Separating cut from uncut vector
enkemans at dc37a.nci.nih.gov
Thu Sep 7 18:27:49 EST 1995
In article <42mc7f$mgi at scotsman.ed.ac.uk>, chrisb at hgu.mrc.ac.uk (Chris
> : 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
> chrisb at hgu.mrc.ac.uk| not for \ Crewe Road / Edinburgh EH4 2XU /
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.
Steven A. Enkemann PhD.
I don't believe that curiosity killed the cat.
I think it just did the wrong experiment.
More information about the Methods