Transformation advice needed? Me too!
tan at aeolus.vmsmail.ethz.ch
Wed Aug 10 13:47:23 EST 1994
In article <sk.1126404122A at inet>, sk at lilly.com (Stuart Kuhstoss) wrote:
> In Article <17008A518S85.UMM500 at ibm.rhrz.uni-bonn.de>,
> UMM500 at ibm.rhrz.uni-bonn.de (Stefan Kahlert) wrote:
> >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.
> 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.
Another possibility for smeared DNA after restriction digestion is that
your plasmid prep is contaminated with low levels of DNase. Your plasmid
is probably stored in TE, I presume. The 0.1 mM or whatever low
concentration of EDTA you have in your TE buffer would be sufficient to
chelate the divalents such as Ca++ or Mg++ that most (all?) DNase enzymes
need as cofactors. However, when you perform the restriction digest, you
add several mM of Mg++ which any contaminating DNase can then use.
I've encountered similar problems of degraded plasmid after restriction
digest, even though the uncut plasmid (not incubated in restriction enzyme
buffer) looked fine, when I tried to modify my plasmid prep protocol. In
particular, I observed the degradation when I removed the
phenol/chloroform extraction that I usually perform. Degradation problem
went away after I restored the phenol/chloroform extraction step.
Hope this helps.
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: tan at aeolus.vmsmail.ethz.ch
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