I asked a similar question about a week ago, thanks for the answers, but what I really need to know is if I can go from digestion to dephosphorylation to ligation WITHOUT a purification step in between. I am using Promega Xba1, Roche CIP and can either use Invitrigen or NEB ligase. The vector is about 14kb long and my bugs don't seem to like it! I get after a mini, about 40ng/microlitre, so I can't afford to lose anything with purification. I can't afford to run any on a gel to see if anything has worked. I've tried gel purification with columns, cleaning through a mini prep column, even the old style 'freeze squeeze' but each time I lose too much. Is purification NECCESSARY and if not, does anyone know if the buffers will be compatible?