uncut vector DNA

Shanthi Adimoolam 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 
first place. 
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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