Separating cut from uncut vector
chrisb at hgu.mrc.ac.uk
Mon Sep 11 05:01:18 EST 1995
Mark W. Trumbore (Trumborm at dc37a.nci.nih.gov) wrote:
: >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:
: >: > 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:
: Actually it¹s not inefficient. It is far more efficient than conventional
: cloning. The major problem with conventional cloning is that the
: palindromic ends generated result in the joining of unwanted fragment
: combinations. For example the reason one dephosphorylates the vector is
: to prevent ligase from sealing the unwanted joining of the two ends of the
: vector because they are complementary and more likely to anneal to each
: other than to another piece of DNA in the mixture. Because the ends
: generated by hapaxoterministic enzymes are unique and non-palandromic the
: correct re-ligation is not blocked by non-productive annealing events.
: The actual re-ligation of a plasmid chopped up with hap. enzymes is quite
Yes, but this is not the procedure in question: what we are talking
about is cloning into a _conventional_ cloning site (e.g. EcoRI) and
using hapaxoterministic enzymes to help purify the pieces of vector
fully cut _at this cloning site_. This is bound to be more inefficient
(in terms of cfu obtained per microgram of input vector DNA used) than
using the cloning enzyme alone. What you say would be relevant to
cloning with a hapaxoterministic enzyme.
: The price of one extra enzyme digest is a lot less than the cost of
: repurifying your DNA every time a restriction enzyme doesn¹t quite cut to
It doesn't circumvent the necessity for gel purification.
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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