Cloning: blunt and sticky - (Aug/04/2008 )
First I was doing the cut with ecoRI and last week I realized it is njot cutting my vector efficinetly. Then I started the first cut with BglII. I cut the vector with BglII, blunt with Klenow. The latest backtracking I did is that I heat inactivated the Klenow. Run it on the gel, compared this band with uncut vector and vector cut once by a different enzyme. I was EtOH ppting the vector initially but was losing quite a bit after blunting, so i did a gel extraction of the linear blunted vector. Then I Cut the linear vector the second time with Xba I.
I had set up two reactions at this point. I checked my vector at the point of blunting by ligating the vector back vs unligated vector and uncut vector followed by a transformation. The unligated vector gave me only 2 colonies vs the ligated vector that gave me about 1000.
Meanwhile, I prepare my insert from it's present vector by cutting 5'end with Nco I, Blunting it, heating the reaction to inactivate Klenow: etoh/NaAcetate pptn, second digestion with XbaI and a second enzyme AseI(it is a unique site in the rest of the vector) to make sure I have cut out my insert band only: the insert and rest of the vector are the same size. I gel purify my insert band. The I set up ligations between the vector and the insert.
Since I see vector religation even after 2nd digestion, what I have done today is use the same vector I made with BglII, blunting, and then XbaI. I am adding more XbaI today and also cutting an internal site between the BglII and XbaI in the MCS: hoping this will work.
Am I on the right track?