The key to successful PCRs lies in the design of appropriate primers. The used primers were arranged to have about the same annealing temperatures and no significant secondary structures. The chosen primers also show one hybridization site only throughout the entire gene. It was also taken care of that oligonucleotide primers did not display significant homology either internally or to one another. The used primer concentrations were determined empirically, as an increased primer concentration may lead to the generation of artifacts (see also section primersel).
The incubation times for the three temperatures corresponding to the three steps in a cycle of amplification - denaturation, annealing, and extension, were shortened. The amplification was performed in a 0.2 ml microblock Omnigene thermal cycler (Hybaid Ltd., England) using sample temperature control and heated lids. Therefore the usual one minute steps could be cut back to 30 seconds and less.
As the concentration of Mg 
ions significantly alters the specificity and
yield of amplification products (see also section PCRbuff), each new primer pair
was routinely tested under a variety of magnesium concentrations prior to
routine use (see figure fig:mg). For all three primer pairs, the standard
Mg concentration of 1.5 mM could be retained.
