. The default primer and salt concentrations are commonly used in real-time PCR. Variations in other typical real-time PCR setups do not significantly affect the Tm values.
Was this article helpful?