Transformation problems

Brian Foley brianf at med.uvm.edu
Thu Aug 25 10:07:49 EST 1994


Emma Macfarlane (emma at HERA.MED.UTORONTO.CA) wrote:

: Dear Netters
: I am having serious problems with some electroporations I am attempting.
: The major problem is a rogue plasmid that is turning up everywhere, even 
: in my negative control (E. coli cells alone - no plasmid). After 
: electroporation these cells will grow on ampicillin plates, before 
: electroporation they won't. 

Because it is turning up in all of your samples, I suspect that the
plasmid may be in your competent cell stock.  Did you make your
own cells?  Did you grow them or centrifuge them in a flask that
was previously used to prepare plasmids?

I had the same problem once, and after that I bought new flasks and
centifuge tubes and labelled them "For Untransformed E.coli Only".
I now grow my E. coli and make them competent in these new flasks
and bottles and have no problems.  An alternative would be to 
carefully clean flasks and bottles prior to using them, but 
just sending them through a standard was and autoclaving was
not enough.  Washing in HCl or something that would degrade
the DNA for sure is needed.

Remember, it only takes a picomole or so of plasmid to give you
lots of colonies, and you may have harvested a milligram or more
of plasmid in a centrifuge bottle a few days ago.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
********************************************************************



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