uncut vector DNA

Georg Kroeger a03m at biologie.uni-bremen.de
Sun Apr 28 11:00:23 EST 1996


Shanthi Adimoolam <shanth at ux5.cso.uiuc.edu> wrote:

>HI,
>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. 


Dear Shanti,

try to transform with cut unligatet vector. If you get lots of clones
you just have to gel-clean your vector! I had similar trouble with
bluescript vector. Large scale preparations of vectors are often
(especcially when using alkaline procedures) contaminated with a
fraction of irreversibly supercoiled DNA (sorry I don't remember the
citation). This fraction runs a little bit faster in agraose than
supercoiled DNA, is  uncutable and transforms bacteria with highest
efficiency.  The only way to get rid of it is to gel purify linerized
vector!
Good luck!
Georg




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