Plasmid restrictions cease working?

JLCruise jlcruise at stthomas.edu
Mon Mar 17 12:51:34 EST 1997


We have suddenly begun miniprepping plasmid DNA that will not cut with
any of the restriction enzymes we normally use, including those that cut
outside the cloning sites.  It is only true of our latest recombinants,
but is happening with some created by T/A cloning of a 250bp PCR product
as well as others appearing from in vitro homologous recombination of a
completely different pair of plasmids.  The empty T/A vector cuts fine,
the recombination substrates and their recombinant "progeny" have always
cut well before.  All these plasmids  (those that cut and those that
don't) have been electroporated into the same host strain, DH10B.  

We assumed that our alkaline miniprep reagents were at fault, even though
our yields and 260:280 ratios for these preps were great. We have remade
all but the phenol, with the same results - no cutting.  We have also
tried re-extracting with phenol/chloroform - still no cuts.

I would appreciate hearing any ideas about what can suddenly change a
working miniprep method into a nonfunctional one.  If it does turn out to
be the phenol, what exactly can bad phenol do to the prep? I'd like to at
least learn something from this bad patch.

Thanks in advance for any comments you can offer.

Jennifer Cruise
Department of Biology
University of St. Thomas
St. Paul, MN 55105
jlcruise at stthomas.edu



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