In Article <32E05F88.596F at sprynet.com>, Louis Geller <bbbbd at sprynet.com> wrote:
>Hi.
> 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.comWe had the same problem using the midi-columns until a Qiagen representative
told us that we were overloading the columns. Overloading Qiagen columns
apparently gives this kind of effect.
Bjorn
