( ctor DNA (50 ng in 0.3 l; purified usin...)Efficiently Insert Unique Restriction Sites into Plasmid Vectors

ctor DNA (50 ng in 0.3 l; purified using the StrataPrep plasmid miniprep kit³), QuikChange 10X reaction buffer (5 l), primers (1 l of each at a concentration of 125 ng/l), and water (40.7 l) were combined and placed in Block 1 of a RoboCycler temperature cycler, at 95C for 2 minutes. The thermal cycler was turned off to allow the samples to cool slowly to room temperature. The reaction mixture was placed on ice while adding 1 l of PfuTurbo DNA polymerase (2.5 U), QuikChange dNTP mix (1 l), and a drop of mineral oil. The reaction mixture was then placed in Block 3 of the cycler at room temperature, and the unit was turned on so the block could slowly heat to 68C. The reaction mixture was incubated at this temperature for 5 minutes followed by 18 cycles of 95C for 1 minute, 55C for 1.5 minutes, and 68C for 12 minutes. At the end of temperature cycling, 10 U (1 l) of Dpn I restriction enzyme were added, and the reaction mixture was incubated at 37C for 1 hour. Following Dpn I digestion of parental DNA, 1- and 4-l volumes of the reaction mixture were used to transform XL1-Blue supercompetent cells. The transformed cells were spread onto LB/ampicillin plates, as described in the QuikChange manual. Random ampicillin-resistant colonies were inoculated into tubes containing 1.5 ml of LB media and 100 g/ml of ampicillin and incubated with shaking for 12 hours at 37C. Plasmid DNA was purified from the cultures using the StrataPrep plasmid miniprep kit.³ Plasmid preparations were digested separately with Stratagenes Bgl II, Pst I, and Xho I restriction enzymes. Plasmid DNA was digested with all 3 enzymes in 9 out of 12 randomly chosen clones. The Bgl II, Pst I, an
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