I continued my digest, starting with “part 2”, using KPnI-HF, which incubates at a lower temperature. After incubating, my lab technician helped at CIP (calf-intestinal alkaline) to the backbone, which removes phosphate groups from the ends of the strand, keeping the split ends separate and the insert “sticky”. Then I used electrophoresis to isolate the strands I want and ligated them. The last step was electroporation (same as Day 1). The last step would be to use a miniprep screen or sequencing to confirm change but I didn’t do that.
