From owner-cellbiol@net.bio.net Wed Jan 22 22:00:00 1997 Path: biosci!agate!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!uunet!in1.uu.net!130.235.20.4!news.lth.se!merkurius.lu.se!news!bolde From: bolde@biogen.wblab.lu.se (Bjšrn Olde) Newsgroups: bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.molbio.yeast,bionet.microbiology Subject: Re: ZERO yield from Qiagen Maxi Prep Date: Thu, 23 Jan 97 15:59:40 GMT Organization: Molecular Neurobiology, Wallenberg Lab., Lund, Sweden Lines: 32 Message-ID: References: <32E05F88.596F@sprynet.com> NNTP-Posting-Host: bjorno.mphy.lu.se X-Newsreader: VersaTerm Link v1.1 Xref: biosci bionet.molbio.methds-reagnts:53826 bionet.cellbiol:6485 bionet.molbio.yeast:6481 bionet.microbiology:8641 In Article <32E05F88.596F@sprynet.com>, Louis Geller 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@csulb.edu or bbbbd@sprynet.com We 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 .