Plasmid prep issues

Pow Joshi via methods%40net.bio.net (by pow.joshi from gmail.com)
Sat Apr 7 12:01:42 EST 2007


On 6 Apr 2007 19:04:31 -0700, peter <peter.ianakiev from gmail.com> wrote:
>
> 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



my understanding is that ampicillin plating tends to  give you suppressor
colonies after a while, surrounding the plasmid containing clone,  when the
ampicillin is used up ...... it is a good idea to incubate the agar plate
for max 12 hrs, maybe less ..... you seem to have lost the plasmid somewhere
along the line ..... if you still have the original plasmid, probably you
could try transforming again....

that's a parsimonious explanation i can come up with.
hope it helps
pow


P.S. these are all similar size plasmids
>
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