Allelic Exchange in Bacteria/RainX for Gels

Christine Ward CVMCKW at VTVM1.CC.VT.EDU
Sat Apr 9 16:55:39 EST 1994


Greetings All,
      I have been attempting to perform allelic exchange in the gram-negative
bacterium, Actinobacillus pleuropneumoniae without much success.  It seems,
lately, that every time I open a journal I see several papers that have
successfully performed this procedure in bacteria. However, I have a feeling
that it is a tricky task to perform.  In brief, I have been introducing
insertions or  deletions into a cloned Ap gene (along with a selectable marker
that I know is expressed in Ap). I then transform the uncut plasmid
(this host plasmid is one that does not replicate in Ap) by electroporation
into Ap and screen for antibiotic resistant colonies.  The few colonies I get
seem to be spontaneous mutants (do not carry the gene for the antibiotic re-
sistance marker).  I know that my transformation efficiency is not great, so
I am working on more efficient ways to get the DNA in (such as conjugation).
I have also thought about propagating my DNA used to initiate the exchange in a
dam-/dcm- E. coli strain in case Ap is restricting the DNA before the exchange
can occur.  Does anyone out there have experience  with allelic exchange in
bacteria?  What is the probability of the exchange occurring once the DNA is in
the cell?  Do I need to screen thousands of transformants??  Any help would be
appreciated.

My second question is in reference to RainX on sequencing gels. I am
considering using RainX on my sequencing gels, however, it seems that it should
not be applied to both plates.  Since I use Long Ranger Gel Solution (from AT
Biochem--no affiliation) I prefer to 'slick' both plates. In that case, should
I avoid using RainX all together?  Does applying RainX to both plates make the
gels more difficult to pour??

Please feel to respond to the net or to me directly. Thanks.

Christine Ward
Virginia Tech
cvmckw at vtvm1.cc.vt.edu



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