( order to insure primarily in the case ...)Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler

order to insure, primarily in the case of genomic DNA, complete denaturation of the target regions on the DNA. After the first PCR products have been synthesized, considerably shorter denaturation times can be selected in the cycles, since only a very short denaturation time is actually required. These short denaturation times, however, may only be adequate if the thermocycler calculates the expired denaturation time on the basis of the temperature in the sample and not in the thermoblock (12).

Figure 1: The effect of the denaturation time on PCR Figure 1 demonstrates how on an Eppendorf Mastercycler denaturation times of 1 second can be sufficient to generate specific PCR products in a PCR reaction volume of 20 l. These tests also revealed that 5 seconds were adequate for a volume of 50 l (data not shown). Amplification of a DNA fragment (length 117 base pairs) located on a plasmid (10.4 kbp) using a standard protocol. Every parameter - except the denaturation time - was maintained at a constant level.

Volume 20 l.
L: 100 bp ladder
1: Denaturation time 30 sec.
2: Denaturation time 20 sec.
3: Denaturation time 10 sec.
4: Denaturation time 5 sec.
5: Denaturation time 1 sec.

This allows shortening the cycle times considerably. In addition, there is evidence that template DNA and PCR products are degraded by extensive denaturation times (13). Such heat damage in the DNA can, during PCR, result in a higher rate of error for nucleotide insertion (14). F
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