Hey all,
I am ligating pBS vector with some insert and they both are cut with EcoRI. Unfortunately, despite dephosporylating my vector wit Quick CIP phosphatase (1h, at 37°C) I still can't get desired clone due to high ratio of religation. According to manufacturer I should heat inactivate all enzymes (phosphatase and restriction) first after dephosphorylation. I am thinking however about heat inactivating EcoRI after digestion, then performing dephosphorylation and immediate running my sample on the gel, without inactivation CIP. What do you think about such solution? Does agarose electrophoresis inactivate restriction enzymes and phosphatase anyway (I don't have SDS in loading dye)? What else would you recommend for increasing dephosphorylation efficiency?