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Using Gradient PCR to determine the optimum annealing temperature.

ificity of a PCR reaction. In most cases, this temperature must be empirically tested. The PCR is normally started at 5C below the calculated temperature of the primer melting point (Tm). However, the possible formation of unspecific secondary bands shows that the optimum temperature is often much higher than the calculated temperature (>12C).

Further PCR reactions with gradually increasing temperatures are required until the most stringent conditions have been found. When a standard PCR cycler is used, this method is the most time-intensive optimization strategy.

The Mastercycler gradient now enables rapid testing of the optimum temperature conditions on one block and in one experiment. During the PCR, a temperature gradient, which can be programmed between 1C and 20C, is built up across the thermoblock. This allows the most stringent parameters for every primer set to be calculated with the aid of only one single PCR reaction.

If the gradient function is not used, the block offers precise homogeneity, thus ensuring first-class reproducible results.

Example for Gradient PCR Program
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