( achieve an optimal result. The gradient...)PCR Optimization

The exposure of the Taq Polymerase to unnecessary high temperatures would decrease the enzyme activity over the PCR run, which would have to be compensated by using more enzyme.

For determining the optimal denaturation temperature of these templates, the gradient function of the Mastercycler can be set from 9099C. An initial denaturation step of 1 to 5 minutes (310) could further optimize the reaction. Harmful nucleases in the sample will be inactivated, ensuring the complete denaturation of complex templates such as genomic DNA.

In special cases the primer annealing temperature may need to be raised as high as the extension temperature. In fact, high-temperature annealing should result in enhanced specificity, because the hybridization of the primer to the template DNA occurs under more stringent conditions. Combining primer annealing and primer extension steps results in a two-step PCR protocol. Primer extension, in most applications, occurs effectively at a temperature of 72C and seldom needs optimization. In contrast, in two-temperature PCR experiments, the annealing-extension temperature may be in the range of 60 to 70C. Again, the optimal temperature can easily be determined with the gradient function of the Mastercycler.

A maximum gradient of 20C can be programmed over the Block, so that every longitudinal row of the block would carry out an individual temperature, either for annealing, elongation, or denaturation.

This figure shows a thermograph of the Mastercycle
'"/>

Source: