Separating cut from uncut vector

Chris Boyd chrisb at hgu.mrc.ac.uk
Fri Sep 8 04:01:51 EST 1995


Steven Enkemann (enkemans at dc37a.nci.nih.gov) wrote:
: In article <42mc7f$mgi at scotsman.ed.ac.uk>, chrisb at hgu.mrc.ac.uk (Chris
: Boyd) wrote:

(snip)

: > 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.
:
: You can cut your vector into smaller pieces that will be easily resolvable
: from uncut fragments that consist of two fragments.  The chopped up vector
: will reassemble into its original form upon ligation because of the ends
: produced by these enzymes.  For cloning purposes: cut and dephosphorylate
: your intended vector, remove or denature your phosphatase, cut the vector
: up into pieces with the hapaxoterministic enzyme (preferably two or three
: pieces), run on a gel and cut out the individual pieces (they can be
: pooled and processed further all together), mix the pieces with a little
: of your insert, ligate, and transform.  You should get the product you
: were trying for initially.

OK, I can see that that would work (albeit very inefficiently), but I
was misled by your earlier statement into thinking you could get away
without gel purification:

: 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

While there are lots of useful things you can do with hapaxoterministic
enzymes, for conventional cloning I still think my earlier suggestion
(about using clean vector DNA in the first place) is more effective!

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



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