Blunting both vector and insert
Posted 09 March 2005 - 06:39 AM
am encountering problems with blunting the vector (EcoRI site) and insert ( EcoRI and SalI ends) using ROCHE Klenow. After I carryout the bluting reaction (for both)and CIP for the vector, I am losing DNA completely. Is Problem with enzyme? or water or many gel extractions? Is their any way in reducing DNA gel extraction steps?(I use freeze thaw extraction)?Please help as it is taking a lot of valuable time.
Posted 09 March 2005 - 08:05 PM
Posted 10 March 2005 - 12:34 AM
i suppose that the better way to extract blunted and "CIPed" fragments is a precipitation with salts and ethanol. A gel extraction reduces strongly the efficiency of recovery.