Separating cut from uncut vector

Steven Enkemann enkemans at dc37a.nci.nih.gov
Thu Aug 31 18:44:49 EST 1995


In article <41fjjj$6sv at Twain.MO.NET>, akravetz at Walden.MO.NET (Andy
Kravetz) wrote:

> John K. Troyer (jtroyer at umabnet.ab.umd.edu) wrote:
> 
> : Non-denaturining acrylamide gels (5-10%) give MUCH better separation than 
> : agarose gels.  The vector can be separated and eluted from the gel in
much the 
> : same way as you do with agarose. 
> 
> John, I am having that very problem. I am trying to do a ligation and 
> well, there is a still a lot of uncut vector. If you do use PAGE, then 
> don't you have to do the crush and soak method of DNA purification. Right 
> ow, I just run it out on the gel and cut the band out, leaving it in the 
> LM agarose. 
> 
> Help, I am at my wit's end trying to do this damn ligation. Thanks,
> 
> andy
> 
> --
> 
> ------------------------------------
> "I love the wild power of language and and the purity of the madness that
> governs it and makes it music" 
>                                         -HST, _Generation of Swine_
> Andy Kravetz
> Freelance Journalist
> St. Louis, Missouri
> akravetz at walden.mo.net
> http://walden.mo.net/~akravetz/


Hey guys why bother trying to isolate the uncut vector from the cut
vector.  Instead chop up the vector further with a Hapaxoterministic
Enzyme.  They are readily available and when used can make these kids of
cloning problems non-existant.  For references on how to use them see:
Berger et al. (1993) Analytical Biochemistry 214: 571-579. and Berger,
S.L. (1994) Analytical Biochemistry 222: 1-8.

Steven A. Enkemann PhD.

I don't believe that curiosity killed the cat.  
I think it just did the wrong experiment.



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