- Jun 19, 2017 -
The three factors of PCR reaction condition are temperature, time and cycle times. The setting of temperature and time is based on the three steps of PCR principle to set up denaturation-annealing-extending three temperature points. In the standard reaction, three temperature point method was adopted, and the two-strand DNA was 90 denatured, then cooled to 40 Seper, the primer was annealed and combined to the target sequence, and then accelerated to 70, which led to the extension of the primer chain along the template under the action of Taq DNA polymerase. For shorter target gene (length is 100), two temperature point method can be used, except denaturation temperature, annealing and elongation temperature can be combined, generally 94 Shan degeneration, 65 Shan left and right annealing and extension (this temperature Taq DNA enzymes still have higher catalytic activity). Shan denaturation Temperature and time: The degeneration temperature is low and the solution chain is not the most important cause of PCR failure. Under normal circumstances, 93 enough to make template DNA degeneration, if less than 93 Shan will need to prolong the time, but the temperature can not be too high, because the high temperature environment on the enzyme activity has an effect. If this step does not make the target gene template or PCR product completely degeneration, it will lead to PCR failure. Shan annealing (complex) temperature and time: annealing temperature is an important factor affecting PCR specificity.