( a method that uses variable ...)Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler

a method that uses variable annealing temperatures (8). Initially, annealing takes place at approx. 15C above the calculated Tm. During the following cycles, the annealing temperature is then gradually reduced by 12C until it has reached a level of approx. 5C below Tm. This method is useful in avoiding nonspecific PCR products, especially in the case of complex genomic DNA, where nonspecific annealing is more probable (9).

Length of amplificate

Ideally, the amplificate should be between 100 and 400 bp long, but recently even considerably longer PCR fragments have been amplified (long PCR, up to 40 kbp) (10).

Hot-start method

Nonspecific primer-template complexes may be generated during sample preparation at room temperature. To prevent extension of these complexes in the first PCR cycles they have to be denaturated. In principle this is done using the hot-start method (11). Taq polymerase is added to the reaction mixture after an initial denaturation step at 95C. This step is followed by the annealing process in the first cycle.

Cycle times

The PCR's temperature/time profile is a particularly critical parameter. The required times selected are often too long. Standard protocols with, for example, 60 seconds for each of the denaturation, annealing and extension phases, can be run on modern devices such as the Eppendorf Mastercycler in less than half those times.

Denaturation

In general, an initial denaturation process of a few minutes duration is carried out in
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