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PCR Optimization

There is obviously no single set of conditions that can be applied to all PCR amplifications. For example, depending on the length and sequence of primer used, the annealing temperatures of the PCRs can differ from each other. Finding the optimal or the most stringent annealing temperature is a prerequisite for reliable results and efficient amplification of a specific target.
The Eppendorf Mastercycler gradient provides a gradient function, that in one single run evaluates up to 12 different annealing, elongation, or denaturation temperatures. During the same run a number of possible concentration parameters can also be tested, row by row. Eppendorf's exclusive SteadySlope Technology allows seamless transfer of gradient experiments to standard PCR protocols.

Multiple Applications for Gradient PCR:
For example, in the presence of 0.8 mM total dNTP concentration, a magnesium chloride titration series in 0.5 mM increments over the 1 mM to 4.5 mM range will determine the magnesium ion concentration, that produces the highest yield of a specific PCR product. In combination with a temperature gradient of 20C across the block for the annealing step, important parameters of a specific PCR can be checked in just one run.

The gradient function of the Mastercycler is not restricted to the annealing temperature. The gradient function can also be used for optimization of the denaturation step, or the elongation temperature. DNA denaturation is a critical step in the PCR process and is often the step where PCR experiments fail. The temperature range for denaturation of most DNA samples is 94C to 96C. However, special target DNAs require a different denaturation temperature to achieve an optimal result. The gradient would determine the lowest possible denaturation temperature providing high yield of amplified DNA.

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 r working in the gradient mode.



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