- Main items required to determine the amplification conditions are as the following.
- (1) Annealing/extention temperature (called medium temperature)
- (2) Annealing/extention time
- (3) Denaturation temperature (=hot start temperature: Called high temperature)
- (4) Hot start time
- (5) Denaturation time
- Although these concrete values differ according to the test object, the following values are usually good enough.
- 95 degeres Celsius usually for (3),
- about 15 seconds for (4),
- and 4 to 5 seconds for (5) would be sufficient.
Therefore, items (1) and (2) require consideration.
The medium temperature level depends on the Tm value of primeres and probes. The level should be several degrees below the Tm value of primeres. Because medium temperature has optimum level, at higher than optimum the reaction would be rapidly suppressed, or at lower be gradually suppressed. For this reason, we recommend that you start at lower temperature, and raise temperature step by step to the optimum.
We also recommend that you start for slightly longer time at medium temperature, and reduce time step by step in order to find the optimum.