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Team:UEA-JIC Norwich/pcr
From 2011.igem.org
1. Program desired cycle into PCR machine. Cycle used was as follows:
94°C - Initial Denaturation - 120 seconds
94°C - 30 seconds
58-68°C - Gradient Cycle - 30 seconds
68°C - Final Extension - 150 seconds
68°C - 300 seconds
4°C - Hold
Second, third and fourth stages were repeated for 30 cycles.
2. Three serial dilutions were conducted, yielding concentrations of 1:10, 1:100, and 1:1000. This was carried out in order to find the optimum concentration of template DNA to use, as we didn't know the concentration yielded from the Miniprep Protocol used to extract the plasmid. We conducted a gradient cycle because even though we knew the annealing temperatures of the primers we'd designed, we wanted to make sure we had the optimum temperature range.
