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==Step 6:USER enzyme digestion== *Add 40ul ddH2O, 5ul USER, 37C 1h *Add 10ul 10x DpnII buffer, 5ul RE-DpnII-ns2 guide oligo, 94C 2min -> 37C 3min -> add 5ul DpnII -> 37C 6h -> 75C 20min -> 4c hold Purified with Qiaquick Incubation with USER enzyme: *Add 5ul DpNII 10x buffer and 2 uL of USER (1K/ml)enzyme incubate for 2 hours on 37C. Annealing of DpnII oligo and incubation with DpnII. *add 6ul of 10X DpnII buffer and 2uL RE-DpnIIs guide oligo (100uM) *Perform 94C 5m > 60C 5m > 0.1C/sec to 37C > add 4ul of DpnII restriction enzyme -> 37C 2 hours > 4C hold *Qiaquick column purification *Elute with 60uL EB Buffer
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