Plasmid prep issues

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Fri Apr 6 21:04:31 EST 2007


Hi group,
I have some bad bacteria recently that won't give me any plasmid on a
miniprep. I tried almost everything and it gets more and more bizarre.
First I tried Qiagen - and I have some controls that I know prep well.
all of them have AmpR, all were grown at the same time, media,
incubator. As a result, the controls had ton of plasmid, and the
bizarre ones had none. Then I plated again on an agar - all grew, the
ones that don't give plasmid were still resistant, with a slightly
bigger colonies than the other. I start questioning the Qiagen kit - I
took some of my cultures and just boiled at 95oC for couple of min.
and then loaded on gel - same result, the ones that always have
plasmid still have it. So I went and I run them on FACS to see if the
"bad guys" are not yeasts - they are not, then I looked under
microscope - they look like each other , but the ones that fail to
give plasmid are much more motile  , compared to the ones that give me
plasmid. At that point I gave up and wrote this asking for your
wisdom.... any ideas why these E. coli won't have a plasmid, but will
have AmpR?
Thanks,
Peter
P.S. these are all similar size plasmids



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