Question

Problem.1: What does a PCR optimization means? Problem.2: You are working on a gradient PCR to determine the optimum annealing temperature for your primer, and you get the following result.

Answer 1

Problem 1: PCR Optimization means the identification of those conditions at which the PCR functions to its best capacity both in terms of yield and specificity. This includes the identification of the optimum concentrations of all individual components of the PCR mix such as Mg²⁺, Primers, dNTPs, Template as well as the determination of the temperature and cycling conditions at which the PCR gives the correct product, in sufficient quantity.

Problem 2: Assuming that the expected product size is around 700 bp, the optimum annealing temperature for this PCR is 57 degrees Celcius. This is because at that temperature the product obtained is specific, (there are no additional bands in the gel) and in sufficient quantity. As the temperature is decreased, the non-specificity increases (additional bands begin to appear) and as the temperature is increased, the correct product decreases in amount.

Problem 5: As mentioned above, the ideal annealing temperature is 57 degrees Celcius because, at temperatures other than 57 degrees, non specific bands begin to appear, and the product yield is also maximal at 57 degrees. Hence that is the optimal annealing temperature.