Separating cut from uncut vector

[Chris Boyd]

Steven Enkemann (enkemans at dc37a.nci.nih.gov) wrote:
: In article <jpcd0-0409951419190001 at macr1-1.welc.cam.ac.uk>,
: jpcd0 at mole.bio.cam.ac.uk (John Dixon) asked:

: > what's a Hapaxoterministic Enzyme?

: in response to my posting:
: >  
: > > 
: > > 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.
: > > 
: > 
: > 
: They are restriction enzymes that cut outside of their recognition
: sequence so that the termini they produce are unique for each cut site. 
: An example is Sfi I that cuts ggccnnnn/nggcc.  A plasmid can be cut into
: several pieces by one of these enzymes and yet it can be ligated back
: together in its original form because the ends only go back together one
: way.  For more information check the references I cited.

Sorry, but I can't see how this would have the desired effect.  If the
cloning site isn't cut to completion, you'll still get a background of
non-recombinants no matter what other sites you cut/ligate.

Best wishes,

--
Chris Boyd          | from,  \MRC Human Genetics Unit / Western General Hospital
chrisb at hgu.mrc.ac.uk| not for \        Crewe Road / Edinburgh EH4 2XU / Scotland