ZERO yield from Qiagen Maxi Prep
anoel at eliot.unil.ch
Fri Jan 24 07:23:51 EST 1997
Louis Geller <bbbbd at sprynet.com> wrote:
> I have used Qiagen's plasmid maxi-preparation many times to amplify a
>yeast genomic library, always with a good yield and no problems. The
>library was in E. coli strain DH5-alpha.
> Now, we are trying to amplify a single plasmid of this library that
>contains a yeast gene we have cloned by complementation. The plasmid
>was removed from yeast and transformed into E. coli strain BMH.
> After 3 tries, we have been unable to get *any* yield whatsoever! I
>have checked the buffers for pH. We made fresh lysis buffer [P2].
>During the last run, samples were removed for the analytical gel and
>will be analyzed. We are using the same exact kit and reagents that
>have been used successafully before. We verified that the starting
>colonies have the plasmid of interest.
> The only thing I can think of is that there may be something different
>about BMH strain that makes such a maxi-pred more difficult. Still,
>getting no yield seems very strange.
> If anyone has any ideas or suggestions, please share them.
> Thanks very much!!
> Louis Geller,
> Graduate Student, Cell & Molecular Biology
> Cal State Long Beach
> lgeller at csulb.edu or bbbbd at sprynet.com
Something weird happend also to me from time to time when I was using
Qiagen columns (esp. for maxipreps). Followed the protocol, all
solutions fresh, knew the bacteria contained the plasmid and at the
end: no pellet. Grr. What I discovered later was that the plasmid was
indeed present but instead of pelleting nicely, it was spread all over
the side of the tube (BTW, tube was not silanized). You could only
barely see it as a veil covering the side. Exactely the same thing
happend to a technician in our lab a few months later. Something to
due with the column batch? Mystery. Anyway, I could easily recover the
plasmid by washing the side of the tube. I would suggest you could try
this, just in case you're facing the same weird problem I had. Hope
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