uncut vector DNA
shanth at ux5.cso.uiuc.edu
Fri Apr 26 11:44:33 EST 1996
I am trying to digest a wild-type fragment out of a vector using two
enzymes KpnI and PstI, and treat it with CIAP. Then i want to put in a
mutant insert digested with the same enzymes and of the same size.
FOr this though, i need to have complete digestion of the vector in the
For some reason, whatever conditions i try i am always left with a little
bit of uncut vector in the digest and as a result, when the colonies are
screened, all are wildtype. I had a control ligation with just the
digested vector and i seem to get about 4-fold more colonies when i
introduce the insert, making it seem like i should have the mutant inserted.
I have screened upto 48 colonies at a time but with no success.
Can anyone suggest a sure way of obtaining complete digestion? Or if you
have had similar problems and solved it please do let me know.
Thank you very much.
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