Because the polymerase chain reaction is capable of amplifying as little as a single molecule of DNA, precautions have to be taken to guard against contamination of the reaction mixture with trace amounts of DNAs that could serve as templates [135]. Contaminations can be controlled in several ways. DNA for PCR was isolated and purified in a different area to the PCR sample preparation. 0.2 ml Eppendorf tubes (sterilised) as well as pre-sterilised 96-well racks were used for the PCR, run in a two block thermocycler (OmniGene Thermocycler, Hybaid) with tube control or a single block thermocycler (GeneAmp PCR System 9700, Perkin-Elmer) with simulated tube control. A negative control was always run with the cycled samples. The water used in PCR was heat sterilised, distilled water subjected to an additional UV sterilisation step. It has to be emphasised that all three major steps of PCR (DNA preparation, sample preparation and PCR) were performed in different rooms.
For details on the used protocols, see section 3.3.3 and 4.2.2.
© 2001 Alexander Binder