Plasmid prep issues

[peter]

On Apr 7, 1:01 pm, "Pow Joshi" <pow.jo... from gmail.com> wrote:
> On 6 Apr 2007 19:04:31 -0700, peter <peter.ianak... 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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I plated same bacteria that did not give plasmids, I did not make new
transformation, so I don't have satellite problem.