( the primers that determine the...)PCR reaction components

the primers that determine the overall assay success. For convenience, several primer design software programs are available. These can be used to ensure that the primer sequences have the following general characteristics:

• Are 1824 bases long.
• Contain no internal secondary structure.
• Contain 4060% G/C.
• Have a balanced distribution of G/C and A/T rich domains.
• Are not complementary to each other at the 3' ends (so primer-dimers will not form).
• Have a melting temperature (Tm) that allows annealing temperatures of 5565C (for maximum specificity use temperatures of 6265C).

Note: Optimal annealing temperatures are often higher than the Tm of the primers (approx. 510C) and have to be determined empirically. Caution: For both primers the Tm should be similar. Bases that do not hybridize to the template may be added at the 5' end of a primer, (e.g. for introducing restriction sites into the amplification product). Primer concentrations between 0.1 and 0.6 M are generally optimal. Higher primer concentrations may promote mispriming and accumulation of nonspecific product. Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yields of desired product. Note: For some systems, a higher primer concentration (up to 1 M) may improve results. When testing new primers, always include a positive control reaction with a template that has been tested for function in PCR. This control shows whether the primers are working. The Human Genomic DNA from Roche Applied Science (Cat. No. 1691112) is a good control template for evaluation of human primer sequen
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