In the 15 years since its introduction [120, 121, 122], the polymerase chain reaction has become a widespread research technique. The popularity of the PCR is primarily due to its apparent simplicity and high probability of success. In fact, the PCR is a relatively complicated and, as yet, incompletely understood biochemical brew; where constantly changing kinetic interactions among the several components determine the quality of the products obtained. Although good results will be obtained in most cases, there are a number of parameters that can be explored if better results are required or if the reaction fails altogether.
The standard PCR is typically done in a 50 µl volume and, in addition to the sample DNA, contains 50mM KCl, 10mM Tris ⋅ HCl (pH 8.4), 1.5mM MgCl2, 100 µg/ml gelatin, 0.25 µM of each primer, 200 µM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), and 2.5 units of Taq polymerase. The type of the DNA sample will be variable, but it will usually have between 102 and 105 copies of template (e.g., 0.1 µg human genomic DNA).
© 2001 Alexander Binder