From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!news.ucdavis.edu!info.ucla.edu!csulb.edu!news.sgi.com!news.msfc.nasa.gov!newsfeed.internetmci.com!paperboy.ids.net!usenet From: rand777@ids.net (Robert Randolph) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Yeast contamination on cell culture:HELP!! Date: Tue, 03 Sep 1996 00:50:45 GMT Organization: Randolph Biomedical Lines: 17 Message-ID: <322b802e.3594072@155.212.1.8> References: <1996Aug26.160056.9676@opalo.etsiig.uniovi.es> <322407CB.55D@pen.gulbenkian.pt> Reply-To: rand777@ids.net (Robert Randolph) NNTP-Posting-Host: dyn007a.nwp-ri.ids.net X-Newsreader: Forte Agent .99e/16.227 >Xose S. Puente wrote: >> >> Could somebody helps me??? >> I`m working on culture of human cell lines and I have some troubles >> with yeast contamination. Somebody knows how I can discart them in my >> cultures? I`ve tried with Nystatin and excess of antibiotic, but It >> doesn`t work. >> Please, I need Help!!! . I would suggest you try Anisomycin...it is often used as a selective inhibitor in culture media to prevent the growth of yeast. RR Robert Randolph Randolph Biomedical 401-826-1407 rrandy@hotmail.com From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!SERVER1.RTC-ATHLONE.IE!asweeney From: asweeney@SERVER1.RTC-ATHLONE.IE ("Antoinette Sweeney") Newsgroups: bionet.molbio.methds-reagnts Subject: test:please ignore Date: 2 Sep 1996 08:11:56 -0700 Organization: rtc-athlone Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <73368209C@server1.rtc-athlone.ie> NNTP-Posting-Host: net.bio.net Sept2/4.10pm From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-feed2.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsfeed.internetmci.com!in3.uu.net!mcsun!EU.net!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!uknet!lyra.csx.cam.ac.uk!warwick!news.ncl.ac.uk!aidan!nddm From: "D.D. Morgan" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen refuses to tell! Date: 2 Sep 1996 14:44:18 GMT Organization: University of Newcastle upon Tyne Lines: 36 Message-ID: <50ers2$mu@whitbeck.ncl.ac.uk> References: <1.5.4.32.19960830122604.006d344c@pop.cwru.edu> <507d0t$2pn@service3.uky.edu> NNTP-Posting-Host: aidan.ncl.ac.uk X-Newsreader: TIN [version 1.2 PL2] Joe Boutell (boutell@cf.ac.uk) wrote > > .....stuff cut out..... > Well i don't know how old the kits you're using are, but i have in front > of me a QIAGEN plasmid handbook (Summer 1993 edition), which on page 23 > contains Appendix A:QIAGEN buffers listing all the ingredients, and even > a little section on how to prepare 1 litre of each. > I wholeheartedly agree that putting this information at the back of the > booklet is totally out of order, i've strained my pipetting thumb on many > an occasion flicking those last few pages.... > Cheerio, > Joe Boutell. Hey Joe How about posting the recipes so that us younger members of the audience who havn't been using Qiagen stuff as long can make up the buffers ;-) Dave ******************************************************************************* * Dave Morgan | **. .***. .***. .***. .** * * Dept of Human Genetics | | | * * | | | * | | | * * | | | * | | * * University of Newcastle | | | | * * | | | * * | | | * * | | | * * | * * 19/20 Claremont Place | * | | | * | | | * * | | | * | | | * * * * Newcastle-Upon-Tyne | '***' '***' '***' '***' * * NE2 4AA | * * Tel: 0191-222-5129 | e-mail: D.D.Morgan@newcastle.ac.uk * * Fax: 0191-222-7143 | * ******************************************************************************* From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-feed2.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsfeed.internetmci.com!in3.uu.net!nntp.inet.fi!news.funet.fi!news.abo.fi!finabo.abo.fi!prappu From: Pekka Rappu TUY Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen: This is ridiculous Date: Mon, 2 Sep 1996 17:08:35 +0300 Organization: Abo Akademi University Lines: 35 Message-ID: References: <1.5.4.32.19960830210110.006c724c@pop.cwru.edu> NNTP-Posting-Host: finabo.abo.fi Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <1.5.4.32.19960830210110.006c724c@pop.cwru.edu> From prappu@finabo.abo.fiMon Sep 2 17:06:42 1996 Date: Mon, 2 Sep 1996 17:04:27 +0300 (EET DST) From: Pekka Rappu TUY Subject: Re: Qiagen: This is ridiculous I have tried to figure out the reasons for occasional poor or no yields of Qiagen gravity-flow based midipreps. I have noticed that the mixing after addition of buffer P3 is very important step. If the mixing is insufficient, SDS doesn't precipitate well, which affects the binding of DNA to the column matrix. At least with 50 ML Oakridge tubes 6-8 quick inversions have usually been enough. The second thing is the growth medium. I have had best results (100 ug or even more, purity: GS-rich DNA sequenced about 600 bp or more with ABI 377 automated sequencer) by using medium containing tryptone 30 g , yeast extract 20 g and MOPS 10 ug in 1 liter, pH about 7. For high copy plasmids I have used 50 ml of culture per column and 200 ml for low copy plasmids (or low copy strain). For 200 ml I have used 10 ml of buffers P1, P2 (made freshly) and P3 instead of 4 ml. Because of larger volume, I have extended the incubation time on ice to 20 min. The strains I have used are DH5 alfa and KE94 (low copy strain). Thirdly, I have noticed that the culture should be grown for more than 15 hours. This can be a case spesific thing, but when I have grown the culture for about 12 hours I have had poor yields. Maybe this helps somebody, maybe not. It's funny that the people at Qiagen have spent months or maybe years in optimizing the kit and then you have to do it all over again. If you want to try the slight modifications above please let me know how they work in your lab. -Pekka From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!daresbury!yama.mcc.ac.uk!loki.cf.ac.uk!news From: Joe Boutell Subject: Re: Qiagen refuses to tell! Sender: news@cf.ac.uk (Usenet News user) Message-ID: Date: Mon, 2 Sep 1996 13:57:30 GMT X-Nntp-Posting-Host: d044.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii References: <1.5.4.32.19960830122604.006d344c@pop.cwru.edu> <507d0t$2pn@service3.uky.edu> Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics, Cardiff Lines: 36 "Michael W. Thompson" wrote: > > >>Well yes, of course. But if you corner the reps, they readily acknowledge >>that it is trivial for the competition to work out the composition of the >>buffers, so its not much of a commercial secret. The real 'secret' of >>Qiagen products is the composition of the _matrix_. There's really no >>reason why they shouldn't reveal the solution compositions. Its incredibly >>irritating to spill/run out of a solution late on Friday and not be able >>to do anything about it even though you've almost certainly got everything >>you need to make a replacement sitting on the shelf. I no longer buy >>Qiagen products for precisely this reason. >> >>David > > Precisely. I have heard many stories regarding this kind of problem, many of them >from this newsgroup. I remember one such story of someone who had run out of an "RNA >resuspension solution" to an RNA isolation kit, and called the company to order some more >of it, and was told that it was about $200 for 500 mL, but the representative then went >on to tell him/her that it was simply DEPC-treated water... > I wonder how many $200 bottles of DEPC-water they sold? > > > Well i don't know how old the kits you're using are, but i have in front of me a QIAGEN plasmid handbook (Summer 1993 edition), which on page 23 contains Appendix A:QIAGEN buffers listing all the ingredients, and even a little section on how to prepare 1 litre of each. I wholeheartedly agree that putting this information at the back of the booklet is totally out of order, i've strained my pipetting thumb on many an occasion flicking those last few pages.... Cheerio, Joe Boutell. From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!mcsun!EU.net!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!uknet!lyra.csx.cam.ac.uk!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Re: Concentrating proteins already in SDS sample buffer? Date: 2 Sep 1996 12:59:58 GMT Organization: University of Leicester, UK (PCFS User) Lines: 9 Message-ID: <50eloe$4lt@falcon.le.ac.uk> References: <507fn2$290@nntp3.u.washington.edu> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Xref: biosci bionet.molbio.methds-reagnts:48678 bionet.molbio.proteins:8637 You may try ultrafiltration. Small volumes are handled in centrifugation devices (Millipore, Centricon, Whatman too, I think), but larger volumes (several ml) are better handled in stirred cells (Amicon). I would not use any precipitation techniques, because it is usually difficult to get proteins into solution again afterwards. I had this problem with both TCA precipitation and Chloroform/Methanol, even Phenol/Ether resulted in most of the protein staying in the wells during electrophoresis (but then I am handling a 160 kDa membrane protein, yours may be less fastidious). From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!mcsun!EU.net!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!uknet!lyra.csx.cam.ac.uk!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Remove SDS from protein preparation Date: 2 Sep 1996 12:50:52 GMT Organization: University of Leicester, UK (PCFS User) Lines: 8 Message-ID: <50el7c$4lt@falcon.le.ac.uk> References: <3226C0B5.692D@mail.ncku.edu.tw> NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) There are special detergent removal columns available (Pierce), but be warned: Flow through these columns is extremly slow. You may want to try the BCA method for protein determination, more sensitive than Lowry or Bradford and reasonably detergent tolerant. Biorad sell the chemicals as kit, but it is cheaper to by them seperately. From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!in3.uu.net!mcsun!EU.net!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!uknet!lyra.csx.cam.ac.uk!mje-mac5.welc.cam.ac.uk!user From: vnp@mole.bio.cam.ac.uk (Venkat Pisupati) Newsgroups: bionet.molbio.methds-reagnts Subject: Electroporation condition for Jurkat cells Date: Mon, 02 Sep 1996 12:54:04 +0000 Organization: Wellcome/CRC Lines: 13 Message-ID: NNTP-Posting-Host: mje-mac5.welc.cam.ac.uk hi! is anyone out there tried electroporating Jurkat cells? If so, can you post the electroporation conditions please. cheers venkat -- Venkat Pisupati Lab -44-01223-334131 Wellcome/CRC Institute Fax -44-01223-334134 Dept. of Genetics E.mail. vnp@mole.bio.cam.ac.uk University of Cambridge UK CB2 1QR From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!daresbury!not-for-mail From: GILLIAN NI BHARLO BOTANY PG Newsgroups: bionet.molbio.methds-reagnts Subject: PLANT RIBOSOMALL RNA SIZES Date: 2 Sep 1996 12:10:34 +0100 Organization: University College Dublin Lines: 6 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <50efba$jdb@mserv1.dl.ac.uk> Original-To: METHODS@dl.AC.UK Hi again! Can anybody out there suggest where I might look to find the sizes of the major plant ribosomal RNAs? Specifically, apple, carrot and maize for starters. Just for quick references on northerns. Thanks in advance Gill From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!daresbury!hgmp.mrc.ac.uk!news From: Mark Hogben Newsgroups: bionet.molbio.methds-reagnts Subject: adding a long polyA tail Date: Mon, 02 Sep 1996 12:07:40 +0100 Organization: NIMR Lines: 8 Message-ID: <322ABFFC.23F7@nimr.mrc.ac.uk> Reply-To: m-hogben@nimr.mrc.ac.uk NNTP-Posting-Host: nimp454.nimr.mrc.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; PPC) Hi, I wish to synthesise mRNA from a plasmid but add a polyA tail of about 200 A's to my gene. Does anyone know of a quick way of doing this? Thanks, Mark Hogben From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!daresbury!hgmp.mrc.ac.uk!news From: Mark Hogben Newsgroups: bionet.molbio.methds-reagnts Subject: 2-hybrid oocyte library? Date: Mon, 02 Sep 1996 12:04:36 +0100 Organization: NIMR Lines: 4 Message-ID: <322ABF44.3733@nimr.mrc.ac.uk> Reply-To: m-hogben@nimr.mrc.ac.uk NNTP-Posting-Host: nimp454.nimr.mrc.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; PPC) Does anyone have a 2-hybrid egg library, preferably mammalian? Please reply via email. Thanks, Mark Hogben. From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!rs.noda.sut.ac.jp!ohba From: ohba@rs.noda.sut.ac.jp (Hiroyoshi Ohba) Newsgroups: bionet.molbio.methds-reagnts Subject: ?: avoid viscous bacterial culture Date: 2 Sep 1996 03:39:08 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609021035.TAA03946@sutnfs.rs.noda.sut.ac.jp> NNTP-Posting-Host: net.bio.net Dear Netters; XL1-Blue which was transformed with pBluescript derived vector got so viscous when cultured that cell was hardly separated from culture medium. I guess that gummy material is mucous polysaccharide. I used Super Broth (1% MOPS pH7.4, 3% bacto-tryptone, 2% yeast extract) supplimented with 1mM IPTG and 50 microgram/ml ampicilin and the culture was maintained at 30 degree. Possibly rich medium has induced production of nongrateful material. This is not caused by the vector itself, but due to expression of inserted DNA because same vector with other insert did not make culture sticky. How can I separate E. coli from viscous culture medium, or avoid viscous culture, or remove or digest viscous staff from the culture? Thank you for your help. regards, ### ### ## ## # # # # # # # # # ## ## ### ### Hiroyoshi Ohba, Ph.D. Department of Biological Science and Technology Science University of Tokyo 2641 Yamazaki, Noda, Chiba 278, JAPAN Phone; +81-471-24-1501 ext. 4429 FAX; +81-471-25-1841 e-mail; ohba@rs.noda.sut.ac.jp ### ### ## ## # # # # # # # # # ## ## ### ### From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!SERVER1.RTC-ATHLONE.IE!dfaller From: dfaller@SERVER1.RTC-ATHLONE.IE ("Don Faller") Newsgroups: bionet.molbio.methds-reagnts Subject: test Date: 2 Sep 1996 03:39:08 -0700 Organization: rtc-athlone Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <29C5A4AD7@server1.rtc-athlone.ie> NNTP-Posting-Host: net.bio.net ignore """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" Dr. Don Faller, Department of Applied Biology, Athlone Regional Technical College, Athlone, Phone: +353-902-24400 Co. Westmeath, Fax: +353-902-24492 IRELAND """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!tank.news.pipex.net!pipex!hole.news.pipex.net!pipex!plug.news.pipex.net!pipex!tube.news.pipex.net!pipex!lade.news.pipex.net!pipex!ggr.co.uk!usenet From: Fraser Dr N J Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen refuses to tell! Date: 2 Sep 1996 10:09:28 GMT Organization: Glaxo Wellcome Lines: 11 Message-ID: <50eboo$93s@ukwsv3.ggr.co.uk> References: <4vv393$sn@merkurius.lu.se> NNTP-Posting-Host: ukwbf56.ggr.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1IS (X11; I; IRIX 5.3 IP22) X-URL: news:4vv393$sn@merkurius.lu.se Fredrik Kamme wrote: >Hi, it's me again, >I called them and they refused to tell. Are they contributing to science >or just profiting from it? > >DISAPPOINTED!! Freddy Are you suprised ? Have you ever 'phone Coca Cola and asked them for their ingredients ;) Neil From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsxfer2.itd.umich.edu!tank.news.pipex.net!pipex!hole.news.pipex.net!pipex!plug.news.pipex.net!pipex!tube.news.pipex.net!pipex!lade.news.pipex.net!pipex!ggr.co.uk!usenet From: Fraser Dr N J Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Rules of thumb for sequencing cloned PCR products? Date: 2 Sep 1996 10:05:36 GMT Organization: Glaxo Wellcome Lines: 17 Message-ID: <50ebhg$93s@ukwsv3.ggr.co.uk> References: <32231624.7189@ic.ac.uk> <505dp0$suv@newsstand.cit.cornell.edu> NNTP-Posting-Host: ukwbf56.ggr.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1IS (X11; I; IRIX 5.3 IP22) X-URL: news:505dp0$suv@newsstand.cit.cornell.edu bjm10@newsstand.cit.cornell.edu (Bryan John Maloney) wrote: >It may be a rule of thumb, but I know that an amplification that a post-doc >in our lab did was worse than that. She used a mixture of Taq and Pfu and >still got two deletions. We know that they're deletions because she was >amplifying a synthetic sequence originally assembled from oligos. >Furthermore, a different clone from the same amplification ended up not >having the deletions. The deletions were confirmed by one machine and >two "by hand" sequencings. Having made numerous synthetic genes in the past, the deletions etc observed are not necessarily due to the amplification step. If you directly clone synthetic sequences assembled from oligo.s without any amplification step, you often find 'mutations'. Neil. From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!ames!enews.sgi.com!news.mathworks.com!newsfeed.internetmci.com!news.dacom.co.kr!news.kreonet.re.kr!news.nuri.net!news From: Geun-woong Noh Newsgroups: bionet.molbio.methds-reagnts,sci.med.immunology,bionet.cellbiol,sci.med.laboratory,sci.research,de.sci.medizin.misc Subject: Re: Help, Interleukine 8 assay Date: Tue, 03 Sep 1996 14:53:46 +0900 Organization: HanNuri Internet Service Lines: 11 Message-ID: <322BC7EA.75F9@nuri.net> References: <19960903000026904752@[141.2.28.26]> NNTP-Posting-Host: sal045.nuri.net Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 2.0b1 (Windows; I; 32bit) Xref: biosci bionet.molbio.methds-reagnts:48706 sci.med.immunology:7332 bionet.cellbiol:5384 sci.med.laboratory:2081 sci.research:9604 Hello! Let me know why you test IL-8. Any way, it may be good for you to test IL-8 mRNA expression simultaneously with IL-8 ELISA assay. According to my experimental experience, there may be time gap between cytokine mRNA expression and cytokine serum level. If you want more information about my experiment, please message me. Thank you. -- BM6´ From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!panix!news.panix.com!not-for-mail From: iayork@panix.com (Ian A. York) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen: This is ridiculous Date: 2 Sep 1996 23:25:58 -0400 Organization: Panix Lines: 48 Message-ID: <50g8g6$p63@panix.com> References: <1.5.4.32.19960830210110.006c724c@pop.cwru.edu> <322B8BAF.376E@unity.ncsu.edu> NNTP-Posting-Host: panix.com In article <322B8BAF.376E@unity.ncsu.edu>, Susan Jane Hogarth wrote: > >At the risk of starting a new set of "kit wars", I just am wondering if >this is really worth all the trouble... was the yeild/purity using the >"old" methods really that bad? No offense meant; I really don't know... For subcloning, no problem; you can do pretty much anything you want so long as you don't actually spit in the tubes. For sequencing, you need clean but not ultraclean DNA - most kits or good-quality minipreps will do. (This is for automated sequencing, I don't do my own.) For transfecting mammalian cells, you need clean DNA; how clean depends on the cells and the transfection protocol. I've run into one or two cases where Qiagen DNA gave good results but the DNA from another company gave a lot of dead cells; but I've also used DNA from The Other Company's kits in the same transfection protocol successfully. I haven't any experience with techniques like gel retardation and so forth; my understanding is that transfection of mammalian cells tends to be the most demanding use of DNA, so I imagine that most other kits would be okay for them. I've rarely done cesium chloride DNA preps; they're considered the 'gold standard' as far as quality, and are high on the list as far as quantity is concerned. Qiagen is about the same in quality, but gives less DNA (if you get any). Other companies' kits - I have limited experience here, I've only tried a couple of others - seem to give larger amounts of DNA than Qiagen with slightly lower quality. It depends very heavily on what you want to do with it. I'm fairly transfection-oriented these days, which is why I've kept the Qiagen around; if I was just subcloning or putting it into yeast, I'd stick with The Other Company entirely, because it's cheaper and gives more DNA more consistently. I should note that there are a handful of restriction enzymes which are very picky and which might work better given Qiagen-quality DNA. For my part, I've had no problems with DNA from The Other Company's kit and some enzymes I've heard of as being somewhat fussy: KpnI, NdeI, and some others - but some of my lab mates, using DNA from a LiCl technique that produces a ton of rather grungy-looking DNA, have had trouble with them. I don't think that Qiagen quality is necessary for subcloning. Ian -- Ian York (iayork@panix.com) "-but as he was a York, I am rather inclined to suppose him a very respectable Man." -Jane Austen, The History of England From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!newsgate.duke.edu!news-server.ncren.net!taco.cc.ncsu.edu!news From: Susan Jane Hogarth Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen: This is ridiculous Date: Mon, 02 Sep 1996 21:36:47 -0400 Organization: North Carolina State University Lines: 25 Message-ID: <322B8BAF.376E@unity.ncsu.edu> References: <1.5.4.32.19960830210110.006c724c@pop.cwru.edu> NNTP-Posting-Host: sparc5c-2203gardnr.ppath.ncsu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; SunOS 5.4 sun4m) Pekka Rappu TUY wrote: > I have tried to figure out the reasons for occasional poor or no yields of > Qiagen gravity-flow based midipreps. I have noticed that the mixing after > addition of buffer P3 is very important step. {...} > The second thing is the growth medium. {...} > Thirdly, I have noticed that the culture should be grown for more than 15 > hours. {...} > Maybe this helps somebody, maybe not. It's funny that the people at Qiagen > have spent months or maybe years in optimizing the kit and then you have to > do it all over again. At the risk of starting a new set of "kit wars", I just am wondering if this is really worth all the trouble... was the yeild/purity using the "old" methods really that bad? No offense meant; I really don't know... -- Susan Jane Hogarth Still trying to figure out the *questions*, never mind the *answers*. http://www4.ncsu.edu/unity/users/s/sjhogart/public/home.html From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!newsgate.duke.edu!async131.async.duke.edu!user From: heifetzp@am.abru.cg.com (Peter Heifetz) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: ECL Western troubles Date: Mon, 02 Sep 1996 22:23:45 -0400 Organization: Ciba Biotechnology Research Lines: 25 Message-ID: References: <50fpck$f9n@dismay.ucs.indiana.edu> NNTP-Posting-Host: async70.async.duke.edu X-Newsreader: Yet Another NewsWatcher 2.0b28 In article <50fpck$f9n@dismay.ucs.indiana.edu>, Jose Bonner wrote: > We're having trouble with Western blots probed by ECL. Films sometimes > come up blank, even though antibodies and reagents are OK. Sometimes a > 5-minute exposure is fine, but a 1-hour exposure (started just after) is > blank--not even any background. What can interfere with ECL? The > problem comes and goes; good blots interspersed with blanks. Reprobe > the blanks, and sometimes they come up good; reprobe the good ones, > sometimes they come up blank. Anyone else have this problem? I had a similar sporadic ECL problem, using Dupont reagents: strong signal at 2-5 min, which rapidly (and completely) disappeared over 30 minutes or so. The manufacturer was stumped as well. I ultimately "solved" the problem by rinsing all glassware (pans, roller bottles, etc.) with concn HCl followed by much water prior to each experiment. I never found out what was quenching the luminescence but it did not recur with the acid-washing. cheers, -- Peter Heifetz Duke University Developmental, Cell, and Molecular Biology Group From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!newsfeed.internetmci.com!demos!news.glas.apc.org!glas!yur77 From: Dmitry Yuryev Newsgroups: bionet.molbio.methds-reagnts Subject: Scatchard Plot Analysys: Widespread Message-ID: Date: Mon, 02 Sep 1996 18:04:16 +0400 (WSU DST) X-Gateway: notes@glas.apc.org Lines: 22 Note my paper describing several widespread errors in performing Scatchard plot analysis. The summary is below. Full text (format MS Word 6.0 for Win) is available from: ftp://garbo.uwasa.fi/pc/science/crsaf_02.zip --------------------- ABSURD TRIVIAL ERRORS IN SCATCHARD PLOT ANALYSIS Dmitriy K.Yuryev yur77@glas.apc.org http://www.glasnet.ru/~yur77 Several types of widespread errors in performing Scatchard plot analysis resulting in fatal distortions of calculated affinities are surveyed more or less systematically. It appears that graphical analysis of non-linear Scathard plot "by eye" is almost impossible; and, moreover, results calculated by computer programs for affinity analysis often require additional "interpretation". Some of reported errors also definitely imply that data fabrication is quite common practice in this field. From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!SBC.KIST.RE.KR!luke From: luke@SBC.KIST.RE.KR ("In-Geol Choi") Newsgroups: bionet.molbio.methds-reagnts Subject: [Q] ssDNA preparation Date: 2 Sep 1996 18:22:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <9609031023.ZM246@sbc.kist.re.kr> NNTP-Posting-Host: net.bio.net Hi, I am planning to prepare ssDNA from pBluescript plasmid using helper phage. When I prepare ssDNA, there are much genomic DNAs in agarose gel. Is it right prep. to get ssDNA with genomic DNA? If it is not, how can I remove genomic DNA from ssDNA? I use NM109 or XL-1-blue as a host cell. Best regards ---- Choi, In-Geol luke@sbc.kist.re.kr From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!news1.erols.com!panix!news.panix.com!not-for-mail From: iayork@panix.com (Ian A. York) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen: This is ridiculous Date: 2 Sep 1996 19:34:56 -0400 Organization: Panix Lines: 41 Message-ID: <50fqv0$chn@panix.com> References: <4vno7a$2qu@panix.com> NNTP-Posting-Host: panix.com In article <4vno7a$2qu@panix.com>, Ian A. York wrote: > >In the last two days, I've run 10 Qiagen DNA preps - 8 midi-preps, two >maxipreps - and got one (1) with detectable DNA. Meanwhile two identical >cultures, run on a different company's resin, gave 160 ug and 175 ug of >DNA from 200 ml bugs (low copy-number plasmid). Here's an update on that story. I did do some troubleshooting, once I cooled off a bit. The problem does indeed seem to be with the Qiagen kit, or at least in the combination of the kit and the particular plasmid/bacterial strain. Specifically, it's with the 'Qiafilters' that are now being flogged. I ran another ten preps today (Qiagen gave me a replacement kit for the last one I went through, which is one reason I gave them another chance; if I'd had to pay for the privilege of doing their troubleshooting instead of just doing it for nothing I wouldn't have bothered). I'm growing a low-copy plasmid, vectors based on pcDNA1. I tried two bug strains (MC1061/P3 and DH10B/P3) and culture volumes of either 200 ml or 100 ml LB; ran them through the Maxi-Prep kit protocol. For two samples, instead of using the Qiafilters, I did the traditional incubate on ice, spin down pellet protocol. These were the only two with detectable DNA - they produced 50 ug and 150 ug from 100 ml cultures, which I consider acceptable for these plasmids. No DNA was detectable from the other cultures, except for a couple which gave a total of less than 20 ug (barely detectable). The moral is, bag the Qiafilters, folks. They've worked for me with other plasmids, but I suspect the amount of DNA you get is generally going to be lower with them. You save 45 minutes with them, which is not worth it. Note that this was not by any means a stringently controlled experiment, but it all I'm going to do for now. If anyone wants to do multiple replicates, be my guest. Ian -- Ian York (iayork@panix.com) "-but as he was a York, I am rather inclined to suppose him a very respectable Man." -Jane Austen, The History of England From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!in3.uu.net!brighton.openmarket.com!decwrl!tribune.usask.ca!rover.ucs.ualberta.ca!elegans.biology.ualberta.ca!user From: Morris_maduro@biology.ualberta.ca (Morris Maduro) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: more Hybond-N problems Followup-To: bionet.molbio.methds-reagnts Date: 2 Sep 1996 23:02:36 GMT Organization: Genetics U of Alberta Lines: 50 Distribution: world Message-ID: References: NNTP-Posting-Host: elegans.biology.ualberta.ca In article , gerry@MORPHEUS.WUSTL.EDU (Gerald B. Downes) wrote: > Hey Netters, > > I've had difficulties trying to strip & reprobe my Southern blots made with > Hybond N. The first hybridization worked great then I stripped the > membrane by pouring boiling .1% SDS onto the blot & letting it shake slowly > until it cooled to room temperature. I repeated this one time then checked > it by exposing the blots o/n. After prehybridization I hybridized again > with a probe of similar size and specific activity and... VIOLA a > completely black blot. I was able to strip the blots but haven't set up > another hyb. Any ideas? > > Gerry > Dear Gerry, My thesis advisor has long used this method for stripping Hybond N, but I never had any luck with it. I use the alternate protocol supplied with the membranes on p.11: 1. Make sure the membrane does not dry out if you plan to strip it. 2. Wash at 45 degrees C in 0.4 M NaOH for 30 minutes. 3. Wash at 45 degrees C in 0.1 x SSC, 0.1% SDS, 0.2M Tris-Cl pH 7.5 for 20 minutes. 4. Verify removal of probe by exposing to film overnight (or however long you normally do). I have used this protocol to strip and reprobe genomic digests, plaque lifts, and pulsed-field gel Southerns of yeast repeatedly (up to six times). It is not efficient, though, if there is a lot of target (i.e. plasmid probe on Southern of plasmid digestions). I have heard, however, that the patented polymer used to make Hybond N is no longer in use by Amersham (or by any other company right now). This means they will soon be substituting alternate membranes for Hybond, and I suspect there will be a number of people with difficulty using their old 'mainstay' protocols. Good luck, Morris Maduro morris_maduro@biology.ualberta.ca From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!vixen.cso.uiuc.edu!news.indiana.edu!news From: Jose Bonner Newsgroups: bionet.molbio.methds-reagnts Subject: ECL Western troubles Date: 2 Sep 1996 23:08:04 GMT Organization: Indiana University Lines: 9 Message-ID: <50fpck$f9n@dismay.ucs.indiana.edu> NNTP-Posting-Host: elm.bio.indiana.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: methods@net.bio.net X-URL: http://www.bio.net:80/hypermail/METHDS-REAGNTS/ We're having trouble with Western blots probed by ECL. Films sometimes come up blank, even though antibodies and reagents are OK. Sometimes a 5-minute exposure is fine, but a 1-hour exposure (started just after) is blank--not even any background. What can interfere with ECL? The problem comes and goes; good blots interspersed with blanks. Reprobe the blanks, and sometimes they come up good; reprobe the good ones, sometimes they come up blank. Anyone else have this problem? From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!bio.indiana.edu!jbonner From: jbonner@bio.indiana.edu (Jose Bonner) Newsgroups: bionet.molbio.methds-reagnts Subject: ECL Western troubles Date: 2 Sep 1996 16:07:46 -0700 Organization: Indiana University Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <9609022303.AA18209@sunflower.bio.indiana.edu> NNTP-Posting-Host: net.bio.net We're having trouble with Western blots probed by ECL. Films sometimes come up blank, even though antibodies and reagents are OK. Sometimes a 5-minute exposure is fine, but a 1-hour exposure (started just after) is blank--not even any background. What can interfere with ECL? The problem comes and goes; good blots interspersed with blanks. Reprobe the blanks, and sometimes they come up good; reprobe the good ones, sometimes they come up blank. Anyone else have this problem? From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!MOLECULE.BIO.UTS.EDU.AU!michelle From: michelle@MOLECULE.BIO.UTS.EDU.AU (Michelle Gleeson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen refuses to tell! Date: 2 Sep 1996 15:30:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net On Mon, 2 Sep 1996, Joe Boutell wrote: > > > Well i don't know how old the kits you're using are, but i have in front > of me a QIAGEN plasmid handbook (Summer 1993 edition), which on page 23 > contains Appendix A:QIAGEN buffers listing all the ingredients, and even > a little section on how to prepare 1 litre of each. > > I wholeheartedly agree that putting this information at the back of the > booklet is totally out of order, i've strained my pipetting thumb on many > an occasion flicking those last few pages.... > > Cheerio, > Joe Boutell. > > Well, Joe, if you have the recipes, please post them for the benefit of everyone. They certainly aren't given in any Qiagen protocol booklet I've seen here in Australia. The recipes will then be archived in this newsgroup and hopefully we'll never see this thread again :-) eagerly awaiting your post, Michelle From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!newsgate.duke.edu!news.duq.edu!newsfeed.pitt.edu!newsflash.concordia.ca!news.nstn.ca!ott.istar!istar.net!van.istar!west.istar!news-w.ans.net!newsfeeds.ans.net!chi-news.cic.net!ddsw1!news.mcs.net!nntp04.primenet.com!nntp.primenet.com!newspump.sol.net!news.inc.net!uwm.edu!cs.utexas.edu!swrinde!elroy.jpl.nasa.gov!news.msfc.nasa.gov!newsfeed.internetmci.com!in3.uu.net!nih-csl!postman From: bernard@elsie.nci.nih.gov (Bernard Murray) Subject: Re: Remove SDS from protein preparation Message-ID: <1996Sep2.215841.3524@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: ermintrude.nci.nih.gov Organization: National Institutes of Health, Bethesda, MD X-Newsreader: WinVN 0.99.7 References: <3226C0B5.692D@mail.ncku.edu.tw> Date: Mon, 2 Sep 1996 21:58:41 GMT Lines: 26 In article <3226C0B5.692D@mail.ncku.edu.tw>, wumolbio@mail.ncku.edu.tw says... > >Dear Netter, >I would grateful if anyone out-there could give any trick to remove SDS >from protein preparation. SDS interfer our protein assay by Bio-Rad >reagent for Braford methods which is the only choice for my protein due >to lack of those amino acids contributes to the absorption of Lowry >methods. Unfortunately, SDS is involved in the preparation of our >nuclear binding protein. I have used ultrafiltration to remove SDS, it >did not work. Thanks for your help in advance. (Quick'n'dirty method) If your samples have SDS present at a known concentration then just make up your standards (eg. BSA) in the presence of SDS at the same concentration. Other methods usually involve TCA or solvent precipitation. Large amounts of detergent can be removed by binding to hydrophobic beads (XAD-2 works, I think) but this does not remove the detergent exhaustively. Bernard Bernard Murray, Ph.D. bernard@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA) From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!news.mathworks.com!fu-berlin.de!news.dfn.de!zeus.rbi.informatik.uni-frankfurt.de!grapool30.rz.uni-frankfurt.de!Ebrecht From: Ebrecht@em.uni-frankfurt.de (Andreas Ebrecht) Newsgroups: bionet.molbio.methds-reagnts,sci.med.immunology,bionet.cellbiol,sci.med.laboratory,sci.research,de.sci.medizin.misc Subject: Help, Interleukine 8 assay Date: Tue, 3 Sep 1996 00:00:26 +0200 Organization: J. W. Goethe-Universitaet Frankfurt/Main Lines: 17 Message-ID: <19960903000026904752@[141.2.28.26]> NNTP-Posting-Host: dialin026.rz.uni-frankfurt.de X-Newsreader: MacSOUP 2.0 Xref: biosci bionet.molbio.methds-reagnts:48694 sci.med.immunology:7329 bionet.cellbiol:5382 sci.med.laboratory:2077 sci.research:9603 Hallo, I would like to do interleukine 1, 6 and 8 assays on plasma probes. Now I am interested in information about different methods. I think that for my intention an ELISA would be the best. Any idea ? Thanks in advance ---------------------------- Andreas Ebrecht Ebrecht@em.uni-frankfurt.de germany ---------------------------- The more you look at the past the more you see the future ---------------------------- From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!mcsun!EU.net!main.Germany.EU.net!fu-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!news From: diabetes@lrz.uni-muenchen.de (Wolfgang Schechinger) Newsgroups: bionet.molbio.methds-reagnts Subject: Q: Sequencing GC rich regions // Looking for sequencing kit/method using a LABELLED TERMINATOR (ddNTP) Date: Mon, 02 Sep 1996 21:30:09 GMT Organization: Institute for Diabetes Research Lines: 32 Distribution: world Message-ID: <50fjm3$db1@sparcserver.lrz-muenchen.de> Reply-To: diabetes@lrz.uni-muenchen.de NNTP-Posting-Host: dial154.ppp.lrz-muenchen.de X-Newsreader: Forte Free Agent 1.0.82 Hi folks! Sorry for the long subject. Actually I wanted to use a kit from Amersham with 33P radiolabelled ddNTPs to overcome my sequencing problem: unspecific terminations -staggering enzyme- in a GC rich sequence. Unfortunately my lab doesn't have the permit to use 33P yet. Thus I'm looking for hints on other kits or methods to overcome this problem. Yet I have tried * cycle sequencing from plasmid DNA with 7-deaza GTP * "convential" plasmid sequencing (dITP, dGTP) * lowering the termination temperature from 37C to 30C to prevent the polymerase from slipping off the DNA * running a 40% formamide gel but always I get these unspecific terminations. Now I want to try another way of radiolabelling the sequencing products by using radioactive ddNTPs in order to see only the correctly terminated DNA transcripts on the gel. But not using 33P. 32P or 35S is OK. (Maybe we're kind of stoneage here?) Who can help or give hints? YOUR opinion is very appreciated! please mail and post your reply. Wolfgang Schechinger Institute for Diabetes Research Kölner Platz 1 80804 Muenchen Germany Phone +49 (89) 30 79 31 24 Fax 30 81 73 3 E-mail: DIABETES@LRZ.UNI-MUENCHEN.DE From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!comp.vuw.ac.nz!ak.netlink.co.nz!auckland.ac.nz!thermo.sbs.auckland.ac.nz!user From: dmorris@sbsnov1.auckland.ac.nz (daniel morris) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: An RT-PCR problem to sink your teeth into Date: Tue, 03 Sep 1996 08:43:41 +1200 Organization: School of Biological Sciences, Auckland University Lines: 82 Message-ID: References: <50cu0f$3um@newsbf02.news.aol.com> NNTP-Posting-Host: thermo.sbs.auckland.ac.nz In article <50cu0f$3um@newsbf02.news.aol.com>, kjc944@aol.com (K J C 944) wrote: > OK, Bionetters, here's a problem you can sink your teeth into: > > I'm doing RT-PCR on mussel larvae total RNA to determine at what > developmental stages a particular gene is transcribed. I tried out my > primers on a cDNA of the gene which I have cloned and sequenced. The > primer set works great giving a single strong band of the correct size. I > then constructed an internal standard by cutting out a piece of the cDNA, > religating and transcribing into RNA. (Yes I transcribed the right way, > etc.) Using the religated cDNA, I tested my primers again and got a > single band of the correct size. I also transcribed my cDNA into RNA > (without deletion) to use as a standard to test the system. Everything was > checked on gels, quantified on the spec, etc. So far, so good. > > I diluted my standards in total RNA from tissue which does not express > the protein (my negative control RNA) and did RT on the following: > > 1. My deletion standard, diluted in negative control RNA. > 2. My standard without deletion, diluted in negative control RNA. > 3. Negative control RNA. > 4. Positive control RNA (total RNA extracted from the tissue where the > gene is expressed). > 5. Positive control RNA without the RT enzyme added. > > After the RT reaction (using random haxamers), I did PCR (hot start, > touchdown) using my gene specific primers. I included a positive PCR > control (the cDNA mentioned above) and a no template negative control. I > got a single strong band for my positive PCR control, no PCR products for > my negative PCR control, my negative control RNA sample, and my positive > control sample without the RT added, as expected. And I got a single > strong band for my deletion standard (correct size). Here's where things > get strange: Both my positive control RNA and my standard RNA without the > deletion gave a very weak band (barely visible) of the correct size and > two even weaker smeary bands that were smaller by 100-200 bp. I should > mention at this point that I have done RT-PCR using random haxamers and > the same RNA samples, but different PCR primers, several times before with > success. > > This morning I pulled out one of my previous (and successful) RT samples > (from the same tissue RNA extraction) to compare to my new RT prep, and > ran two sets of PCRs on each RT prep: One with a set of primers for the > same gene but further upstream. (I've used these primers before.) And the > second with the primers described above. Using the upstream primers, I > got good amplification for both templates. Using the downstream primers, > I got the same results on both templates; same as before: three very weak > bands. > > At this point, I know my primers are no good, but why? Can anyone give me > a good explanation as to why the RNA template works ok with these primers > if a small section has been cut out, but not if left intact? Any Ideas on > how to solve this problem, without going through the trouble of testing > new primers, making new standards, etc. (The upstream primer set is not > suitable due to polymorphism in the upsteam gene sequence.) > > I apologize if this is too long, just trying to provide as many details as > possible. > > thanks in advance, > > Kathryn J. Coyne > College of Marine Studies > University of DE > Lewes, DE 19958 > > >Hi, Just a quick thought. I have no direct experience with RT PCR but quite a lot of experience with In-Situ hybridisations targeted at rRNA. Occasionally we have problems with the accessiability of probes to certain regions of the rRNA genome which is probably due to the secondary structure. Although in PCR you are heating up to high temperatures during denaturation, at annealing temperature the secondary structure of the RNA is probably folding up faster than the annealing of primers not allowing access. To overcome this problem you could try adding DSMO at varying concentrations (helps to relax secondary structure) and increasing the anealing temperature if possible. John Tyrrell Centre for Marine Science University of Auckland Auckland New Zealand From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!faseb.org!cpk-news-feed1.bbnplanet.com!ox.mc.edu!usenet From: Robert Hamilton Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen refuses to tell! Date: 2 Sep 1996 20:31:06 GMT Organization: Mississippi College Lines: 12 Message-ID: <50fg6a$f87@ox.mc.edu> References: <1.5.4.32.19960830122604.006d344c@pop.cwru.edu> <507d0t$2pn@service3.uky.edu> <50ers2$mu@whitbeck.ncl.ac.uk> NNTP-Posting-Host: mobo.mc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) I bought a kit last year, and the book that came with it has the recipes as well, on page 23! "D.D. Morgan" wrote: >Hey Joe > How about posting the recipes so that us younger members of the >audience who havn't been using Qiagen stuff as long can make up the >buffers > ;-) From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!J002.BCHE.UIC.EDU!U55850 From: U55850@uic.edu (Arthur Wohlwill) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Disappearing plasmids Date: Sat, 31 Aug 1996 15:12:12 Organization: University of Illinois at Chicago Lines: 24 Message-ID: References: <50dk6r$a1u@flood.weeg.uiowa.edu> NNTP-Posting-Host: j002.bche.uic.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] In article <50dk6r$a1u@flood.weeg.uiowa.edu> cmstoltz@vaxa.weeg.uiowa.edu (Marty Stoltzfus) writes: >From: cmstoltz@vaxa.weeg.uiowa.edu (Marty Stoltzfus) >Subject: Disappearing plasmids >Date: 2 Sep 1996 03:27:23 GMT >Off and on our laboratory has experienced an annoying problem only with >pUC vector clones but never with other high copy plamids such as >Bluescript or low copy plasmids with the same size inserts. When we >screen our minipreps, we have high amounts of plasmid DNA (bact. strain is >HB101 in LB + 200 mic./ml AMP). Then when we grow up maxipreps we find >that sometimes even though the bacteria grow normally in AMP we have lost >most or all the plasmid. This happens even though we have streaked for >single colony isolation. Why is this happening? We would appreciate >any help we can get to solve this frustrating problem. > Marty Stoltzfus I have had problems with HB101 large preps that had to do with Dnase's. Like you, I had no problems with minipreps, but did have problems with larger scale preps. The problems went away when I added a proteinase K step to my protocol. I cant recall whether I had problems with some vectors and not others--It was over 10 years ago. Good luck, Arthur Wohlwill Adwohlwi@UIC.EDU From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!news.ucdavis.edu!info.ucla.edu!nnrp.info.ucla.edu!usenet From: mcatania@ucla.edu Newsgroups: bionet.molbio.methds-reagnts Subject: TUNEL and PI staining - apoptosis vs necrosis Date: Mon, 02 Sep 1996 19:17:13 GMT Organization: University of California, Los Angeles Lines: 11 Sender: Michael catania Message-ID: <50fc07$1joa@uni.library.ucla.edu> NNTP-Posting-Host: ts5-13.wla.ts.ucla.edu X-Newsreader: Forte Free Agent 1.0.82 Can the difference between apoptotic and necrotic cell death be determined using only these two methods, or is something else needed to give a clear answer, such as morphology/ultrasructural EM confirmation? Thanks in advance Michael Catania mcatania@ucla.edu From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!unsw.EDU.AU!R.Passey From: R.Passey@unsw.EDU.AU (Robert Passey) Newsgroups: bionet.molbio.methds-reagnts Subject: Make Your Own Vacuum Pump for Gel Blotting-WEB PAGE Date: 2 Sep 1996 11:06:06 -0700 Organization: UNSW Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <322A477C.6098@pop3.unsw.edu.au> Reply-To: r.passey@unsw.EDU.AU NNTP-Posting-Host: net.bio.net A couple of weeks ago I posted an article in this newsgroup relating to a home made vacuum pump suitable for vacuum blotting gels and slot/dot blots. It is very easy to make and costs less than $70.00 Australian. The design for this pump is now availabe on a web page at ftp://sirronald.wustl.edu/pub/mbmiller/vacuum.htm care of Mike Miller at the Washington University School of Medicine, thanks Mike. Comments, suggestions etc can be sent to R.Passey@UNSW.edu.au. Cheers, Robert Passey. From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!mcsun!EU.net!Norway.EU.net!nntp.uio.no!nntp-oslo.UNINETT.no!nntp-trd.UNINETT.no!daresbury!not-for-mail From: "Denni Schnapp" Newsgroups: bionet.molbio.methds-reagnts Subject: Munster: library/computer access Date: 2 Sep 1996 11:26:15 +0100 Lines: 29 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <50eco7$gfh@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk Hi folks, I have to ask a favour from those of you who may be following this newsgroup from Munster (Westfalen, Germany). I have to go to Munster for ca. 3 weeks during September and I wonder if there is a possibility to get access to some general use computers (ie not password-protected) at the university which would make it possible to access my homebase here at St. Andrews. I do not require access often (< once a week). Also, does anybody know where to find a library/depart- mental coffee-room, which keeps current issues of "Nature"? Any assistance will be greatly appreciated! Many regards, ///////////////////////////////////////////////////////////////////////////// Denni Schnapp Gatty Marine Laboratory Fax: (0)1334-463443 University of St. Andrews e-mail: ds4@st-and.ac.uk St. Andrews Fife KY16 8LB Scotland http://www.st-and.ac.uk/~www_sa/personal/ds4/ "If you can do - do, If you can't do - teach, If you can't teach - administrate" (Anyone need a manager?) //////////////////////////////////////////////////////////////////////////// From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!vixen.cso.uiuc.edu!uwm.edu!cs.utexas.edu!news.sprintlink.net!news-stk-200.sprintlink.net!news.sprintlink.net!news-pen-14.sprintlink.net!mcsun!EU.net!main.Germany.EU.net!fu-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!uni-regensburg.de!news From: *@alf1.ngate.uni-regensburg.de (*) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: NH4Ac-Miniprep: The Protocol Date: Mon, 02 Sep 1996 10:46:40 GMT Organization: University of Regensburg, Germany Lines: 21 Message-ID: <50ea6j$gn3@rrzs3.uni-regensburg.de> References: <506o54$fos@rrzs3.uni-regensburg.de> <507cu2$25e4@piglet.cc.uic.edu> NNTP-Posting-Host: pc3210.klinik.uni-regensburg.de X-Newsreader: Forte Free Agent 1.0.82 levenson@uic.edu (Victor Levenson) wrote: >Hi, Mike, >Is your protocol good for auto-sequencing? >Thanks, >Victor ...it is !!! I usually use between 1 and 5ul per reaction. Good luck Mike From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!rs.noda.sut.ac.jp!ohba From: ohba@rs.noda.sut.ac.jp (Hiroyoshi Ohba) Newsgroups: bionet.molbio.methds-reagnts Subject: Help needed: viscous bacterial culture Date: 2 Sep 1996 02:50:57 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 25 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609020950.SAA34652@sutnfs.rs.noda.sut.ac.jp> NNTP-Posting-Host: net.bio.net Dear Netters; Culture of XL1-Blue which was transformed with pBluescript derived vector got so viscous that cell was hardly separated from culture medium. I guess that gummy material is mucous polysaccharide. I used Super Bloth (1% MOPS pH7.4, 3% bacto-tryptone, 2% yeast extract) supplimented with 1mM IPTG and 50 microgram/ml ampicilin and the culture was maintained at 30 degree. Possibly rich medium tends to induce production of nongrateful material. This is not caused by the vector itself, but due to its insert DNA because same vector with other insert did not make culture sticky. How can I separate E. coli from viscous culture medium, or avoid viscous culture, or remove or digest viscous staff from the culture? Thank you for your help. regards, ### ### ## ## # # # # # # # # # ## ## ### ### Hiroyoshi Ohba, Ph.D. Department of Biological Science and Technology Science University of Tokyo 2641 Yamazaki, Noda, Chiba 278, JAPAN Phone; +81-471-24-1501 ext. 4429 FAX; +81-471-25-1841 e-mail; ohba@rs.noda.sut.ac.jp ### ### ## ## # # # # # # # # # ## ## ### ### From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!math.ohio-state.edu!jussieu.fr!oleane!in2p3.fr!swidir.switch.ch!swsbe6.switch.ch!news.belnet.be!news.fundp.ac.be!chokotoff.biocell.fundp.ac.be!user From: madi@biocell.fundp.ac.be (Marc DIEU) Newsgroups: bionet.molbio.methds-reagnts Subject: Help, we need informations about Duriquinone reduction. Date: Mon, 02 Sep 1996 10:03:12 +0100 Organization: F.U.N.D.P - Cellular Biochemistry Lines: 12 Message-ID: NNTP-Posting-Host: chokotoff.biocell.fundp.ac.be we are working on mitochondrial dysfunction in ischemia injury. For our work, we will assessed complexe 3 's activity with duriquinol. Duriquinol is not on market and we will reduce duriquinone. We have no protocols. Thanks for your help. -- Marc DIEU Laboratory of Cellular Biochemistry, Facultes Universitaires ND de la Paix, 61, rue de Bruxelles, B-5000 Namur (Belgium). Fax: ++/32/81/72.43.26. Email: madi@biocell.fundp.ac.be From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!SUN1.UKL.UNI-FREIBURG.DE!fweber From: fweber@SUN1.UKL.UNI-FREIBURG.DE (Friedemann Weber) Newsgroups: bionet.molbio.methds-reagnts Subject: H7 PKC Inhibitor Date: 2 Sep 1996 02:19:20 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <322AA6FC.6164@sun1.ukl.uni-freiburg.de> NNTP-Posting-Host: net.bio.net Dear netters, I want to inhibit PKC in cell culture by adding H7 (an isoquinoline sulphonamide) to the medium prior to transfection. Does anybody have any experience with this? Especially, I want to know at which timepoint it is best to add the inhibitor and how long the cells survive under H7. Are they susceptiple to a transfection at all? Thanx in advance, Friedemann -------------------------------------------------- fweber@sun1.ukl.uni-freiburg.de(Friedemann Weber) From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!newsfeed.internetmci.com!demos!news.glas.apc.org!glas!yur77 From: Dmitry Yuryev Newsgroups: bionet.molbio.methds-reagnts Subject: Inhibition Curves: one more trouble Message-ID: Date: Mon, 02 Sep 1996 18:05:01 +0400 (WSU DST) X-Gateway: notes@glas.apc.org Lines: 27 Note my paper which proves that upgrades of traditional Cheng-Prusoff procedure (ncluding computer approaches) can not be used for analysis of inhibition experiment beyond "Cheng-Prusoff conditions". The summary is appended below. Full text (MS Word 6.0 f.W. format) is available from: ftp://garbo.uwasa.fi/pc/science/crsaf_02.zip ----------------------- REFINING CHENG-PRUSOFF EQUATION Dmitriy K. Yuryev yur77@glas.apc.org http://www.glasnet.ru/~yur77 A generalisation of the Cheng-Prusoff formula relating affinity constant and the concentration of inhibitor giving 50% inhibition has been derived for the case when concentrations of ligands are not in a great excess. It shows that methods ensuring precise solving of binding equations (including computer approaches and equations derived in the present work) can not be used if these "Cheng-Prusoff conditions" of ligands' excess are not observed. The trouble is that beyond these conditions analysis becomes unreliable: the error contained in estimations of necessary parameters appears abruptly multiplied in the calculated affinity constant From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!ames!enews.sgi.com!decwrl!tribune.usask.ca!rover.ucs.ualberta.ca!news From: Colin Rasmussen Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen refuses to tell! Date: Mon, 02 Sep 1996 11:46:56 -0600 Organization: University of Alberta Lines: 18 Message-ID: <322B1D90.231C@fungus.biochem.ualberta.ca> References: <4vv393$sn@merkurius.lu.se> <50eboo$93s@ukwsv3.ggr.co.uk> Reply-To: colin@fungus.biochem.ualberta.ca NNTP-Posting-Host: mac03.biochem.ualberta.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; 68K) Fraser Dr N J wrote: > > Fredrik Kamme wrote: > >Hi, it's me again, > >I called them and they refused to tell. Are they contributing to science > >or just profiting from it? > > > >DISAPPOINTED!! Freddy > > Are you suprised ? Have you ever 'phone Coca Cola and asked them for their > ingredients ;) > Neil Actually you don't have to. They're listed on the side of the can ! They just don't mention exactly how much of everything there is. In answer to Freddy, they are profiting from science. Colin From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!news.ucdavis.edu!info.ucla.edu!csulb.edu!news.sgi.com!esiee.fr!jussieu.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!01-newsfeed.univie.ac.at!Austria.EU.net!mcsun!EU.net!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!uknet!lyra.csx.cam.ac.uk!warwick!bham!bcm20.bham.ac.uk!user From: N.A.Hotchin@bham.ac.uk (Neil Hotchin) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: looking for a method to strip gel plates Date: Mon, 02 Sep 1996 18:39:01 +0000 Organization: The University of Birmingham, UK. Lines: 14 Distribution: world Message-ID: References: NNTP-Posting-Host: bcm20.bham.ac.uk In article , gshuh@garnet.berkeley.edu (Gene Huh) wrote: > > From previous discussions on this topic here, it was something like 5% KOH > in methanol. I tried it and it worked very nicely, which was a good > thing, since I have come to HATE siliconized plates. > My previous lab gave up on siliconizing plates and started using Windolene (window cleaning spray available in the UK - don't know about US) instead. It seems to work fine, is safe, cheaper and smells good too. Neil Hotchin From owner-methds-reagnts@net.bio.net Sun Sep 01 23:00:00 1996 Path: biosci!agate!howland.erols.net!cam-news-hub1.bbnplanet.com!news3.near.net!sol.caps.maine.edu!dartvax.dartmouth.edu!usenet From: Jun.Chen@dartmouth.edu (Jun Chen) Newsgroups: bionet.molbio.methds-reagnts Subject: Help: How deoxycholate (DOC) affect the size of membrane protein analized by gel filtration or velocity cantrifugation? Date: 2 Sep 1996 15:38:40 GMT Organization: Dartmouth College, Hanover, NH Lines: 12 Message-ID: <50ev20$i9b@dartvax.dartmouth.edu> NNTP-Posting-Host: atgw2-ksp-1-6.dartmouth.edu X-Posted-From: InterNews 1.1@dartmouth.edu Hello, I am studying a membrane protein complex whose activity can be solubilized by Deoxycholate (DOC). I am trying to determine the size of the complex, but couldnŐt figure out what is DOCŐs effect on the complex behave in either gel filtration or velocity centrifugation. Does anyone know if there is a constant binding ratio of DOC/protein like SDS does? Or if there are some way I can count out the DOCŐs effect? Please include references if you can. Thanks a lot. jun From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!in3.uu.net!nntp.inet.fi!news.funet.fi!news.abo.fi!finabo.abo.fi!prappu From: Pekka Rappu TUY Newsgroups: bionet.molbio.methds-reagnts Subject: Re: QIAGEN refuses to tell Date: Tue, 3 Sep 1996 15:53:35 +0300 Organization: Abo Akademi University Lines: 30 Message-ID: References: NNTP-Posting-Host: finabo.abo.fi Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: On 3 Sep 1996, Roberto Cappai wrote: It seems to me there is a misunderstanding regarding the buffer recipes of Qiagen Kits. According to my experience, the Qiagen Plasmid MidiPrep (and also Maxi?) contains the Plasmid handbook including the recipes for all the buffers you need to purify plasmids with gravity-flow columns. The handbook also contains useful backround information of the method. On the other hand, for example the Qiaprep Spin Plasmid Mini Kit doesn't contain the recipes for buffers. Especially the contents of buffers PB, PE and N3 seems to be a secret. P1 and P2 are apparently the same as in the Qiagen Plasmid MidiPrep Kit. Similarily, there are no recipes available for Qiagen Gel Extraction Kit (buffers QX1 and PE) and for Qiagen PCR Purification Kit (PB and PE), to mention just a few examples. I think it was the PB buffer of PCR Purification Kit that started the whole discussion about recipes. Tell me if I am wrong. -Pekka Pekka Rappu Biochemistry, University of Turku FIN-20014 Turku, Finland ++358-2-333 6856 FAX ++358-2-333 6860 pekrappu@utu.fi pekka.rappu@utu.fi From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!howland.erols.net!panix!news.panix.com!not-for-mail From: iayork@panix.com (Ian A. York) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen: This is ridiculous Date: 3 Sep 1996 09:09:21 -0400 Organization: Panix Lines: 31 Message-ID: <50ham1$k56@panix.com> References: <1.5.4.32.19960830210110.006c724c@pop.cwru.edu> <322B8BAF.376E@unity.ncsu.edu> <50g8g6$p63@panix.com> <50gpsl$iu9@ukwsv3.ggr.co.uk> NNTP-Posting-Host: panix.com In article <50gpsl$iu9@ukwsv3.ggr.co.uk>, Fraser Dr N J wrote: >iayork@panix.com (Ian A. York) wrote: > >>if I was just subcloning or putting it into yeast, I'd stick with >>The Other Company entirely, because it's cheaper and gives more DNA more >>consistently. > >... and the other company was ???? Primm Labs' DNA kit. The maxi-prep kit has given me consistently good yields. The quality is acceptable - I routinely do an ethanol precipitation on the final product, both to concentrate it a little bit (the final volume otherwise is about 2 ml; that's okay if you're using a high-yield plasmid, where you'll end up with around 1 mg, but with a low-copy plasmid and particularly the expression vectors I've been using you're looking at a total of maybe 250 - 500 ug DNA) and also to clean it up a little more. I've no doubt there are other kits that are just as good, but Primm labs has an arrangement with our Central Stores, so I just have to walk downstairs to get new kits. No affiliation. Ian -- Ian York (iayork@panix.com) "-but as he was a York, I am rather inclined to suppose him a very respectable Man." -Jane Austen, The History of England From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!SERVER1.RTC-ATHLONE.IE!asweeney From: asweeney@SERVER1.RTC-ATHLONE.IE ("Antoinette Sweeney") Newsgroups: bionet.molbio.methds-reagnts Subject: Baekon-200 electroporator... Date: 3 Sep 1996 05:31:43 -0700 Organization: rtc-athlone Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <1C882D7D50@server1.rtc-athlone.ie> NNTP-Posting-Host: net.bio.net Hi, Has anybody used (or even heard of ) the Beakon 2000 electropermeabilizer. We obtained one mainly because it could provide a non-contact pulse delivery system, which we hoped would minimise cell death in the vicinity of the electrodes. Voltage, # of pulses, burst time, cycles, pulse time and electrode distance are the adjustable parameters (its very different to other electroporators) ..and while we have extensively tested for optimum permeabilizing conditions for both somatic and spermatogonial cells, we have had limited success.I would be interested to see if anyone is successfullt using this piece of equipment...or even know of any publications featuring it. Many thanks Antoinette Sweeney Toxicology Unit RTC Athlone Ireland From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!newspump.sol.net!spool.mu.edu!usenet.eel.ufl.edu!warwick!lyra.csx.cam.ac.uk!drm21 From: drm21@mole.bio.cam.ac.uk (David Micklem) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Q: Sequencing GC rich regions // Looking for sequencing kit/method using a LABELLED TERMINATOR (ddNTP) Date: Tue, 03 Sep 1996 13:04:24 +0000 Organization: Wellcome/CRC Institute Lines: 68 Distribution: world Message-ID: References: <50fjm3$db1@sparcserver.lrz-muenchen.de> NNTP-Posting-Host: drm-mac1.welc.cam.ac.uk X-Newsreader: Yet Another NewsWatcher 2.2.0b6 In article <50fjm3$db1@sparcserver.lrz-muenchen.de>, diabetes@lrz.uni-muenchen.de wrote: >Hi folks! Sorry for the long subject. >Now I want to try another way of radiolabelling the sequencing >products by using radioactive ddNTPs in order to see only the >correctly terminated DNA transcripts on the gel. But not using 33P. >32P or 35S is OK. (Maybe we're kind of stoneage here?) >Who can help or give hints? YOUR opinion is very appreciated! > >please mail and post your reply. > >Wolfgang Schechinger Hi Wolfgang, If I understand you correctly, then you think that the bands you are getting are the result of non-ddNTP termination. If this is the case, you could try one of three methods that I have heard alluded to on this newsgroup recently (I haven't got round to trying them yet). They all sound simpler (and cooler!) than termination-labelling your reactions: 1) Daniel Mytelka and Michael Chamberlain NAR1996, vol24, pp2774-2781 - they find that addition of 2M betaine to the termination reactions can cure T7 polymerase pauses. 2) Addition of a terminal transferase reaction step after extension/termination: the terminal transferase will extend only chains NOT terminated with a dideoxy nucleotide. Incorrectly terminated chains get so large that they no longer interfere (or maybe its just that their size distribution is large enough that they don't form a visible band...). I'm sorry, I don't have the ref, or a detailed protocol. 3) Felix Guerrero posted a Klenow co-sequencing ref (Biotechniques Vol 17, pg. 286 (1994)), which I think works much the same way as terminal transferase, but is simpler. He kindly posted the protocol: >The Klenow co-sequencing is surprisingly very simple. I use the standard >Sequenase Ver. 2.0 protocol with the only change being that after adding >the Sequenase enzyme to the DNA-annealled primer/buffer solution, one adds >1 unit of Klenow. Then the only difference is to allow the termination >reaction step to proceed at 37 degrees C for 45 minutes instead of the >usual 2-4 minutes. >Pretty easy and I think there is a noticeable improvement in band >uniformity from difficult templates. At times, I have had to resort to >dITP sequencing, but now I add the Klenow routinely! I'm going to try this method first, next time I have a problem template. I can't see any reason why the Klenow shouldn't be incorporated into the master mix that I distribute, rather than added seperately. I'd be very interested to hear whether/how you manage to solve your problem. Cheers, David _____________________________________________________________ D.R.Micklem, Wellcome/CRC Institute, Time flies like an arrow... Tennis Court Road, Cambridge CB2 1QR Fruit flies like a banana. UK Tel: [+44] (0)1223 334129 Email:drm21@mole.bio.cam.ac.uk Fax: [+44] (0)1223 334089 _____________________________________________________________ From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!howland.erols.net!news.sprintlink.net!news-stk-200.sprintlink.net!news.sprintlink.net!news-stk-11.sprintlink.net!dublin.bitwise.net!usenet From: jlitt@capecod.net (Jerry Litt) Newsgroups: bionet.molbio.methds-reagnts,sci.med.immunology,bionet.cellbiol,sci.med.laboratory,sci.research,de.sci.medizin.misc Subject: Re: Help, Interleukine 8 assay Date: Tue, 03 Sep 1996 14:41:35 GMT Organization: Unknown Lines: 38 Message-ID: <50h5a8$arc@dublin.bitwise.net> References: <19960903000026904752@[141.2.28.26]> NNTP-Posting-Host: ost59.capecod.net X-Newsreader: Forte Free Agent 1.0.82 Xref: biosci bionet.molbio.methds-reagnts:48716 sci.med.immunology:7339 bionet.cellbiol:5386 sci.med.laboratory:2083 sci.research:9605 Some references: -Kauma, SW. J.Clin.Endocrinol.Metab.76:701-703(1993)......IL1beta -van der Zee, AGJ et al. Cancer 75:1004-1009 (1995)....IL-6 -Ullum, H. et al. J. Appl. Physiol. 77:93-97(1994).....IL1alpha and beta, IL-6 -van Leeuwen, MA et al. Ann. Rheum. Dis. 54:33-38(1995)...IL-6 -Woodroffe, SB eta al. Arch. Virol. 133:295-308(1993)...IL-1 alpha and beta -Helle, M. et al. J. Immunol. Meth. 138:47-56(1991)....IL-6 -Hack, CE et al. Infection and Immunity 60:2835-2842(1992)....IL-8 Virtually all ELISA formats, most amplified with Catalyzed Reporter Deposition. Jerry Ebrecht@em.uni-frankfurt.de (Andreas Ebrecht) wrote: >Hallo, >I would like to do interleukine 1, 6 and 8 assays >on plasma probes. Now I am interested in >information about different methods. I think >that for my intention an ELISA would be >the best. Any idea ? >Thanks in advance >---------------------------- >Andreas Ebrecht >Ebrecht@em.uni-frankfurt.de >germany >---------------------------- >The more you look at the past >the more you see the future >---------------------------- From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!daresbury!not-for-mail From: Newsgroups: bionet.molbio.methds-reagnts Subject: Furosine Date: 3 Sep 1996 08:40:09 +0100 Lines: 1 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <50gncp$red@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk Does anybody know where it is possible to buy furosine? In addition has someone produced MAbs or PAbs directed against furosine? Please answer Marinis@utovrm.it From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!spool.mu.edu!usenet.eel.ufl.edu!warwick!bris.ac.uk!usenet From: "Andrew J. Doherty" Subject: Re: An RT-PCR problem to sink your teeth into X-Nntp-Posting-Host: anaajd.ana.bris.ac.uk Content-Type: text/plain; charset=us-ascii To: K J C 944 Message-ID: <322BDF68.5039@bsa.bristol.ac.uk> Sender: usenet@fsa.bris.ac.uk (Usenet) Content-Transfer-Encoding: 7bit Organization: University of Bristol, UK References: <50cu0f$3um@newsbf02.news.aol.com> Mime-Version: 1.0 Date: Tue, 3 Sep 1996 07:34:00 GMT X-Mailer: Mozilla 3.0b5a (Win16; I) Lines: 90 K J C 944 wrote: > > OK, Bionetters, here's a problem you can sink your teeth into: > > I'm doing RT-PCR on mussel larvae total RNA to determine at what > developmental stages a particular gene is transcribed. I tried out my > primers on a cDNA of the gene which I have cloned and sequenced. The > primer set works great giving a single strong band of the correct size. I > then constructed an internal standard by cutting out a piece of the cDNA, > religating and transcribing into RNA. (Yes I transcribed the right way, > etc.) Using the religated cDNA, I tested my primers again and got a > single band of the correct size. I also transcribed my cDNA into RNA > (without deletion) to use as a standard to test the system. Everything was > checked on gels, quantified on the spec, etc. So far, so good. > > I diluted my standards in total RNA from tissue which does not express > the protein (my negative control RNA) and did RT on the following: > > 1. My deletion standard, diluted in negative control RNA. > 2. My standard without deletion, diluted in negative control RNA. > 3. Negative control RNA. > 4. Positive control RNA (total RNA extracted from the tissue where the > gene is expressed). > 5. Positive control RNA without the RT enzyme added. > > After the RT reaction (using random haxamers), I did PCR (hot start, > touchdown) using my gene specific primers. I included a positive PCR > control (the cDNA mentioned above) and a no template negative control. I > got a single strong band for my positive PCR control, no PCR products for > my negative PCR control, my negative control RNA sample, and my positive > control sample without the RT added, as expected. And I got a single > strong band for my deletion standard (correct size). Here's where things > get strange: Both my positive control RNA and my standard RNA without the > deletion gave a very weak band (barely visible) of the correct size and > two even weaker smeary bands that were smaller by 100-200 bp. I should > mention at this point that I have done RT-PCR using random haxamers and > the same RNA samples, but different PCR primers, several times before with > success. > > This morning I pulled out one of my previous (and successful) RT samples > (from the same tissue RNA extraction) to compare to my new RT prep, and > ran two sets of PCRs on each RT prep: One with a set of primers for the > same gene but further upstream. (I've used these primers before.) And the > second with the primers described above. Using the upstream primers, I > got good amplification for both templates. Using the downstream primers, > I got the same results on both templates; same as before: three very weak > bands. > > At this point, I know my primers are no good, but why? Can anyone give me > a good explanation as to why the RNA template works ok with these primers > if a small section has been cut out, but not if left intact? Any Ideas on > how to solve this problem, without going through the trouble of testing > new primers, making new standards, etc. (The upstream primer set is not > suitable due to polymorphism in the upsteam gene sequence.) > > I apologize if this is too long, just trying to provide as many details as > possible. > > thanks in advance, > > Kathryn J. Coyne > College of Marine Studies > University of DE > Lewes, DE 19958 > > Hi Kathryn, is this a secondary structure problem? The fact that the primers work well on the deletion standard but not the full length one would suggest that cut something out which prevents the amplification and/or RT process. This may be a loop in the RNA, especially as you are also getting a couple of smaller bands amplifying (maybe RT "jumping" across the junction?). Just don't ask why this should not be a problem with your standard RNA!! Hope it helps and I'll keep chewing the cud. TTFN -- *********************************************** Dr Andrew Doherty Department of Anatomy School of Medical Sciences University Walk Bristol UK BS8 1TD e-mail Doherty@bsa.bristol.ac.uk ************************************************ From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!in3.uu.net!fu-berlin.de!holmes.rz-berlin.mpg.DE!not-for-mail From: wegloehner@mpimg-berlin-dahlem.mpg.de Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Q: Sequencing GC rich regions // Looking for sequencing kit/method using a LABELLED TERMINATOR (ddNTP) Date: 3 Sep 96 09:25:34 +0100 Organization: MPI f. Molekulare Genetik, Berlin Lines: 51 Distribution: world Message-ID: <1996Sep3.092534@crick.rz-berlin.mpg.de> References: <50fjm3$db1@sparcserver.lrz-muenchen.de> NNTP-Posting-Host: holmes.rz-berlin.mpg.de (141.14.129.124) X-Access: 16 25 816 In article <50fjm3$db1@sparcserver.lrz-muenchen.de>, diabetes@lrz.uni-muenchen.de (Wolfgang Schechinger) writes: > Hi folks! Sorry for the long subject. > > Actually I wanted to use a kit from Amersham with 33P radiolabelled > ddNTPs to overcome my sequencing problem: unspecific terminations > -staggering enzyme- in a GC rich sequence. Unfortunately my lab > doesn't have the permit to use 33P yet. Thus I'm looking for hints on > other kits or methods to overcome this problem. Yet I have tried > * cycle sequencing from plasmid DNA with 7-deaza GTP > * "convential" plasmid sequencing (dITP, dGTP) > * lowering the termination temperature from 37C to 30C to prevent the > polymerase from slipping off the DNA > * running a 40% formamide gel > > but always I get these unspecific terminations. > > Now I want to try another way of radiolabelling the sequencing > products by using radioactive ddNTPs in order to see only the > correctly terminated DNA transcripts on the gel. But not using 33P. > 32P or 35S is OK. (Maybe we're kind of stoneage here?) > Who can help or give hints? YOUR opinion is very appreciated! > > please mail and post your reply. > > Wolfgang Schechinger > Institute for Diabetes Research > Kölner Platz 1 > 80804 Muenchen > Germany > Phone +49 (89) 30 79 31 24 > Fax 30 81 73 3 > E-mail: DIABETES@LRZ.UNI-MUENCHEN.DE > Why don't you try cycle sequencing on an automated sequencer using labelled ddNTPs. And if there isn't a ABI machine in your institute phone Applied Biosystems, they should be able to give you a hint were in muenchen you can find one. We had exactly the same problem - GC contents of more then 90% in a stretch of over 200 bp. The only way to get the sequence was cycle sequencing with labelled ddNTP. At least in our hands this worked fine - even without any experience - we used a borrowed kit and gave the samples to the technician who was running the machine. hope this helps wolf From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!spool.mu.edu!usenet.eel.ufl.edu!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!swsbe6.switch.ch!news.belnet.be!news.fundp.ac.be!immuno-wawa.sciences.fundp.ac.be!user From: Pascal.Mertens@fundp.ac.be (Pascal Mertens) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: [Q] ssDNA preparation Date: 3 Sep 1996 06:54:25 GMT Organization: Immunologie Microbiologie, FUNDP Namur Lines: 21 Distribution: world Message-ID: References: <9609031023.ZM246@sbc.kist.re.kr> NNTP-Posting-Host: immuno-wawa.sciences.fundp.ac.be In article (Dans l'article) <9609031023.ZM246@sbc.kist.re.kr>, luke@SBC.KIST.RE.KR ("In-Geol Choi") wrote (écrivait) : > Hi, > I am planning to prepare ssDNA from pBluescript plasmid using helper phage. > When I prepare ssDNA, there are much genomic DNAs in agarose gel. > Is it right prep. to get ssDNA with genomic DNA? > If it is not, how can I remove genomic DNA from ssDNA? > I use NM109 or XL-1-blue as a host cell. > You should not have such genomic DNA in your preps! The way you can make pure ssDNA is to precipitate twice with PEG-NaCl or precipitate once with PEG-NaCl and once with acetic acid. You should then obtain pure phage that you can than use for your ssDNA extractions. Before extracting ssDNA, you can check the purity of your phage (ie the absence of genomic DNA) by scanning your prep in a spectro; DO max shoul be at 269nm and you should not see any secondary peak at 280nm. Hope this helps Pascal From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!pathology.unimelb.edu.au!r.cappai From: r.cappai@pathology.unimelb.edu.au (Roberto Cappai) Newsgroups: bionet.molbio.methds-reagnts Subject: QIAGEN refuses to tell Date: 3 Sep 1996 00:19:50 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 50 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net In article , michelle@MOLECULE.BIO.UTS.EDU.AU (Michelle Gleeson) wrote: > On Mon, 2 Sep 1996, Joe Boutell wrote: > > > > > Well i don't know how old the kits you're using are, but i have in front > > of me a QIAGEN plasmid handbook (Summer 1993 edition), which on page 23 > > contains Appendix A:QIAGEN buffers listing all the ingredients, and even > > a little section on how to prepare 1 litre of each. > > > > CUT > > > Well, Joe, if you have the recipes, please post them for the benefit of > everyone. They certainly aren't given in any Qiagen protocol booklet > I've seen here in Australia. The recipes will then be archived in this > newsgroup and hopefully we'll never see this thread again :-) > > eagerly awaiting your post, > > Michelle Dear Michelle, I have in front of me the February 1995 QIAGEN plasmid handbook that I got with our latest kit from BRESATEC (Australian distributor for overseas readers). On page 30 (Appendix A) are the buffer compositions and preparation guidelines. In some kits we get they include the Summer 1993 Handbook and this info is on page 23 as stated by others. Maybe people in your lab throw out the handbook without realizing its value. I can fax it to you if you have no luck (sorry, but a lot to type in). Regards Roberto Cappai Dept of Pathology University of Melbourne Australia r.cappai@pathology.unimelb.edu.au From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!arclight.uoregon.edu!usenet.eel.ufl.edu!warwick!yama.mcc.ac.uk!loki.cf.ac.uk!news From: Joe Boutell Subject: Re: Qiagen refuses to tell! Sender: news@cf.ac.uk (Usenet News user) Message-ID: Date: Tue, 3 Sep 1996 10:10:15 GMT X-Nntp-Posting-Host: d023.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii References: <1.5.4.32.19960830122604.006d344c@pop.cwru.edu> <507d0t$2pn@service3.uky.edu> <50ers2$mu@whitbeck.ncl.ac.uk> Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics, Cardiff Lines: 57 OK, here goes, hope this puts an end to it all.... QIAGEN buffers. Buffer P1 (Resuspension buffer): 100microg/ml RNase A, 50mM Tris/HCl, 10mM EDTA, pH 8.0. Storage 4 C. Buffer P2 (Lysis buffer): 200mM NaOH, 1% SDS. Storage room temp. Buffer P3 (Neutralization buffer): 3.0M KAc, pH 5.5. Storage 4 C. Buffer QBT (Equilibration buffer): 750mM NaCl, 50mM MOPS, 15% ethanol, pH 7.0, 0.15% Triton X-100. Storage room temp. Buffer QC (Wash buffer): 1.0M NaCl, 50mM MOPS, 15% ethanol, pH7.0. Storage room temp. Buffer QF (Elution buffer): 1.25M NaCl, 50mM Tris/HCl, 15% ethanol, pH 8.5. Storage room temp. TE: 10mM Tris/HCl, 1mM EDTA, pH 8.0. Storage room temp. STE: 100mM NaCl, 10mM Tris/HCl, 1mM EDTA, pH8.0. Storage room temp. Preparation of buffers (per litre): P1 Dissolve 6.055g Tris base, 3.722g EDTA.2H20 in 800 ml H2O. Adjust pH to 8.0 with HCl. Allow the solution to cool to room temp before making final pH adjustments. Adjust volume to 1 litre with H2O. P2 Dissolve 8.0g NaOH pellets in 950 ml H2O, 50ml 20% SDS solution. P3 Dissolve 294.45g KAc in 500ml H2O. Adjust pH to 5.5 with glacial acetic acid (approx 110ml). Adjust volume to 1 litre with H2O. QBT Dissolve 43.83g NaCl, 10.46g MOPS (free acid) in 800ml H2O. Adjust pH to 7.0. Add 150ml pure ethanol and 15ml 10% triton X-100 solution. Adjust volume to 1 litre. QC Dissolve 58.44g NaCl and 10,46g MOPS (free acid) in 800ml H2O. Adjust the pH to 7.0. Add 150ml pure ethanol.Adjust the volume to 1 litre. QF Dissolve 73.05g NaCl and 6.055g Tris base in 800 ml H2O. After addition of 150 ml pure ethanol adjust the pHto 8.5 with HCl. Allow the solution to cool to room temp before making final pH adjustments, Adjust volume to 1 litre. STE Dissolve 5.844g NaCl, 1.211g Tris base and 372.2mg of EDTA.2H2O in 800ml H2O. Adjust ph to 8.0 with HCl. Adjust volume to 1 litre with H2O. That's it, as i said it can be found near the back of any QIAGEN plasmid handbook. Cheers, Joe Boutell. From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!newsfeed.internetmci.com!in3.uu.net!news00.sunet.se!sunic!news99.sunet.se!news.uni-c.dk!news.daimi.aau.dk!biobase!wind From: wind@biobase.dk (Troels Wind) Newsgroups: bionet.molbio.methds-reagnts Subject: Ni-chelation and Mg-binding Date: 30 Aug 1996 13:27:58 GMT Organization: The Danish BioBase Lines: 13 Message-ID: <506q8u$ku5@bioalp.biobase.dk> NNTP-Posting-Host: biobase.dk X-Newsreader: TIN [version 1.2 PL2] Hi all, I have a question regarding Ni-chromatography of His-tagged proteins. What if your protein has a Mg-binding site, wont it stick via that as well, i.e. wont the Ni ions on the column be able to replace Mg in the site? Just wondering... Cheers, Troels From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!tank.news.pipex.net!pipex!dish.news.pipex.net!pipex!lade.news.pipex.net!pipex!ggr.co.uk!usenet From: Fraser Dr N J Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen: This is ridiculous Date: 3 Sep 1996 08:22:45 GMT Organization: Glaxo Wellcome Lines: 21 Message-ID: <50gpsl$iu9@ukwsv3.ggr.co.uk> References: <1.5.4.32.19960830210110.006c724c@pop.cwru.edu> <322B8BAF.376E@unity.ncsu.edu> <50g8g6$p63@panix.com> NNTP-Posting-Host: ukwbf56.ggr.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1IS (X11; I; IRIX 5.3 IP22) X-URL: news:50g8g6$p63@panix.com iayork@panix.com (Ian A. York) wrote: >I've run into one or two cases where >Qiagen DNA gave good results but the DNA from another company gave a lot >of dead cells; but I've also used DNA from The Other Company's kits in the >same transfection protocol successfully. >if I was just subcloning or putting it into yeast, I'd stick with >The Other Company entirely, because it's cheaper and gives more DNA more >consistently. > >I should note that there are a handful of restriction enzymes which are >very picky and which might work better given Qiagen-quality DNA. For my >part, I've had no problems with DNA from The Other Company's kit > ... and the other company was ???? (It might be useful to know !) - Neil From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!in3.uu.net!mcsun!EU.net!Belgium.EU.net!news From: r.carreer@eurogentec.be (carreer) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Electroporation condition for Jurkat cells Date: Tue, 03 Sep 1996 09:38:15 GMT Organization: EUnet Belgium, Leuven, Belgium Lines: 40 Message-ID: <50gn2k$dhb@news.Belgium.EU.net> References: NNTP-Posting-Host: 195.0.8.1 X-Newsreader: Forte Free Agent 1.0.82 Conditions : 5 million exponential growing cells spin down resuspend in 250 microliter of culture medium (not DMEM) RPMI is okay with 10% FCS place cells in cuvette of 4 mm Keep on ice add 20 microgramme of plasmid Perform Electroporation 250 Volt Capicity : 1200 microFarad shunt : infinite half time : about 45 to 55 mseconds Place directly electroporated cells in the corresponding culture medium at 37°C vnp@mole.bio.cam.ac.uk (Venkat Pisupati) wrote: >hi! >is anyone out there tried electroporating Jurkat cells? >If so, can you post the electroporation conditions please. >cheers >venkat >-- >Venkat Pisupati Lab -44-01223-334131 >Wellcome/CRC Institute Fax -44-01223-334134 >Dept. of Genetics E.mail. vnp@mole.bio.cam.ac.uk >University of Cambridge >UK CB2 1QR From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!hri.ac.uk!trevor.fenning From: trevor.fenning@hri.ac.uk (trevor fenning) Newsgroups: bionet.molbio.methds-reagnts Subject: Qaigen + kits etc Date: 3 Sep 1996 06:19:53 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <9608038417.AA841785024@HRI.BBSRC.AC.UK> NNTP-Posting-Host: net.bio.net Well folks, we find the Qaigen kits usually work (and with Agrobacterium, which most don't), but if all you want is plasmid miniprep DNA from E. coli why not just do rapid boils on them ? - Holmes and Quigley (1981), or find the method in Sambrook. The only catch is the method does not work well with E. coli HB101, but is fine with DH5a lines. They honestly don't take much longer, usually work and cost next to nothing. I have even used them for sequencing without problem, SO, the take home message is only use those blasted kits for things that really need them ! Trevor Fenning, HRI Wellesbourne, Warwick, UK. PS Why don't Qaigen answer all these queries themselves ? Don't they read what everyone is saying about them on the Bionet ?! From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!howland.erols.net!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!usenet From: Keld Sorensen Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen refuses to tell! Date: Tue, 03 Sep 1996 08:25:50 +0000 Organization: University of Illinois, College of Medicine Lines: 17 Message-ID: <322BEB8E.4E84@uic.edu> References: <4vv393$sn@merkurius.lu.se> <507cg7$2ku@service3.uky.edu> NNTP-Posting-Host: sliprock-03.dialin.uic.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Macintosh; I; PPC) To: "Michael W. Thompson" [snip - some whining about students using kits and not knowing chemistry] - I seem to remember the same whining when calculators where first used!! - I guess I just gave my age away, but WHY is it important to mix your own reagents ? - What IS important is that you understand what the hell you are doing - Example: I purchase my coffee already roasted and ground and at times even brewed. Would I be a better coffee drinker if I roasted my own beans, ground them and then brewed my coffee?? I would say NO not necessarily - I can go to Starbucks and get much better coffee, because they spend the time to do everything just right! And yes - I know I do pay for that convenience, but it does not make me an inferior coffee drinker / hope I did not start the kit-wars again / Keld. From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!spool.mu.edu!uwm.edu!news.cse.psu.edu!news.cc.swarthmore.edu!netnews.upenn.edu!news.tju.edu!norton1.jmc.tju.edu!user From: pnorton@lac.jci.tju.edu (Pamela Norton) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen refuses to tell! Date: Tue, 03 Sep 1996 17:35:43 -0500 Organization: Thomas Jefferson University Lines: 45 Message-ID: References: <1.5.4.32.19960830122604.006d344c@pop.cwru.edu> <507d0t$2pn@service3.uky.edu> <50ers2$mu@whitbeck.ncl.ac.uk> NNTP-Posting-Host: norton1.jmc.tju.edu X-Newsreader: Yet Another NewsWatcher 2.0.1 In article <50ers2$mu@whitbeck.ncl.ac.uk>, "D.D. Morgan" wrote: > Joe Boutell (boutell@cf.ac.uk) wrote > > > Well i don't know how old the kits you're using are, but i have in front > > of me a QIAGEN plasmid handbook (Summer 1993 edition), which on page 23 > > contains Appendix A:QIAGEN buffers listing all the ingredients, and even > > a little section on how to prepare 1 litre of each. > > > I wholeheartedly agree that putting this information at the back of the > > booklet is totally out of order, i've strained my pipetting thumb on many > > an occasion flicking those last few pages.... ... > > Hey Joe > How about posting the recipes so that us younger members of the > audience who havn't been using Qiagen stuff as long can make up the > buffers.... This is directed at those who are complaining that Qiagen doesn't reveal the composition of their solutions: What are you guys talking about? Have you LOOKED at the manual? I knew that Joe was absolutely correct in his reply above, because we have indeed prepared our own replacement buffers, but thought that perhaps the information had been removed from more recent manuals. In our new kit (midi size), the handbook is dated Feb 1995,and has the words "New Edition" in white in a red field on the cover. On p 30 appears: Appendix A: Composition of Buffers. Table indicates composition and appropriate storage conditions. Below, Preparation of Buffers. Specific instructions for the preparation of 1 l of each. What more do you want? No affiliation with Qiagen, but they have done the right thing, don't give them grief unnecessarilty. Pam Norton -- Pamela A. Norton, Ph.D. Assistant Professor of Medicine Thomas Jefferson University Philadelphia, PA 19107 p_norton@lac.jci.tju.edu From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!mcsun!EU.net!main.Germany.EU.net!fu-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!news From: diabetes@lrz.uni-muenchen.de (Wolfgang Schechinger) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Detergent for Tissue Culture glassware Date: Tue, 03 Sep 1996 21:36:28 GMT Organization: Institute for Diabetes Research Lines: 10 Distribution: world Message-ID: <50i8dt$8se@sparcserver.lrz-muenchen.de> References: <4vqj2r$3354@news.doit.wisc.edu> Reply-To: diabetes@lrz.uni-muenchen.de NNTP-Posting-Host: dial154.ppp.lrz-muenchen.de X-Newsreader: Forte Free Agent 1.0.82 What are you afraid for that you must use chromic acid? We have *best* experince made in our lab with the ordinary procedure: We collect all our glass and plasticware we want to re-use to be cleaned by a dishwasher (using a sort of hypochlorite/hydroxide containing liquid). Glassware is strerilized then at 150C, plastics are with gas (I assume ethylene oxide). We don't re-use culture plates but pipettes and so. Leave the chrome in its bottle! Wolfgang From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!bunyip.cc.uq.oz.au!marlin.jcu.edu.au!news From: Lynn Hughes Subject: E.coli rare codons Content-Type: text/plain; charset=us-ascii Message-ID: <322E1166.3687@jcu.edu.au> Sender: news@marlin.jcu.edu.au (USENET News System) Content-Transfer-Encoding: 7bit Organization: James Cook University Mime-Version: 1.0 Date: Wed, 04 Sep 1996 16:31:50 -0700 X-Mailer: Mozilla 2.01 (Win16; I) Lines: 10 Hi all, I am attempting to clone my DNA fragment into an E.coli expression system. My DNA contains quite a high percentage of 'E.coli rare codons'. Will this lead to low expression levels of my protein, if so any ideas how I can get around this problem, for example any special feature I should look for in the expression system I choose. Thanks for your help Lynn Hughes From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!spool.mu.edu!newspump.sol.net!www.nntp.primenet.com!nntp.primenet.com!news.sgi.com!esiee.fr!jussieu.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!swsbe6.switch.ch!news.belnet.be!news.fundp.ac.be!immuno-wawa.sciences.fundp.ac.be!user From: Pascal.Mertens@fundp.ac.be (Pascal Mertens) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Westerns-blotting-BSA Date: 4 Sep 1996 05:44:07 GMT Organization: Immunologie Microbiologie, FUNDP Namur Lines: 25 Message-ID: References: NNTP-Posting-Host: immuno-wawa.sciences.fundp.ac.be In article (Dans l'article) , Steve wrote (écrivait) : > Hey all! > > I'm doing a normal western blot, but our polyclonal antibodies seem to be > binding to many of the E. Coli proteins. It is possible that the > antibodies were raised to our protein, as well as any bacterial proteins > present in the blood during an infection in the rabbit. > > Is there any protocol to add cell extract to the BSA blocking buffer, or > the primary antibodies, which will prevent all this binding? > > Or is there a better way to block? > > Thanks. > > STEVE You can try this latter one: "Alternative method to remove antibacterial antibodies from antisera used for screening of expression libraries" in: Biotechniques 19:28-30 (1995) Pascal From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!news.Stanford.EDU!nntp-hub2.barrnet.net!cam-news-hub1.bbnplanet.com!www.nntp.primenet.com!nntp.primenet.com!news.sgi.com!news-out.microserve.net!news-in.microserve.net!news.sprintlink.net!news-dc-2.sprintlink.net!rebecca!newserve!news.cc.geneseo.edu!uno.cc.geneseo.edu!KXP97 From: kxp97@uno.cc.geneseo.edu Newsgroups: bionet.molbio.methds-reagnts Subject: biological journals Date: 3 Sep 1996 18:48:24 GMT Organization: SUNY Geneseo, Geneseo, NY 14454 Lines: 9 Message-ID: <50huho$be@news.cc.geneseo.edu> Reply-To: kxp97@uno.cc.geneseo.edu NNTP-Posting-Host: uno.cc.geneseo.edu Senior Seminar, My journal is the Journal of Molecular Biology. It is a primary journal. It has alot of really interesting articles in the last edition. It is also in the Miline library, but can also be read on the www (address: URL:http://www.hbuk.co.uk/jmb). Most of the articles in this edition dealt with TF's there were also a few dealing with NMR studies of domains and the secondary structure of proteins using the known info. of the tertiary structure. Katie Petko From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!newsgate.duke.edu!solaris.cc.vt.edu!homer.alpha.net!uwm.edu!chi-news.cic.net!newspump.sol.net!www.nntp.primenet.com!nntp.primenet.com!mr.net!news-out.microserve.net!news-in.microserve.net!news.sprintlink.net!news-dc-2.sprintlink.net!rebecca!newserve!news.cc.geneseo.edu!uno.cc.geneseo.edu!KXP97 From: kxp97@uno.cc.geneseo.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Senior Seminar Library Info Date: 3 Sep 1996 19:03:53 GMT Organization: SUNY Geneseo, Geneseo, NY 14454 Lines: 6 Message-ID: <50hvep$mn@news.cc.geneseo.edu> Reply-To: kxp97@uno.cc.geneseo.edu NNTP-Posting-Host: uno.cc.geneseo.edu Senior Seminar, My journal is the Journal of Molecular Biology. It is a primary journal and can be found in Milne and on www. It contains alot of interesting articles concerning TF's and the structure of specific protein domains. Katie Petko From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!newspump.sol.net!spool.mu.edu!torn!resunix.sickkids.on.ca!news From: Marc Visconti Newsgroups: bionet.molbio.methds-reagnts Subject: scintillation counter Date: Tue, 03 Sep 1996 13:56:08 -0400 Organization: The Hospital For Sick Children Lines: 8 Message-ID: <322C7138.24CC@resunix.ri.sickkids.on.ca> NNTP-Posting-Host: 142.20.6.168 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Macintosh; I; 68K) Can someone please direct me to a scintillation counter made by either LKB or Packard that is able to count microplates instantaneously? Contacts in the US or Canada and approximate cost would be most beneficial. Thank you in advance. Marc Visconti The Hospital for Sick Children Toronto, Canada From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!PUBLIC.CC.JL.CN!ouyhs From: ouyhs@PUBLIC.CC.JL.CN Newsgroups: bionet.molbio.methds-reagnts Subject: plasmid p205 Date: 3 Sep 1996 21:56:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609040356.MAA07626@public.cc.jl.cn> NNTP-Posting-Host: net.bio.net Does anybody know where it is possible to buy plasmid p205? my email is : ouyhs@public.cc.jl.cn From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!nature.berkeley.edu!lhom From: lhom@nature.berkeley.edu (Louis Hom) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: ECL Western troubles Date: 4 Sep 1996 04:56:34 GMT Organization: University of California, Berkeley Lines: 11 Distribution: na Message-ID: <50j262$iaj@agate.berkeley.edu> References: <50fpck$f9n@dismay.ucs.indiana.edu> NNTP-Posting-Host: nature.berkeley.edu As with most other folks, I have a strong preference for Pierce's chemiluminescent HRP substrate. One source of variation that I found with DuPont's kit (which I started with) was how wet I had the membrane before wrapping it in cellophane. I usually left a fair amount of substrate on the membrane (drippy to drip-drippy). Of course it can increase your background, but usually it's not too bad. -- _______________________________________________________________________________ Lou Hom >K '93 "Putney is confusing obscenity lhom@nature.berkeley.edu with originality." http://www.ocf.berkeley.edu/~lhom -- Myron X in "Putney Swope" From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!ames!enews.sgi.com!news.uoregon.edu!neonlights.uoregon.edu!cyberbro.uoregon.edu!user From: meneghini@molbio.uoregon.edu (Marc Meneghini) Newsgroups: bionet.molbio.methds-reagnts Subject: northwesterns Date: Tue, 03 Sep 1996 21:34:30 -0800 Organization: University of Oregon Lines: 7 Message-ID: NNTP-Posting-Host: cyberbro.uoregon.edu Does anybody have a good protocol for northwesterns? If so could you direct me to it, post it or email it to me?? thanks Marc Meneghini Institute of Molecular Biology University of Oregon Eugene OR, 97402 From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!aol.com!WSchick From: WSchick@aol.com Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Deep well slides - looking for a supplier Date: 3 Sep 1996 21:09:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <960903235052_515482901@emout17.mail.aol.com> NNTP-Posting-Host: net.bio.net In a message dated 96-09-03 12:44:23 EDT, das1002@cam.ac.uk (David) writes: << I would be very grateful if anyone could suggest a supplier for deep-well slides. >> These should be available from Nalge/Nunc distributors. These are the LabTek tissue culture chamber slide system shown on page 1872 of the Fisher catalog. Both plastic and glass slides are available with 8 wells of 400ul and 4 wels of 800ul. If you want 48 wells in a microtiter format, Polyfiltronics has PS, PP and Teflon plates, culture treated and plain sterilized. Some labs use them to store tissue slices in a library. Try Whatman in the UK--they should be distributing these. Walt Schick From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!aol.com!WSchick From: WSchick@aol.com Newsgroups: bionet.molbio.methds-reagnts Subject: QPCR-Analyse DNA Melting Point Curves? Date: 3 Sep 1996 20:48:29 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <960903232122_193855545@emout15.mail.aol.com> NNTP-Posting-Host: net.bio.net The new QPCR Lite cycler with real-time monitoring can produce DNA product melting point curves for each sample. For simple (short length) products, the curve has one inflection point; for longer products, multiple melting point domains characteristic of the product appear on the curve. These curves could identify a product without complex probe chemistry. As the raw data is available from the instrument, does anyone know of software that can analyse (maybe compare to a library) complex curve patterns? Maybe IR, HPLC, electrochemical, ?? software has functions that could be adapted for this? I don't want to reinvent algorithms or software that could be modified to identify PCR products by their characteristic melting point curves. If you can point me in the right direction, thanks a whole bunch! Walt Schick From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!U.ARIZONA.EDU!jim From: jim@U.ARIZONA.EDU (Jin-seon Im) Newsgroups: bionet.molbio.methds-reagnts Subject: re: concentrating protein already in SDS sample buffer Date: 3 Sep 1996 20:28:47 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <199609040329.UAA74527@aruba.ccit.arizona.edu> NNTP-Posting-Host: net.bio.net you wrote : We used to concentrate solutions by loading them into dialysis tubing and burying them under dry Sephadex for an empirically-determined period of time. It seems to me that the salts would migrate from the dialysis tubing along with the water. but how do you assume the salts would migrate from the dialysis tubing along with the water ? and is that reducing SDS sample buffer?? Thanks in advance jsim From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.unimelb.EDU.AU!news From: "Sandra Verschoor" Newsgroups: bionet.molbio.methds-reagnts Subject: Testing Date: Wed, 4 Sep 1996 09:51:42 +1000 Organization: Peter MacCallum Cancer Institute Lines: 1 Message-ID: <01bb99f2.db5a8aa0$9da404cb@mckay1.petermac.unimelb.edua.u> NNTP-Posting-Host: 203.4.164.157 X-Newsreader: Microsoft Internet News 4.70.1080 Test From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!UC.EDU!Richard.Converse From: Richard.Converse@UC.EDU (Richard Converse) Newsgroups: bionet.molbio.methds-reagnts Subject: removing mineral deposits from water baths? Date: 3 Sep 1996 16:41:33 -0700 Organization: University of Cincinnati Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <322C89B2.619B@ucbeh.san.uc.edu> NNTP-Posting-Host: net.bio.net Hello everyone! Does anyone have a good method to remove mineral deposits (from tap water) from a stainless steel water bath? (Fisher)--please e-mail any reponses! Thanx, Richard-- From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!spool.mu.edu!newspump.sol.net!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!newsfeed.internetmci.com!netnews.nwnet.net!news.u.washington.edu!NewsWatcher!user From: ziping@u.washington.edu (Ziping Yang) Newsgroups: bionet.molbio.methds-reagnts Subject: Denaturing dsDNA in formaldehyde-agarose gel Date: Tue, 03 Sep 1996 16:38:48 -0700 Organization: University of Washington Lines: 6 Message-ID: NNTP-Posting-Host: 128.95.185.233 Hi: Does anybody out there know that do I need to denature double stranded DNA fragment by boiling before loading onto 1% formaldehyde-agarose gel. I want to use the dsDNA as probe control. I'm not quite sure the dsDNA, like RNA, is also denatureed in formaldehyde buffer and gel? Thank. email me better. From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news.sgi.com!enews.sgi.com!decwrl!tribune.usask.ca!rover.ucs.ualberta.ca!elegans.biology.ualberta.ca!user From: Morris_maduro@biology.ualberta.ca (Morris Maduro) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: E. coli codon usage table Followup-To: bionet.molbio.methds-reagnts Date: 3 Sep 1996 23:11:36 GMT Organization: Genetics U of Alberta Lines: 8 Message-ID: References: NNTP-Posting-Host: elegans.biology.ualberta.ca Hello again, OK, sorry. For the codon usage table to work you must 'strip off' the extra information added by your newsgroup browser, up until to the " E. coli Codon Usage

Codon Usage in E. coli
from a reference by HÉNAUT and DANCHIN

Amino
Acid		 Codon Class Amino
Acid		 Codon Class
IIIIII IIIIII
Phettt 55.0929.0867.14 Leuctt 9.705.5619.00
ttc 44.9170.9232.86 ctc 10.408.039.04
Leutta 10.993.4420.09 cta 3.090.836.81
ttg 13.025.4715.05 ctg 52.7976.6729.99
Sertct 13.2632.4119.63 Procct 13.7111.2328.30
tcc 15.0226.5611.34 ccc 11.191.6316.26
tca 10.834.7922.09 cca 18.6315.2531.50
tcg 16.887.3910.60 ccg 56.4771.8923.94
Tyrtat 54.4235.2369.60 Hiscat 56.8029.7761.69
tac 45.5864.7730.40 cac 43.2070.2338.31
TERtaa     Glncaa 33.4018.6537.06
tag     cag 66.6081.3562.94
Cystgt 40.9038.8555.71 Argcgt 38.9964.2526.05
tgc 59.1061.1544.29 cgc 42.2332.9721.94
TERtga     cga 5.521.0712.80
Trptgg 100.00100.00100.00 cgg 8.970.8013.62
Ileatt 51.2033.4947.57 Valgtt 23.7439.7734.33
atc 44.3765.9426.65 gtc 22.4813.4518.95
ata 4.430.5725.78 gta 14.8619.9721.78
Metatg 100.00100.00100.00 gtg 38.9226.8124.94
Thract 14.8529.0826.83 Alagct 14.5227.5422.86
acc 46.8353.6024.45 gcc 27.6216.1423.67
aca 10.524.6727.93 gca 19.6324.0131.27
acg 27.8112.6520.80 gcg 38.2332.3022.19
Asnaat 40.8717.2564.06 Aspgat 62.8346.0570.47
aac 59.1382.7535.94 gac 37.1753.9529.53
Lysaaa 75.4478.5572.21 Glugaa 68.3375.3566.25
aag 24.5621.4527.79 gag 31.6724.6533.75
Seragt 13.964.5218.73 Glyggt 32.9150.8431.79
agc 30.0424.3317.61 ggc 43.1742.8324.51
Argaga 1.750.6215.63 gga 9.191.9724.75
agg 1.540.299.96 ggg 14.744.3618.95


Genes are clustered by using factorial correspondence analysis into three classes. Class I contains genes involved in most metabolic processes. Class II genes correspond to genes highly and continuously expressed during exponential growth. Class III genes are implicated in horizontal transfer of DNA. One can see that the distribution of codons in class III genes is more or less even, whereas it is exteremely biased in class II genes (in particular, codons terminated in A are selected against).
M. Maduro
From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!MORPHEUS.WUSTL.EDU!gerry From: gerry@MORPHEUS.WUSTL.EDU (Gerald B. Downes) Newsgroups: bionet.molbio.methds-reagnts Subject: (none) Date: 3 Sep 1996 16:08:39 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >This is directed at those who are complaining that Qiagen doesn't >reveal the composition of their solutions: What are you guys talking >about? Have you LOOKED at the manual? As Qiagen has pointed out they have only provided the composition of their solutions with the plasmid prep kits- the Qiaex and Qiaquick kits are a different story. You could argue for days whether it is right or wrong for Qiagen to patent some presumably "common" parts of their kit but that's the way it stands. If that displeases you then don't use the kit. ----------------------------------------- Gerald B. Downes, Gautam Lab NIGMS Pre-doctoral Fellow Neuroscience Program Washington University Medical School Box 8054, St. Louis MO. 63110 Ph. (314)362-8526 Fax (314)362-8571 From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!spool.mu.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@facstaff.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts Subject: re: Ni-chelation and Mg-binding Date: 3 Sep 1996 17:30:52 GMT Organization: UW-Madison Lines: 28 Message-ID: <50hq0c$p9e@news.doit.wisc.edu> References: <322C5FF7.43D@visar.wustl.edu> NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 X-No-Archive: Yes In article <322C5FF7.43D@visar.wustl.edu>, nikolaic@VISAR.WUSTL.EDU (Nikolai Chitaev) wrote: ->> Hi all, ->> ->> I have a question regarding Ni-chromatography of His-tagged proteins. ->> ->> What if your protein has a Mg-binding site, wont it stick via that ->> as well, i.e. wont the Ni ions on the column be able to replace ->> Mg in the site? ->> ->> Just wondering... ->> ->> Cheers, ->> Troels Shouldn't. Mg is way smaller and isn't transition metal. -> ->One of the most abundant contominating proteins found on Ni-NTA ->chromatography was identified as SOD (superoxide dismutase), which binds ->Mg (?). -> Probably has a lot of clustered aromatic residues exposed. - Dima From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!agate!news.ucdavis.edu!info.ucla.edu!nnrp.info.ucla.edu!csulb.edu!news.sgi.com!swrinde!cs.utexas.edu!howland.erols.net!newsfeed.internetmci.com!in3.uu.net!mcsun!EU.net!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!uknet!lyra.csx.cam.ac.uk!news.ox.ac.uk!oxpath!sbutcher From: sbutcher@molbiol.ox.ac.uk Newsgroups: bionet.molbio.methds-reagnts Subject: looking for Cuno Inc. Meriden Date: 3 Sep 96 18:15:15 BST Organization: Oxford University Molecular Biology Data Centre Lines: 15 Message-ID: <1996Sep3.181515@molbiol.ox.ac.uk> NNTP-Posting-Host: ania.path.ox.ac.uk Dear all, I am looking for a way of contacting Cuno Inc. Meriden Conneticut. I am trying to locate a source of their positively charged filter membranes in the UK - Zetapore AMF and Virosorb 1MDS - to be exact. I have tried catalog searching, and a quick web company search without any success. Any suggestions gratefully received Sarah ------------------------------------------------------------------------------- Dr Sarah Butcher sbutcher@worf.molbiol.ox.ac.uk NERC IVEM Oxford instant .sig file - just add beer ------------------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!newsfeed.internetmci.com!in3.uu.net!news.u.washington.edu!root From: Tori Brophy Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Yeast contamination on cell culture:HELP!! Date: 3 Sep 1996 21:25:10 GMT Organization: University of Washington Lines: 25 Message-ID: <50i7nm$jt7@nntp3.u.washington.edu> References: <1996Aug26.160056.9676@opalo.etsiig.uniovi.es> <322407CB.55D@pen.gulbenkian.pt> <322b802e.3594072@155.212.1.8> NNTP-Posting-Host: 128.95.12.81 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) To: rand777@ids.net snip >>> I`m working on culture of human cell lines and I have some troubles >>> with yeast contamination. snip >. >I would suggest you try Anisomycin...it is often used as a selective >inhibitor in culture media to prevent the growth of yeast. RR >Robert Randolph >Randolph Biomedical >401-826-1407 >rrandy@hotmail.com How much anisomycin? I've used anisomycin at 10 ug/ml on my murine B cells to activate the MAPK p38. p38 activity is implicated in inducing apoptosis. What concentration of anisomycin is used to kill yeast and do you know that the cells are not activated? Tori Brophy Dept. of Genetics University of Washington Seattle, WA From owner-methds-reagnts@net.bio.net Mon Sep 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!mcsun!EU.net!enews.sgi.com!news.uoregon.edu!kaiwan.kaiwan.com!kaiwan212.kaiwan.com!user From: qiagen@kaiwan.com (QIAGEN) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Qiagen refuses to tell! Date: Tue, 03 Sep 1996 13:52:18 +0100 Organization: QIAGEN, Inc Lines: 52 Distribution: world Message-ID: References: <4vv393$sn@merkurius.lu.se> NNTP-Posting-Host: kaiwan212.kaiwan.com sbc4@PO.CWRU.EDU (Steven Cohen) and drm21@mole.bio.cam.ac.uk (David Micklem) wrote: >>It always surprises me when people think that companies will/should give away thief intellectual property. If they spent the time(and Money) developing a product the secret belongs to them. If that bothers you (and it does me at times too) than don't use kits. Or figure out what's in them by using the literature or find the next bes