Changes to the PCR buffer will usually affect the outcome of the amplification. In particular, the concentration of MgCl2 can have a profound effect on the specificity and yield of an amplification. Concentrations of about 1.5 mM are usually optimal (with 200 µM each dNTP), but in some circumstances, different amounts of Mg2+ may prove to be necessary. For that reason, every established PCR in this thesis was preceded by an MgCl2 optimisation assay. Generally, excess Mg2+ will result in the accumulation of non-specific amplification products and insufficient Mg2+ will reduce the yield.
The deoxynucleotide triphosphates are usually present at 50 to 200 µM of each. Higher concentrations may tend to promote misincorporations by the polymerase and should be avoided [131]. At 50 and 200 µM, there is sufficient precursor to synthesise approximately 6.5 to 25 µg of DNA, respectively.
Taq polymerase is available from a number of vendors. For amplification reactions involving DNA samples with high sequence complexity, such as genomic DNA, there is an optimum concentration of Taq polymerase, usually 1 to 4 units per 100 µl. Increasing the amount of enzyme beyond this level can result in greater production of non-specific PCR products and reduce yield of the desired target fragment.
© 2001 Alexander Binder