Transformation advice needed? Me too!
sk at lilly.com
Thu Aug 4 11:08:02 EST 1994
In Article <17008A518S85.UMM500 at ibm.rhrz.uni-bonn.de>,
UMM500 at ibm.rhrz.uni-bonn.de (Stefan Kahlert) wrote:
>Hi everybody in this astonishing Newsproup,
>I got problem with a CMV-Luc plasmid (don't ask me what the backbone-vector
>is, no one told us).
>First we tried to Transform it in DH5 alpha. When we isolated it by
>Quiagen or alkaline-lysis it really looked like a plasmid on the gel.
>But cutting out the Luc-fragment by BamH1 and HindIII caused problems:
>We got only one large smear.
>First we thought this is problem of the enzymes but when we tested the
>same enzymes they did fine even on the CMV-Luc plasmid we used for
>transformation of the DH5-cells.
>Transformation in MC1061 already failed (but we have to do more testing
>on these bacteria, they are recommended for this plasmid but we know, that
>it should work in DH5 too).
>Does anyone know about such problems and can tell us how to go on?
>Stefan Kahlert, Universitaet Bonn
If you get a smear when you cut your "plasmid prep," you probably don't have
plasmid DNa at all but rather sheared chromosomal DNA. As to why you're
having trouble getting transformants, thats hard to say.
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