Primer annealing time

Each step of the cycle should be optimized for each template-primer combination. In certain cases in which the temperature during the annealing and extension steps are similar, the two steps can be combined into a single step in which both primer annealing and extension take place. After 20 to 45 cycles, the products amplified may then be analyzed for size, quantity, sequence, and so on, or used in further experimental procedures. A typical PCR temperature-time profile is given in Table 2.2 and a standard PCR setup in Table 2.3. 

Fig. 6.7. Probe based real-time PCR (a) During primer annealing the probe hybridises to a region within the target sequence and fluorescence is quenched, (h) and (c) As the primer extension proceeds, the exonuclease activity of the polymerase cleaves the reporter from the rest of the probe, (d) The reporter now fluoresces freely as it is in solution and no longer...

As a rule of thumb, PCR primer annealing temperatures should be about 5°C lower than the calculated melting temperature. Elongation times should be about 1 min/kb to reduce the number of new mutations. 

The procedure recommended by the Sequenase supplier (USB) (using the reagents contained in the kit) is adequate for most sequencing reactions. However, the reaction conditions for heat-denatured templates can be further optimized with regard to the following set of parameters (17) a primer annealing temperature of -70°C, (b) a labeling reaction time between 15 and 45 sec, and (c) a primer/ template ratio of 20. 

As long as the concentrations of primers remain substantially higher than that of the template, an annealing time of 20 sec is adequate. As the primers become exhausted, a longer time should be allowed so that the primer annealing is maximized with respect to the template self-annealing. 

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