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Calculation of optimal annealing temperature
Optimal annealing temperature for PCR calculated using our modified formula from Rychlik et.al.1990:
According to our experience with PCR, almost all high quality value primers designed by FastPCR in default conditions can be use at the annealing temperature from 60 to 68 without loosing PCR efficiency.
Differences in values of melting temperature calculated using different nearest neighbour thermodynamic parameters do not play important role in calculation of the PCR optimal annealing temperature.
The “excellent” primers show stable efficient amplification in vary PCR annealing temperature. The longer PCR product needs to amplify the higher annealing temperature use with the same primer’s combination.
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