Screning E.coli transformants with an antibody?

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Mon Feb 8 15:45:48 EST 1999


In article <79mhij$nb3$2 at bubba.NMSU.Edu>, D. KIM <dkim at NMSU.Edu> wrote:

> Not to contradict the eminent Dr. Roychowdhury, but if your goal is to
> detect E. coli that have  been transformed with a recombinant plasmid, as
> opposed to recircularized plasmid, you might be able to do the following:
 
[trimmed to placate my fussy news reading software]

> Before the culture reaches late log phase, pellet the bacteria and prepare
> the plasmid DNA.  Cut with a restriction enzyme that is expected to cut
> your insert-bearing plasmid only once.  Run the digested DNA on a gel.
> 
> Your recombinants should run in a distinctly higher mw band.  Extract the
> DNA from the band and re-ligate.  Re-transform.  your plated colonies will
> contain an overwhelming percentage of recombinant clones.
> 
> Daniel Kim
> dkim at nmsu.edu

If the insertion is very inefficient compared to empty
vector formation will the latter transformants simply
overgrow the desired transformants?  Do you have any
idea how efficient the insert ligation must be for
this method to work?  I have a couple of really nasty
ligations to perform at the moment and I am very tempted
to try this as I know the desired clone is there somewhere
(PCR) but many screeninge later I haven't found it
(one more round and it will have to be colony hybridisation).
     Thanks for any input,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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