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PCR reaction components

Template The quality of the template influences the outcome of the PCR. For instance, large amounts of RNA in a DNA template can chelate Mg2+ and reduce the yield of the PCR. Also, impure templates may contain polymerase inhibitors that decrease the efficiency of the reaction. Note: To get the purest template, always use a purification product specifically designed to purify DNA such as the High Pure PCR Template Preparation Kit from Roche Applied Science (Cat. No. 1796828). The integrity of the template is also important. Template DNA should be of high molecular weight. To check the size and quality of the DNA, run an aliquot on an agarose gel. When testing a new template, always include a positive control with primers that amplify a product of known size and produce a good yield.
Example: For testing human genomic DNA, the Human tPA Control Primer Set from Roche Applied Science (Cat. No. 1691104, for amplification of 4.8 kb, 9.3 kb, and 15.0 kb targets) can serve as a positive control. The amount of template in a reaction strongly influences performance in PCR. The recommended amount of template for standard PCR is:
  • a maximum of 500ng of human genomic DNA
  • 110 ng bacterial DNA
  • 0.11 ng plasmid DNA
Low amounts of template, e.g. <10 ng human genomic DNA, will require specific reaction modifications, such as changes in cycle number, redesign of primers, use of Hot Start, etc. Primers In most PCR applications, it is the sequence and the concentration of
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