From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!agate!spool.mu.edu!howland.reston.ans.net!math.ohio-state.edu!uwm.edu!fnnews.fnal.gov!nntp-server.caltech.edu!nntp-server.caltech.edu!anita From: anita@accord.cco.caltech.edu (Anita Gould) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Mitochondrial DNA extraction? Date: 02 Mar 1996 05:31:34 GMT Organization: California Institute of Technology CCO Unix cluster Lines: 23 Message-ID: References: <4g0cvp$2pb@post.its.mcw.edu> NNTP-Posting-Host: accord.cco.caltech.edu In-reply-to: kpleyte@post.its.mcw.edu's message of 15 Feb 1996 16:44:09 -0600 From: kpleyte@post.its.mcw.edu (Kay A. Pleyte) Newsgroups: bionet.molbio.methds-reagnts Date: 15 Feb 1996 16:44:09 -0600 Organization: Medical College of Wisconsin; Milwaukee Wisconsin I'm looking for a method for extraction of DNA from mitochondria. Will a plasmid prep. protocol work? Any suggestions welcome. Kay Pleyte kpleyte@post.its.mcw.edu Yes, I've done this on mammalian cells or homogenized tissue (using resin-based plasmid preps, either Promega or roll-your-own Celite/silica gel), and it works just fine. I actually use it for isolating extrachromosomal circular DNA, but the mitochondrial DNA (which is much more plentiful) comes along for the ride. It may be that you'd get better yield or quality by first doing a mitochondrial isolation (as Martin Leach suggested) -- I can't speak to that. Good luck! -Anita Gould anita@cco.caltech.edu From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!eworld.com!Microplate From: Microplate@eworld.com Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Dye and terminator chemistry Date: 2 Mar 1996 07:40:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <960302073739_26453195@hp1.online.apple.com> NNTP-Posting-Host: net.bio.net Suggest you call John Seed at AGTC 1800 323 2685. He probaly has a lot of refrences. From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!chi-news.cic.net!nntp.coast.net!swidir.switch.ch!swsbe6.switch.ch!scsing.switch.ch!elna.ethz.ch!retrograde.ethz.ch!user From: mantei@neuro.biol.ethz.ch (Ned Mantei) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: nicking DNA plasmids on purpose Date: Sat, 02 Mar 1996 15:45:06 +0100 Organization: Dept. of Neurobiology, ETH-Zurich Lines: 21 Message-ID: References: <4h66p1$ht2@agate.berkeley.edu> <31374522.58D1@simmons.swmed.edu> NNTP-Posting-Host: retrograde.ethz.ch In article <31374522.58D1@simmons.swmed.edu>, walberg@simmons.swmed.edu wrote: > Sorry I missed the beginning of this, but if you want to isolate open > circles (form II) then there are some published methods to do this. > An interesting one was published in NAR in 1975 or 1976 or so > by Phil Martens and David Clayton. They nicked circular DNA in the > presence of ethidium using a strong visible light- a slide projector I > think. I think they got it to work with very good efficiency with > very little form III. The procedure was more or less to have DNA plus fairly concentrated EtBr (perhaps 10--50 ug/ml?) in a cuvette. Illuminate with a slide projector about 10 cm away, meanwhile cooling with a fan or hairdrier. Times to get nicked circles were in the range of minutes. The method was around at least a year or two before 1975. -- Ned Mantei Dept. of Neurobiology, Swiss Federal Institute of Technology CH-8093 Zurich, Switzerland mantei@neuro.biol.ethz.ch Fax: +41-1-633-1046 From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!SFSU.EDU!jmorlan From: jmorlan@SFSU.EDU (John Morlan) Newsgroups: bionet.molbio.methds-reagnts Subject: Northern controls Date: 2 Mar 1996 13:15:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hello: I am looking for information on a good internal control for studying mRNA expression in rice (Oryza sativa). We have heard that EF1a, actin 1, a member of the acitn gene family in rice, or 17S rRNA might be good candidates. Does anyone have experience with these or other genes as controls in studies of rice gene expression? Thanks, John Morlan SFSU From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!news-feed.iguide.com!uunet!in2.uu.net!pravda.aa.msen.com!news.eecs.umich.edu!umn.edu!newsstand.tc.umn.edu!biosci!wcheung From: wcheung@biosci.cbs.umn.edu (Gabriel Cheung) Newsgroups: bionet.molbio.methds-reagnts Subject: In situ hybridization Date: 2 Mar 1996 09:23:23 GMT Organization: University of Minnesota Lines: 24 Message-ID: <4h942b$scf@epx.cis.umn.edu> NNTP-Posting-Host: biosci.cbs.umn.edu X-Newsreader: TIN [version 1.2 PL2] Dear everyone, I have been using DIG In Situ Protocol for couple months, however, the results have not been consistant. The background usually was high, also whenever I use RNase A during post hybridization wash, poor results were given. I first thought it was my probe, turned out the probe works fine in a Northern blot. I am really puzzled by all these strange results everytime I changed the conditions. I would love to have some of your expert opinion on what I can do and how I can check my protocol. Thank you very much in advance! Gabe Prince of Wales Hospital Department of Orthopaedics and Traumatology Hong Kong p.s. I am working on two bone tumors and trying to pin point some genes expression. -- == Cheung wan-cheung Gabriel wcheung@biosci.cbs.umn.edu http://biosci.cbs.umn.edu/~wcheung/gabriel.html University of Minnesota Biochemistry From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!ennfs.eas.asu.edu!gatech!newsxfer2.itd.umich.edu!news.itd.umich.edu!pm037-05.dialip.mich.net!user From: davidlee@umich.edu (David S. Lee) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DH5 alpha Date: Sat, 02 Mar 1996 03:03:19 -0500 Organization: University of Michigan Lines: 13 Message-ID: References: <4h7tqh$n31@taco.cc.ncsu.edu> NNTP-Posting-Host: pm037-05.dialip.mich.net X-Newsreader: Yet Another NewsWatcher 2.2.0b4 In article <4h7tqh$n31@taco.cc.ncsu.edu>, chip peele wrote: > Does anyone know if DH5 alpha has the capabilities of natural selection. Don't all cells/organisms have that capability...or did I misunderstand your question? -------------------------------------------------------------------------- David S. Lee (internet: davidlee@umich.edu) Department of Otolaryngology University of Michigan Ann Arbor, MI 48109 http://www-personal.umich.edu/~davidlee/ From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!swen.emba.uvm.edu!emba-news!aquilla From: aquilla@salus.med.uvm.edu (Tracy Aquilla) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: rat cardiomyocyte cell line, who has one? Date: Thu, 29 Feb 96 19:30:39 GMT Organization: EMBA Computer Facility, The University of Vermont Lines: 15 Message-ID: References: <31334DB2.4D3F@physci.ucla.edu> NNTP-Posting-Host: 132.198.80.155 X-Newsreader: VersaTerm Link v1.1 In Article <31334DB2.4D3F@physci.ucla.edu>, Ed Castro wrote: >Hello everyone, >I am wondering whether anyone has information concerning the >availability of a rat cardiomyocyte cell line. > >Any information is greatly appreciated. Thanks in advance, > >Edmundo Since cardiomyocytes are terminally differentiated and non-proliferative, there is no immortal cardiocyte cell line that I'm aware of. That's why we must make primary cultures for our in vitro experiments. If you did happen to produce such a cell line, you could probably make a lot of money! Tracy From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!daresbury!yama.mcc.ac.uk!peer-news.britain.eu.net!tank.news.pipex.net!pipex!newsxfer2.itd.umich.edu!chi-news.cic.net!nntp.coast.net!fu-berlin.de!news.belwue.de!news.uni-stuttgart.de!uni-regensburg.de!lrz-muenchen.de!ipp-garching.mpg.de!usenet From: Gabriele Kerber Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Alkaline Phosphatase substrates Date: 1 Mar 1996 12:49:35 GMT Organization: mpi fuer psychatrie Lines: 8 Message-ID: <4h6rp1$2b1k@sat.ipp-garching.mpg.de> References: NNTP-Posting-Host: 141.61.35.148 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.2N (Windows; I; 32bit) To: botbml@luff.latrobe.edu.au Hi Ben! You can need HNPP/Fast Red TR as a substrate for alkaline phosphatase. There is a kit from Boehringer called HNPP Fluorecent Detection Set. Bye Gabriele From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!nntp.coast.net!fu-berlin.de!news.belwue.de!news.uni-stuttgart.de!news.ruhr-uni-bochum.de!news.rwth-aachen.de!newsserver.rrzn.uni-hannover.de!news From: bmay Newsgroups: bionet.molbio.methds-reagnts Subject: Re: SYBR Green staining of PAGE/urea gels Date: 2 Mar 1996 21:11:19 GMT Organization: RRZN - Newsserver Lines: 18 Message-ID: <4hadhn$8pu@newsserver.rrzn.uni-hannover.de> References: <4gn9jg$2rm@mserv1.dl.ac.uk> NNTP-Posting-Host: h15.ts1.uni-hannover.de I use SybrGreen II for SSCP in neutral 10%PAGE and for denaturing PAGE in 8%PAGE 10M urea. I load 20µl PCR-product on a 0.75 or 1.5 mm Gel. I prepare a 1:10000 dilution of SybrGreenII in 1x TBE (always dark, store @ -20°, only reuse 1-2x) stain for 20 min with out any pretreatment and examine the gel on a usual 320 nm transilluminator. I never failed, but using old SybrGreen gives weak signals. I'd reread the data sheet for hints what could have harmed the dye, for that's what I think is the reason. As a control experiment load 1µg dsDNA whatsoever on a neutral gel and stain. dsDNA is stained weaker but strong enough to see 1µg. Bernhard Mayr Medizinische Hochschule Hannover Dept. Clin. Endocrinology Hannover, Germany From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!cc.usu.edu!nntp Newsgroups: bionet.molbio.methds-reagnts Subject: recombinant thrombin Message-ID: <1996Mar2.134904.75652@cc.usu.edu> From: jmorrey@sleepy.usu.edu (jmorrey) Date: 2 Mar 96 13:49:04 MDT Organization: USU Nntp-Posting-Host: johnpm.biotec.usu.edu X-Posted-From: InterNews 1.1@johnpm.biotec.usu.edu X-Authenticated: jmorrey on VMS host sleepy.usu.edu Lines: 3 Is there a source of recombinant thrombin as compared to thrombin isolated from plasma. I don't want ANY other plasma contaminants, so I need recombinant. Thanks in advance! From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!daresbury!not-for-mail From: Andrei Popov Newsgroups: bionet.molbio.methds-reagnts Subject: RE: purification of 140 kbp from agarose Date: 2 Mar 1996 14:50:21 -0000 Lines: 51 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <4h9n7d$ros@mserv1.dl.ac.uk> Importance: High Sensitivity: Company-Confidential Original-To: methods@dl.ac.uk (IPM Return Requested) : Hi, : lately I have been trying to isolate a pulse-field gel : fragment from agarose. GeneClean didn't work at all so I went on : with an agarase digestion of the agarose followed by a dialysis : over a millipore membrane sitting on a becher full of TE. This : dialysis usually work very well for smaller fragment. But after : this step, I precipitate with 1/10 volume of 3M NaoAc and 3 volume : of cold 95% Ethanol, rinse with 80% ethanol and resuspend in 10 ul : of dH2O, run on a gel and there is nothing. I even hybridised it to : see if there was a trace amount. But there was nothing. I also tried : a phenol extraction with no dialysis followed by the precipitation, : and still nothing. I also tried this thing called GeneCapsule, which : uses electrophoresis to get the DNA out of the agarose and onto a : membrane. But the after an overnight run at 100 volts, the DNA was : still in the plug.?????. So if any of you out there have any idea : please share them with me. : Many thanks : Ghis : P.S. I used low-melt agarose, but my pulse-field gel would be much : nicer if I could use a non-low-melt agarose. Hi Ghis, you described a standard protocol that should work. Here are some suggestions: 1. make plugs with as high DNA concentration as practicaly possible 2. Dr. A.Schedl et. al (see NAR 21:4783) used to run normal preparative gel than another one at 90 degr angle to concentrate DNA in 4% LMA agarose. This gives you 5-fold concentration of your sample. 3. I missed the point of membrane dialysis. Normally people do it when they want to microinject large DNA w/o precipitation 4. If the amount of DNA after agarase digestion is really low try co-precipitation with glycogen 5. If the fragment is a YAC, beware that you co-purify some amount of the 2-mu plasmid (which has multimeric forms)- it can interfere with some applications (probe) 6. check the presense of DNA after agarase- some batches are no good at all, or the buffer might be contaminated with nucleases 7. You did not specify your final goal, but sometimes LMA does not interfere with further enzymatic protocols (Klenow, REs, T4 ligase etc...) Of course there is no need to stain ALL the gel with EthBr- only outer lanes. Having your DNA in agarose even has some advantages- you can change the buffer by washing the block good luck Andrei Popov From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!daresbury!not-for-mail From: Andrei Popov Newsgroups: bionet.molbio.methds-reagnts Subject: RE: SfiI enzyme Date: 2 Mar 1996 13:10:45 -0000 Lines: 15 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <4h9hcl$mol@mserv1.dl.ac.uk> Importance: High Sensitivity: Company-Confidential Original-To: methods@dl.ac.uk (IPM Return Requested) >Has anyone had difficulties ligating PCR products following Sfi1 >digestion? We are using Geneclean to gel purify PCR products following >digestion with various restriction enzymes. All products seem to ligate >efficiently with the exception of those cut with Sfi1. Hi I suggest to have a close look at the SfiI site sequence ggccNNNNNggcc Unless you have indentical NNNNN in your primers and THE vector it will not fit. regards Andrei From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!swrinde!tank.news.pipex.net!pipex!dish.news.pipex.net!pipex!soap.news.pipex.net!pipex!usenet From: Dr G R Taylor Newsgroups: bionet.molbio.methds-reagnts Subject: Re: ***Multiplex PCR Hell****** Date: 2 Mar 1996 23:33:30 GMT Organization: UnipalmPIPEX server (post doesn't reflect views of UnipalmPIPEX) Lines: 8 Message-ID: <4halsa$ods@soap.news.pipex.net> References: <4h75ai$psg@cville-srv.wam.umd.edu> NNTP-Posting-Host: an205.du.pipex.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1 (Windows; I; 16bit) We found that the concentration and purity of the template DNA to be critical for high order multiplex PCR. Good luck Graham Taylor Leeds, UK From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!vixen.cso.uiuc.edu!chi-news.cic.net!news.math.psu.edu!news.iag.net!news From: mannv@ocps.k12.fl.us (JCM) Newsgroups: bionet.molbio.methds-reagnts Subject: Anyone have indepth info on annealing temps and unzipping temp or equation for PCR components? Date: 2 Mar 1996 23:13:11 GMT Organization: Internet Access Group, Orlando, Florida Lines: 6 Message-ID: <4hakm7$nql@news.iag.net> NNTP-Posting-Host: pm4-orl27.iag.net Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.7 I am hoping someone will have info on annealing temp calculations for oligo primers and unzipping temps or equation for calculating time temp happenings in DNA used in PCR and info on Taq. I have not been able to find any indepth research on the topic. Indepth meaning equations or time-temp. relationship info. Any help would be appreciated. From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!lhc.nlm.nih.gov!nih-csl!NewsWatcher!user From: sscherer@helix.nih.gov (Steve Scherer) Subject: Re: Ribonuclease Protection Assay (RPA) Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: 128.231.222.96 Organization: LCMN/NICHD/NIH References: Date: Sun, 3 Mar 1996 21:18:28 GMT Lines: 34 This sounds like either too much salt in the loaded samples or differing ionic strengths between the gel and the running buffer or both. Can you give a few more hints as to protocol? Steve sscherer@helix.nih.gov In article , Osama wrote: > Dear All, > > We are running into trouble with RPA. The problem lies in the loading of > the gel. We start off by loading the individual lanes. However, the lanes > sort of converge to a one point or taper to a one point as the gel runs on?? > > We have done it more than once and we always get the same thing. What > could be wrong? Any ideas or suggestions.. > > I would really appreciate any comment. Thank you. > > > Regards, > > Osama > > ......................................................................... > Osama A. Al-Assar . ||\ * * *. > N. Ireland, UK . ||'\ * * *. > EDQ424@dsets.ulst.ac.uk . / |''\ * * *. > Research Student . \ |''/ * * *. > . / |'/ * * *. > . /..|/ * * . > .......................................................................... From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!lhc.nlm.nih.gov!nih-csl!NewsWatcher!user From: sscherer@helix.nih.gov (Steve Scherer) Subject: Re: 5' RACE Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: 128.231.222.96 Organization: LCMN/NICHD/NIH References: <4h77iq$7dh@tribune.usask.ca> Date: Sun, 3 Mar 1996 21:12:09 GMT Lines: 37 I just finished using the Marathon-Ready cDNA from Clontech (a rat brain cDNA library with fancy adaptors ligated to the ends). You'll need to generate a few gene-specific primers and try all of them both individually or in combinations as nested primers where appropriate. I recommend using touchdown PCR and then blotting and screening an aliquot of your PCR reactions to find appropriate bands or smears to cut out and gel purify before cloning and sequencing. A word of caution though: I found that whoever constructed this particular library at Clontech didn't do a very good jump of removing the original cDNA primers and found a number of clones containing at least parts of this sequence ligated between the adaptor and the 5' end of the message. Other than that, a fine system. Hope this helps - email me if you have any other questions. Happy sequencing, Steve sscherer@helix.nih.gov In article <4h77iq$7dh@tribune.usask.ca>, bonhamp@duke.usask.ca (Peta C. Bonham-Smith) wrote: > I need to carry out some 5' RACE to locate the 5' end of a message of > interest - do I need Poly A+ RNA for this procedure? If so what is the > recommended procedure these days - I really don't want to go back to > oligo-dT columns!!! Secondly, which kit or procedure could you recommend > for the 5' RACE itself? > > Please reply to > bonhamp@duke.usask.ca > > Thank you > > Peta Bonham-Smith > Department of Biology > University of Saskatchewan > Saskatoon > Canada From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!miwok!bdt.com!usenet From: "R. Scott Jokerst" Newsgroups: bionet.molbio.methds-reagnts Subject: Biological Data Transport info Date: Sat, 02 Mar 1996 14:29:00 -0800 Organization: Biological Data Transport Lines: 30 Message-ID: <3138CBAC.B11@data-transport.com> Reply-To: scott_jokerst@data-transport.com NNTP-Posting-Host: jokerst.dial-up.bdt.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; PPC) CC: "R. Scott Jokerst" I wanted to let you know about a service we offer to the life sciences community on the Web. Our site and is emerging as a great source for links to bioinformatics resources, company products and services information, etc. We're becoming more comprehensive daily. Our growth has been quite rapid. We're at: http://www.data-transport.com The entire collection of product, services, and public information links integrated within our system may be found by searching at our site. This allows you to FOCUS on just the information you need, reducing the effort needed to find what you want. The site is exceptionally fast, and we have a track record of being up virtually all of the time. Everything we offer at the site works, (we do not post services under construction) and we have more coming soon. We are eager to receive feedback from you regarding how well the site serves you, and what you would like to see to help you be more productive. Thanks ahead of time. It's worth a look, Scott (P.S. I have cross posted to other lists, wanting to get the info out to you.) -- R. Scott Jokerst Biological Data Transport scott_jokerst@data-transport.com 510-648-8229 http://www.data-transport.com 510-648-8279 (FAX) From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!CS.Arizona.EDU!noao!ennfs.eas.asu.edu!gatech!newsfeed.internetmci.com!chi-news.cic.net!uwm.edu!msunews!netnews.upenn.edu!dsinc!ub!newsstand.cit.cornell.edu!news.tc.cornell.edu!newsserver.sdsc.edu!newshub.csu.net!charnel.ecst.csuchico.edu!csusac!csus.edu!news.ucdavis.edu!chip.ucdavis.edu!fznewt From: Jeff 'Newt' Sekelsky Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Sfi 1 enzyme Date: Sat, 2 Mar 1996 09:18:57 -0800 Organization: University of California, Davis Lines: 19 Message-ID: NNTP-Posting-Host: chip.ucdavis.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: fznewt@chip.ucdavis.edu In-Reply-To: <4h7frv$jjn@news3.cts.com> On 1 Mar 1996, tera wrote: > Has anyone had difficulties ligating PCR products following Sfi1 > digestion? We are using Geneclean to gel purify PCR products following > digestion with various restriction enzymes. All products seem to ligate > efficiently with the exception of those cut with Sfi1. > Are you sure the ends are comaptible? The SfiI recognition sequence is: * 5' G G C C N N N N N G G C C 3' C C G G N N N N N C C G G * If the ends you're trying to ligate are indeed compatible, then I'm not much help to you. JS From owner-methds-reagnts@net.bio.net Fri Mar 01 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!cssun.mathcs.emory.edu!gatech!newsfeed.internetmci.com!howland.reston.ans.net!nntp.coast.net!fu-berlin.de!news.belwue.de!news.uni-stuttgart.de!news.ruhr-uni-bochum.de!news.rwth-aachen.de!newsserver.rrzn.uni-hannover.de!news From: bmay Newsgroups: bionet.molbio.methds-reagnts Subject: Re: New life for old PCR templates? Date: 2 Mar 1996 21:28:14 GMT Organization: RRZN - Newsserver Lines: 8 Message-ID: <4haehe$90r@newsserver.rrzn.uni-hannover.de> References: NNTP-Posting-Host: h15.ts1.uni-hannover.de I reaed in a QIAGEN protocol that DNA undergoes acid hydrolysis at neutral pH, they recommend TE pH 9.0 for that reason. Seems your DNA's degraded a little bit... Bernhard Mayr Medizinische Hochschule Hannover Dept. Clin. Endocrinology Hannover, Germany From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!news.math.psu.edu!psuvax1!uwm.edu!newsspool.doit.wisc.edu!night.primate.wisc.edu!nntp.msstate.edu!fiona.umsmed.edu!fiona.umsmed.edu!hutchins From: hutchins@fiona.umsmed.edu (Jim Hutchins) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: EtOH + PBS = precipitation? Followup-To: bionet.molbio.methds-reagnts Date: 3 Mar 1996 15:44:17 GMT Organization: University of Mississippi Medical Center Lines: 15 Message-ID: <4hceoh$4v7@fiona.umsmed.edu> References: <4gviri$50i@ustsu10.ust.hk> NNTP-Posting-Host: fiona.umsmed.edu X-Newsreader: TIN [version 1.2 PL1] LU Pin (bolupin@uxmail.ust.hk) wrote: : Recently when I tried to prepare 95% ethanol in PBS, I always got the : same problem. When PBS was added into abs. ethanol, the solution became : turbid. Later, some white precipitate formed. Shaking or vortexing during : PBS addition did not help. The PBS I used contains NaCl, KCl, Na2HPO4, : KH2PO4. Anyone knows what's wrong or how to avoid this? Thank you very much. Why are you trying to prepare 95% EtOH/PBS? As far as I'm aware, phosphate precipitates under the conditions described. If you tell us the intended use, maybe we can suggest a substitute. -- Jim Hutchins Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr http://fiona.umsmed.edu/~hutchins/ *** E-mail: hutchins@umsmed.edu From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!news.math.psu.edu!psuvax1!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!tank.news.pipex.net!pipex!peer-news.britain.eu.net!yama.mcc.ac.uk!news.iscm.ulst.ac.uk!dsets.ulst.ac.uk!bcmm14 From: "Ashraf R. Dallol" Newsgroups: bionet.molbio.methds-reagnts Subject: C127I cells & 6-TG Date: Sun, 3 Mar 1996 16:01:55 +0000 Organization: Interactive Systems Centre, University of Ulster Lines: 17 Message-ID: NNTP-Posting-Host: dsets.ulst.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Have any body used or heard about the use of C127I cells to estimate mutation frequency following UV-irradiation using 6-thioguanine? Please advise me as soon as possible. /\ _______________________________****____________________________________________ A s h r a f D a l l o l || "Ultimate Consistancy Lies in being Dept. Biomedical Sciencess || Consistantly Inconsistant" University of Ulster, BT52 1SA || http://www.zakat.org.uk/palestine/ N. Ireland || trail.htm || http://www.zakat.org.uk/mominet/ashraf || /pers.htm ________________________________||_____________________________________________ [] From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!nntp.coast.net!news.sprintlink.net!paperboy.ids.net!usenet From: rand777@ids.net (Robert Randolph) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Superbroth for EVERYONE! (was NOT, but now IS) Date: Sun, 03 Mar 1996 15:49:21 GMT Organization: Randolph Biomedical Lines: 39 Message-ID: <4hcesj$115@paperboy.ids.net> References: <4gtg62$7he@news.tamu.edu> Reply-To: rand777@ids.net (Robert Randolph) NNTP-Posting-Host: 155.212.203.82 X-Newsreader: Forte Agent .99b.113 On Tue, 27 Feb 1996 19:47:36 +0000, horowicz@icrf.icnet.uk (David Ish-Horowicz) wrote: >SUPER BROTH APPARATUS AND PROTOCOL > > >Prepare the desired amount of Super Broth Media (SB). > > > Super Broth Media Recipe: > > 32 g Tryptone > 20 g Yeast Extract > 5 g NaCl > 5 mL 1.0 M NaOH > > Bring to 1 liter with house distilled water. > > > >Add antibiotic (100 ug/mL ampicillin) to and pre-warm the SB media. "SUPER BROTH 10X" Sterile concentrate - ready to use ...just add to sterile DI water in culture system...with or without antibiotics ... available: http://www2.ids.net/~rand777/home.htm R.Randolph -- Robert Randolph Randolph Biomedical 21 McElroy Street West Warwick, RI 02893 401-826-1407 rand777@ids.net http://www2.ids.net/~rand777/home.htm From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!newsfeed.internetmci.com!tank.news.pipex.net!pipex!peer-news.britain.eu.net!yama.mcc.ac.uk!news.iscm.ulst.ac.uk!dsets.ulst.ac.uk!bcmm14 From: "Ashraf R. Dallol" Newsgroups: bionet.molbio.methds-reagnts Subject: Thymidine kinase antibody Date: Sun, 3 Mar 1996 16:04:48 +0000 Organization: Interactive Systems Centre, University of Ulster Lines: 17 Message-ID: NNTP-Posting-Host: dsets.ulst.ac.uk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I am in urgent need for anti-mouse thymidine kinase 1 antibody. Can anybody advise me about the availability of this antibody? If not does the human antibody work instead? /\ _______________________________****____________________________________________ A s h r a f D a l l o l || "Ultimate Consistancy Lies in being Dept. Biomedical Sciencess || Consistantly Inconsistant" University of Ulster, BT52 1SA || http://www.zakat.org.uk/palestine/ N. Ireland || trail.htm || http://www.zakat.org.uk/mominet/ashraf || /pers.htm ________________________________||_____________________________________________ [] From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!news.itd.umich.edu!umich.edu!jchristn From: jchristn@umich.edu Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: Help! Contract protein sequencing and phage display Date: Fri, 1 Mar 1996 15:51:28 Organization: University of Michigan Lines: 15 Distribution: world Message-ID: NNTP-Posting-Host: pm002-05.dialip.mich.net X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Xref: biosci bionet.molbio.methds-reagnts:41284 bionet.molbio.proteins:7221 I was hoping that you people could send me information on the experiences you have had with contract protein sequencing companies. We need to get some N-terminals done from proteins transferred to PVDF and would appreciate any info on who is and isn't reliable, best deals, etc. Also, does anyone know if any decent general purpose phage display vectors (not including those from Pharmacia or Stratagene) are or ever will be made commercially available? Please E-mail me directly since I do not have regular access to Usenet. Thanks in advance, Rob Christner igglab@opus.mco.edu rchristner@opus.mco.edu From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!news.itd.umich.edu!umich.edu!jchristn From: jchristn@umich.edu Newsgroups: bionet.immunology,sci.med.immunology,bionet.molbio.methds-reagnts Subject: ELISA reagent supplier help needed! Date: Fri, 1 Mar 1996 15:41:10 Organization: University of Michigan Lines: 16 Distribution: world Message-ID: NNTP-Posting-Host: pm002-05.dialip.mich.net Keywords: ELISA, immunoglobulin, substrate, allotype X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Xref: biosci bionet.immunology:8069 sci.med.immunology:5356 bionet.molbio.methds-reagnts:41283 Our laboratory is developing an ELISA for human IgG subclasses and we need to know the allotype specificity of our reagents. Does anyone know of commercial suppiers of human IgG allotypes or how to acquire these reagenst from the WHO? Also, we recently had a ploblem with KPL laboratories refusing to replace a suspect batch of substrate that they sent us (we didn't get it for almost 3 weeks!). Does anyone know of companies that offer substrates comparable to their 2-component TMB and 1-component BCIP/NBT liquid substrates? Please E-mail me directly since I do not have regular access to usenet. Thanks in advance, Rob Christner Igglab@opus.mco.edu rchristner@opus.mco.edu From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!daresbury!not-for-mail From: Andrei Popov Newsgroups: bionet.molbio.methds-reagnts Subject: RE : purification of a 140 kb fragment Date: 3 Mar 1996 13:03:40 -0000 Lines: 66 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <4hc5bc$bcv@mserv1.dl.ac.uk> Importance: High Sensitivity: Company-Confidential Original-To: methods@dl.ac.uk (IPM Return Requested) ------------------------------ Start of body part 1 ------------------------------ Start of forwarded message 1 From: Andrei Popov Message-ID: <0548441402031996/A20727/IAPC/11A313883300*@MHS> To: methods@daresbury.ac.uk (IPM Return Requested) Subject: RE: purification of 140 kbp from agarose Importance: High Sensitivity: Company-Confidential : Hi, : lately I have been trying to isolate a pulse-field gel : fragment from agarose. GeneClean didn't work at all so I went on : with an agarase digestion of the agarose followed by a dialysis : over a millipore membrane sitting on a becher full of TE. This : dialysis usually work very well for smaller fragment. But after : this step, I precipitate with 1/10 volume of 3M NaoAc and 3 volume : of cold 95% Ethanol, rinse with 80% ethanol and resuspend in 10 ul : of dH2O, run on a gel and there is nothing. I even hybridised it to : see if there was a trace amount. But there was nothing. I also tried : a phenol extraction with no dialysis followed by the precipitation, : and still nothing. I also tried this thing called GeneCapsule, which : uses electrophoresis to get the DNA out of the agarose and onto a : membrane. But the after an overnight run at 100 volts, the DNA was : still in the plug.?????. So if any of you out there have any idea : please share them with me. : Many thanks : Ghis : P.S. I used low-melt agarose, but my pulse-field gel would be much : nicer if I could use a non-low-melt agarose. Hi Ghis, you described a standard protocol that should work. Here are some suggestions: 1. make plugs with as high DNA concentration as practicaly possible 2. Dr. A.Schedl et. al (see NAR 21:4783) used to run normal preparative gel than another one at 90 degr angle to concentrate DNA in 4% LMA agarose. This gives you 5-fold concentration of your sample. 3. I missed the point of membrane dialysis. Normally people do it when they want to microinject large DNA w/o precipitation 4. If the amount of DNA after agarase digestion is really low try co-precipitation with glycogen 5. If the fragment is a YAC, beware that you co-purify some amount of the 2-mu plasmid (which has multimeric forms)- it can interfere with some applications (probe) 6. check the presense of DNA after agarase- some batches are no good at all, or the buffer might be contaminated with nucleases 7. You did not specify your final goal, but sometimes LMA does not interfere with further enzymatic protocols (Klenow, REs, T4 ligase etc...) Of course there is no need to stain ALL the gel with EthBr- only outer lanes. Having your DNA in agarose even has some advantages- you can change the buffer by washing the block good luck Andrei Popov ------------------------------ End of forwarded message 1 From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!usenet.kornet.nm.kr!news.kreonet.re.kr!xpat.postech.ac.kr!usenet From: kim myung jong Newsgroups: bionet.molbio.methds-reagnts Subject: Daudi-Permanent cell line Date: Sun, 03 Mar 1996 23:53:26 -0800 Organization: Lab of SignalTransduction, Dep.of Life Science, POSTECH Lines: 7 Message-ID: <313AA176.1160@vision.postech.ac.kr> Reply-To: Lab, of, SignalTransduction, Dep.of, Life, Science, POSTECH, San31, HyoJaDong, PoHang, South, Korea NNTP-Posting-Host: hybrid.postech.ac.kr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0b5 (Macintosh; I; PPC) I am writting to ask which expression vectors is usful to establish permanent cell line overexpressing cDNA Daudi cells. If anyone have performed such a experiment and have got good result, please tell me the vector,transfection method and selection condition. Thanks in advance. M,J,Kim From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!nntp.coast.net!news.sprintlink.net!news.interserv.net!news1.interserv.net!NewsWatcher!user From: voellerj@gunet.georgetown.edu (Jim Voeller) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Anyone successfully doing RNAse protection Assays? Date: 3 Mar 1996 18:07:46 GMT Organization: Lombardi Cancer Center, Georgetown University Lines: 23 Message-ID: References: <3135F21D.DA0@unixg.ubc.ca> NNTP-Posting-Host: 141.161.120.30 X-Newsreader: Yet Another NewsWatcher 2.0.6b4 In article <3135F21D.DA0@unixg.ubc.ca>, genes@unixg.ubc.ca wrote: >I have started doing RNAse protection assays using an Ambion kit(RNAse >II). I can't seem to get it to work. The probe is 600 bp and is made >from a pGEM plasmid containing an insert. The target is made from a >longer sequence that is in another plasmid (probe was actually >subcloned from this longer sequnce). I am trying to titre the reaction >so as not to use up my total RNA sample that is limited. The control >for the kit works fine. Can anyone give me some pointers on making the >probe or the target sample and getting this to work. >Any help will be greatly appreciated. >Craig I do not understand titreing the reaction. What are you trying to accomplish? Your probe should always be in large excess. Also a simple thing to check is that your probe and target are complimentary, ie. one is sense and the other antisense. -- Jim Voeller Lombardi Cancer Center Georgetown University Washington, DC USA From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!in2.uu.net!news1.digital.com!decwrl!news.alaska.edu!news From: Reese Bolinger Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Magnetic RNA isolation Date: 3 Mar 1996 08:15:06 GMT Organization: University of Alaska Computer Network Lines: 24 Message-ID: <4hbkea$4h6@news.alaska.edu> References: <31372C69.1AA@leeds.ac.uk> NNTP-Posting-Host: uaa-du-04-02.alaska.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: BMB6MWS@leeds.ac.uk X-URL: news:31372C69.1AA@leeds.ac.uk BMB6MWS@leeds.ac.uk ("M.W. SMITH") wrote: >Can anybody comment on their experiences with mRNA isolation using >magnetic beads e.g. from Promega or Dynal. I need to make high quality >poly a+ RNA for construction of a cDNA library. These manufacturers claim >that their kits can be used for direct isolation of poly A+ from tissue, >without the need to prepare total RNA. Does this provide high quality >poly a+? Any comments would be gratefully received. >Thanks. >Mark Smith (m.w.smith@leeds.ac.uk) >University of Leeds. >UK. Hi: I've used the Promega PolyAttract 1000 kit for isolating polyA(+) RNA directly from whole Xenopus embryos with good results in both = 5' RACE and cDNA library construction. The one CRITICAL thing I know of with the magnetic particle technology is that the magnetic = ferritin/oligo dT particles must not be frozen in shipment or they will not work at all. Depending on where you are (I'm in Alaska,= so you can imagine), that may be a consideration. The only problem that I've observed with them other than that of freezing is tra= ce contamination of the RNA with magnetic particles after isolation, so an additional spin to pellet out the remaining particles is = probably a good idea, though they supposedly don't cause problems in small quantities in most procedures. Reese From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!newsfeed.internetmci.com!in2.uu.net!gateway.sequent.com!newsfeed.orst.edu!news.orst.edu!news From: chend@ucs.orst.edu (Don Chen) Newsgroups: bionet.molbio.methds-reagnts Subject: Ps fluorescens biovars? Date: Sat, 02 Mar 1996 03:30:08 GMT Organization: Oregon State University Lines: 21 Message-ID: <4hb3pg$sg5@news.orst.edu> NNTP-Posting-Host: slip198.ucs.orst.edu X-Newsreader: Forte Free Agent 1.0.82 Hi: I have read that there are five biovars of Ps. fluorescens, but they are not described. Does anyone what are the type strains and where they can be obtained (eg ATCC?)? Is there specific references for these strains which are published and available. I wish to use them as reference to soil pseudomonads which I am trying to characterize. Thanks for any help you can offer. Don ********************************************************* Don Chen * Standard disclaimers apply here. USDA-ARS-NFSPRC * 3450 SW Campus Way * Corvallis, OR 97331 * * 503-750-8721 * ******************************************************** From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!news.ohsu.edu!merza From: merza@ohsu.edu (Alex Merz) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Superbroth for EVERYONE! (was NOT, but now IS) Date: 4 Mar 1996 05:35:24 GMT Organization: Oregon Health Sciences University Lines: 10 Message-ID: <4hdves$ldp@fremont.ohsu.edu> References: <4gtg62$7he@news.tamu.edu> <4hcesj$115@paperboy.ids.net> NNTP-Posting-Host: 137.53.1.40 In article <4hcesj$115@paperboy.ids.net>, Robert Randolph wrote: >On Tue, 27 Feb 1996 19:47:36 +0000, horowicz@icrf.icnet.uk (David >Ish-Horowicz) wrote: [...] _That_ is what workstudy students are for. -Alexey From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!nntp.coast.net!harbinger.cc.monash.edu.au!news.uwa.edu.au!newsman.murdoch.edu.au!usenet From: Jim Cummins Newsgroups: bionet.molbio.methds-reagnts,bionet.immunology,bionet.agroforestry,bionet.celegans,bionet.cellbiol,bionet.drosophila,bionet.general,bionet.microbiology,bionet.molbio.hiv,bionet.molbio.proteins,bionet.molbio.yeast,bionet.mycology,bionet.plants,bionet.virol Subject: Re: PCR Primers Database - UPDATE. Date: 4 Mar 1996 04:29:54 GMT Organization: Murdoch University Lines: 16 Message-ID: <4hdrk2$ljd@newsman.murdoch.edu.au> References: NNTP-Posting-Host: vetmac3.murdoch.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: bshomer@ebi.ac.uk X-URL: news:DnEqGE.LFJ@ebi.ac.uk Xref: biosci bionet.molbio.methds-reagnts:41296 bionet.immunology:8073 bionet.agroforestry:2828 bionet.celegans:826 bionet.cellbiol:4232 bionet.drosophila:1892 bionet.general:20208 bionet.microbiology:5166 bionet.molbio.hiv:1945 bionet.molbio.proteins:7223 bionet.molbio.yeast:4871 bionet.mycology:3701 bionet.plants:10461 Wallace et al's searchable database on human mtDNA is now up on WWW page http://www.gen.emory.edu/mitomap.html Kogelnik AM, Lott MT, Brown MD, Navathe SB, Wallace DC. Mitomap - a human mitochondrial genome database. Nucleic Acids Research 1996;24(1):177-179. YOu may care to cross-reference Jim Cummins, Associate Professor in Veterinary Anatomy, Murdoch University, Western Australia 6150. TEL +61-9-360 2668 FAX +61-9-310 4144 From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!nntp.coast.net!harbinger.cc.monash.edu.au!news.mel.connect.com.au!munnari.OZ.AU!news.uwa.edu.au!newsman.murdoch.edu.au!usenet From: Jim Cummins Newsgroups: bionet.molbio.methds-reagnts Subject: mitochondrial DNA database Date: 4 Mar 1996 04:27:13 GMT Organization: Murdoch University Lines: 15 Message-ID: <4hdrf1$ljd@newsman.murdoch.edu.au> NNTP-Posting-Host: vetmac3.murdoch.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.methds-reagnts Hi: Wallace et al's searchable database on human mtDNA is now up on WWW page http://www.gen.emory.edu/mitomap.html Kogelnik AM, Lott MT, Brown MD, Navathe SB, Wallace DC. Mitomap - a human mitochondrial genome database. Nucleic Acids Research 1996;24(1):177-179. Jim Cummins, Associate Professor in Veterinary Anatomy, Murdoch University, Western Australia 6150. TEL +61-9-360 2668 FAX +61-9-310 4144 From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!rockyd!rutgers!mcrcr6!vilimf01 From: vilimf01@mcrcr6.med.nyu.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Amersham Hybond N problem Message-ID: <1996Mar2.165022.8215@mcrcr6> Date: 2 Mar 96 16:50:22 EDT References: Organization: NYU Medical Center, New York, NY 10016, USA Lines: 28 In article , schulzb@pz350.ipsp.uni-hohenheim.de (BRITTA SCHULZ) writes: > Who can help me to find out, which company produces the uncharged membrane > formerly sold by Amersham as Hybond N ? > The problem is , they are now selling a new membrane, calling it Hybond N as > well, but it is very different. However, since we have the new Hybond N all > we get is black autoradiographs ( we are using the Dig-system) The new > membrane is very thick and stiff, it doesn't look like nylon > at all, and it is produced by another manufacturer. Since I would like to go > on with my work without too much delay and the Amersham people did not want > to tell me their former supplier, maybe someone here knows something. > > Thank you very much in advance > > Britta I just recieved something from MSI (http://www.msifilters.com) saying that Amersham had replaced its supplier and implied that that its MAGNA membrane would give the same results as the the old Amersham Hybond-N membranes. But MSI has changed its manufacturing protocol recently, and several netters noted problems with this switch. It changed its protocols due to a patent infringement lawsuit by Pall corp, so pall may be the best source of a replacement since they probably supplied Amersham and use the old protocol for manufacture. It was also posted on this newsgroup recently that they were a favored supplier. Usual Disclaimers, SVEN From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!daresbury!nntp-trd.UNINETT.no!newsfeed.sunet.se!news00.sunet.se!sunic!mn6.swip.net!plug.news.pipex.net!pipex!tube.news.pipex.net!pipex!lade.news.pipex.net!pipex!tank.news.pipex.net!pipex!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!newsxfer.itd.umich.edu!news.join.ad.jp!news.imnet.ad.jp!ripspost.aist.go.jp!news.tisn.ad.jp!gan!usenet From: Subject: test Content-Type: text/plain; charset=ISO-8859-1 Message-ID: X-Xxdate: Sat, 2 Mar 1996 14:08:48 GMT Sender: usenet@gan.ncc.go.jp (Usenet) Content-Transfer-Encoding: 8bit Organization: National Cancer Center JAPAN X-Newsreader: Nuntius 2.0.4_PPC Mime-Version: 1.0 Date: Sat, 2 Mar 1996 14:10:15 GMT Lines: 1 This article was probably generated by a buggy news reader. From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!agric.nsw.gov.au!gillinm From: gillinm@agric.nsw.gov.au (Michael Gillings) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: freeze-squeeze Date: 3 Mar 1996 14:58:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 38 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net I have used a method from low melt agarose that we used to call the "Freeze-squeeze". Cut your band from the agarose and place it into a folded piece of parafilm in the freezer. When it is completely frozen place the agarose parafilm sandwich between two sterile microscope slides. Make sure that some of the parafilm sticks out the sides of the setup. Form a "V" with the microscope slides that is in the same orientation as the "V" of the parafilm. Slowly squeeze the slides together, and as the slice defrosts under the pressure, the buffer and some of the DNA will come out of the agarose. Collect the droplet of buffer from the parafilm fold. This works best for small molecular weight fragments (naturally). Phenol extraction will remove the bromophenol blue. Good luck, Michael Dr Michael Gillings Senior Research Scientist Biological and Chemical Research Institute PMB 10, Post Office Rydalmere NSW 2116 AUSTRALIA Ph 61-2-843-5709 Fax 61-2-630-4475 On Thu, 29 Feb 1996 vsood@acs.ucalgary.ca wrote: > I remember seeing some advice on retrieving DNA from agarose slices via a > method of freezing the slices and then mashing them, but don't recall the > details. Does anyone use this method who can refresh my memory? Also, what > about any EtBr or bromophenol blue (my band runs at the same place as the > dye front) present in the slice? > > thanks a lot. > bb. > > From owner-methds-reagnts@net.bio.net Sat Mar 02 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!natinst.com!news-relay.us.dell.com!swrinde!cssun.mathcs.emory.edu!hobbes.cc.uga.edu!news From: phillip buckhaults Newsgroups: bionet.molbio.methds-reagnts Subject: Biotinylated Primer for S1 probe? Date: Sun, 03 Mar 1996 16:27:14 +0800 Organization: University of Georgia, Athens Lines: 21 Message-ID: <313957E2.5507@bscr.uga.edu> NNTP-Posting-Host: l-pha.biochem.uga.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) Would someone post advice on S1 nuclease protection to map transcriptional start sites? I would like to make use of a biotinylated primer i have to synthesize a probe, either by using klenow or PCR, and then blot the protected products for avidin-AP detection. I have no experience with this kind of assay, so there may be an obvious reason why using a biotinylated primer will not work. Any comments on this type of approach, or a pointer to a good traditional protocol would be helpful. thanks in advance. Phillip Joe Buckhaults Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center Life Science Building, Room B316 University of Georgia Athens Georgia 30602 Phone (706) 542-1701 Fax (706) 542-1759 "Let us not confuse effort with achievement" From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!rutgers!csn!news-1.csn.net!magnus.acs.ohio-state.edu!yulee From: yulee@magnus.acs.ohio-state.edu (Yuandan Lee) Newsgroups: bionet.molbio.methds-reagnts Subject: large-scale prep of ssDNA Date: 4 Mar 1996 21:09:26 GMT Organization: The Ohio State University Lines: 11 Message-ID: <4hfm66$8mb@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: bottom.magnus.acs.ohio-state.edu Dear netters, I'm looking for a method to prepare a large amount of single-strand DNA (several hundred micrograms). I have subcloned the DNA fragment into the Bluescript vector. The protocol I have in hand is basically for the sequence reaction, therefore will give very small amount of ssDNA. Your help world be appreciated. Dan Lee lee.634@osu.edu 614-261-1467 From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!nntp.coast.net!swidir.switch.ch!swsbe6.switch.ch!scsing.switch.ch!news.rediris.es!news.cica.es!obelix.cica.es!not-for-mail From: claros@obelix.cica.es (Manuel G. CLAROS) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Unspecific hybridization to plasmid-vector Date: 4 Mar 1996 19:29:12 GMT Organization: Universidad de Malaga y el CICA por su apoyo Lines: 19 Distribution: world Message-ID: <4hfga8$cga@erika.cica.es> References: <4h19ul$ru2@rzunews.unizh.ch> NNTP-Posting-Host: obelix.cica.es Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8895-1 Content-Transfer-Encoding: 8bit X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Se me ponen los ojos a cuadros cuando David Grant nos cuenta: > > Hybridization of my probe to the vector is weaker than to a true positive, > > but quite strong, so that I can see clear bands after 2h exposition. ... > phenol extraction - the DNA was then run again on a 1% gel, the bands cut > out and DNA purified and run AGAIN on a 1% gel - this gel was blotted and ... > I have no good explanation for this but have seen it for years DNA bands in agarose are contaminated with DNA from other bands. Try tu PURIFY TWICE your probe and you will diminish dramatically the plasmid hybridztion -- Manuel G. CLAROS -. .-. .-. .-. . -> claros@obelix.cica.es <- Biologia Molecular ||X|||\ /|||X|||\ /| No quiero cambiar, no puedo, Facultad Ciencias |/ \|||X|||/ \|||X|| no quiero ser una frase mas. E-29071 Malaga ' `-' `-' `-' `- (Esclarecidos) From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!daresbury!bioftp.unibas.ch!infobiogen.fr!jussieu.fr!math.ohio-state.edu!uwm.edu!msunews!netnews.upenn.edu!Lehigh.EDU!Lehigh.EDU!not-for-mail From: mjb1@Lehigh.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: Anyone have pcDNA II? Date: 4 Mar 1996 14:13:39 -0500 Lines: 16 Message-ID: <4hffd3$3ac5@ns1-1.CC.Lehigh.EDU> NNTP-Posting-Host: ns1-1.cc.lehigh.edu Folks- Does anyone have the plasmid pcDNA II? It contains a superlinker region that we need for some experiments we're planning. We tried the manufacturer (InVitrogen) but they are no longer selling it because of some patent dispute. Also, if someone has the map for the plasmid, we could use that too. Thanks very much. Mike -------------------------------------------------------------- Michael J. Behe Phone: 610-758-3474 Department of Biological Sciences FAX: 610-758-4004 Iacocca Hall #111 Secretary: 610-758-3680 Lehigh University e-mail: mjb1@Lehigh.edu Bethlehem, PA 18015 From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!ns1.faseb.org!lamarck.sura.net!fconvx.ncifcrf.gov!cockleberry!pnh From: pnh@cockleberry.ncifcrf.gov (Paul N Hengen) Subject: Re: Sequencing after G run Message-ID: Sender: usenet@ncifcrf.gov (C News) Nntp-Posting-Host: cockleberry.ncifcrf.gov Organization: Frederick Biomedical Supercomputer Center X-Newsreader: TIN [version 1.2 PL2] References: Date: Mon, 4 Mar 1996 20:25:42 GMT Lines: 43 Michael Gorry (mcgorry@med.pitt.edu) wrote: : I am having a problem sequencing a clone. Using the taq-FS : dyedeoxy terminal cycle sequencing kit, I have reached a point : with 21 G's and get 1 more base call before the reaction quits. : A manual sequencing kit was also used with the same results. : Does anyone have any advice on how to get through this stop? Not surprising! Have you tried a potassium-free buffer? @article{Woodford1995, author = "K. Woodford and M. N. Weitzmann and K. Usdin", title = "The use of {K+}--free buffers eliminates a common cause of premature chain termination in {PCR} and {PCR} sequencing", journal = "Nucleic Acids Res.", volume = "23", number = "3", pages = "539", comment = "cycle sequencing through high GC content", year = "1995"} @article{Hengen1996Jantibs, author = "P. N. Hengen", title = "Methods and reagents - Cycle sequencing through {GC}--rich regions", journal = "Trends in Biochemical Sciences", volume = "21", number = "1", pages = "33-34", month = "jan", year = "1996"} ******************************************************************************* Paul N. Hengen, Ph.D. (pnh@ncifcrf.gov) National Cancer Institute--FCRDC--Frederick, Maryland 21702-1201 USA phone:(301) 846-5581 fax:(301) 846-5598 ******************************************************************************* Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/ Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html ******************************************************************************* From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!chi-news.cic.net!mr.net!umn.edu!newsstand.tc.umn.edu!biosci!horton From: horton@biosci.cbs.umn.edu (Robert Horton) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR probes by incorporation of 32P ? Date: 4 Mar 1996 19:45:58 GMT Organization: University of Minnesota Lines: 35 Distribution: world Message-ID: <4hfh9m$ee8@epx.cis.umn.edu> References: NNTP-Posting-Host: biosci.cbs.umn.edu X-Newsreader: TIN [version 1.2 PL2] Geoffrey Neale (geoffrey.neale@STJUDE.ORG) wrote: : Hello everyone. : I am looking for a method for making 32-P labeled probes during PCR. The : information that I specifically need is the concentration of cold : nucleotide in the reaction so that you generate sufficient full length : probes, but still retain high specificity of the isotope label. : Other considerations are the amount of starting template, the : concentration of primers, and the number of cycles. : Thanks for your input. : Geoff Neale : PS Could you please email me your advice since our news reader is not yet : operational on our system. E-mail is my only access at this time. Thanks. (Yeah, I'll e-mail it, too. Posting here for the general audience...) See: Schowalter DB. Sommer SS. The generation of radiolabeled DNA and RNA probes with polymerase chain reaction. Analytical Biochemistry. 177(1):90-4, 1989 Feb 15. You can achieve higher specific activities with lower total [dNTP], but you need to increase the extension time. --- Robert M. Horton http://134.84.47.3 Have a :) day "Scotty, try flushing the radioactive waste into the ventilation system!" From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!natinst.com!news-relay.us.dell.com!swrinde!tank.news.pipex.net!pipex!lade.news.pipex.net!pipex!news00.sunet.se!sunic!news99.sunet.se!news.uni-c.dk!news From: soren.rasmussen@risrms1.risoe.dk (Soren K. Rasmussen) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Plasmid Drawing Programme Date: 4 Mar 1996 19:22:42 GMT Organization: Risoe National Laboratory Lines: 38 Message-ID: <4hffu2$qog@news.uni-c.dk> References: <4h4o49$g3d@is.bbsrc.ac.uk> <4heskn$1nd@crick.bms.com> NNTP-Posting-Host: riso-remote-1.risoe.dk Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 In article <4heskn$1nd@crick.bms.com>, Doughert@synapse.bms.com says... > >In article <4h4o49$g3d@is.bbsrc.ac.uk>, Your EMail address (HARVEY@BBSRC.AC >.UK) (HARVEY) says: >> >>I am looking to buy a plasmid drawing programme for use on a PC, something >>that will take a list of features and their location and produce a nice >>map. I would like to know what programmes are available and any advice >>or opinions as to which might be the best option. >>Many thanks >>Alison Harvey >>HARVEY@BBSRC.AC.UK > >Alison- > We have been using a PC plasmid program called "Enhance", and are very >happy with the results. It produces a very high quality plasmid (or linear >) map >on a laser printer. Our version is DOS, but a Windows version is due short >ly. >It is very easy to learn and use. You can view some of the output in J. Ba >cteriol. >175, pages 113 and 114. The program is put out by Scientific & Educational >Software, PO Box 440, State Line, Pa, USA. Phone is (717)597-5307. > Tom Dougherty > Dept. Microbiology > Bristol-Myers Squibb >***Opinions strictly my own*** we are happy users of the Enhance plasmid drawing program for DOS. Yuo can make colormaps as well soren From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!KIDS.WUSTL.EDU!haviland From: haviland@KIDS.WUSTL.EDU ("David L. Haviland, Ph.D.") Newsgroups: bionet.molbio.methds-reagnts Subject: COS-cells Date: 4 Mar 1996 11:37:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Greetings: Does anyone have any direct experience with protein expression (cDNA constructs) in the COS cells? The Harvard manual states that COS-7's are best for this purpose however, as most know, the description of COS-1 and COS-7 cells are virtually identical in the ATCC catalog. My only test thus far has been a beta-gal vector that serves as a control for transfection (DEAE). Using this vector, transfection and expression are effectively the same as far as I can tell. Many thanks in advance. Any thoughts? David =========================================================================== + David L. Haviland, Ph.D. .***. .***. .***. + + Washington Univ. School of Med * | | | * * | | | * | | | * + + Dept. of Peds./Pulm. Box 8116 *| | | * * | | | * * | | | * + + One Children's Place * | | | * | | | * * | | | * + + St. Louis, MO 63110 '***' '***' '***' + + Internet:"haviland@kids.wustl.edu" + + Voice: 314-454-6076 FAX: 314-454-6076 + =========================================================================== From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!newsfeed.sunet.se!news00.sunet.se!sunic!mn6.swip.net!plug.news.pipex.net!pipex!tube.news.pipex.net!pipex!lade.news.pipex.net!pipex!tank.news.pipex.net!pipex!newsfeed.internetmci.com!in2.uu.net!newsflash.concordia.ca!news.mcgill.ca!news From: Graham Dellaire Newsgroups: bionet.molbio.methds-reagnts Subject: Alpha Satellite Probes for Mice??? Where can you get them? FOR FISH Date: 4 Mar 1996 18:29:55 GMT Organization: McGill University Computing Centre Lines: 18 Message-ID: <4hfcr3$ia9@sifon.cc.mcgill.ca> NNTP-Posting-Host: b-03.das.mcgill.ca X-Newsreader: AIR News 3.X (SPRY, Inc.) Help! I am trying to karotype a mouse cell line (LTA derived from L M Tk- cells) and I think I might need paints for murine alpha satellite DNA (or equivalent centromer specific paints) for FISH analysis. I am seeing a lot of metacentrics etc and more than 40 chrms. Any info would be appreciated Graham Dellaire McGill Exp. medicine dellaire@odyssee.net From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!ra.nrl.navy.mil!news.math.psu.edu!chi-news.cic.net!nntp.coast.net!howland.reston.ans.net!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!usenet From: Keld Sorensen Newsgroups: bionet.molbio.methds-reagnts Subject: Re: fluorescently labelled protein Date: 4 Mar 1996 18:47:32 GMT Organization: Univ. Illinois / College of Med. Lines: 11 Message-ID: <4hfds4$od0@piglet.cc.uic.edu> References: <4hf86d$i7t@news.tamu.edu> NNTP-Posting-Host: sliprock-01.dialin.uic.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: davesmith@bioch.tamu.edu X-URL: news:4hf86d$i7t@news.tamu.edu [snipped Q about fluorescent labeling of protein] - Fluorescein, Rhodamine and AMCA are all available as NHS compounds, i.e. they will react spontaneously with proteins (the NH2 groups). This is identical to the biotinylation reagents most researchers use. Check the Pierce catalog for furhter info and pricing etc. Keld. From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!MSMTP.IDDE.SACI.ORG!brett_beitzel From: brett_beitzel@MSMTP.IDDE.SACI.ORG ("Brett Beitzel") Newsgroups: bionet.molbio.methds-reagnts Subject: Polymerases, anyone!?!?!? Date: 4 Mar 1996 08:33:38 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Subject:Polymerases, anyone!?!?!? Date: 3/4/96 Anyone know of a themostable DNA polymerase that can use an RNA oligo as a primer, instead of a DNA oligo? A lot of the tech support people at the different companies I have talked to seem to think that some of their polymerases will, but they have never actually tried it. Any help would be appreciated! Thanks, Brett Beitzel brett_beitzel@msmtp.idde.saci.org Cancer Therapy and Research Center San Antonio, TX From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!newsfeed.internetmci.com!in2.uu.net!fdn.fr!jussieu.fr!citi2.fr!not-for-mail From: mayaux@bisance.citi2.fr (Jean-Francois Mayaux) Newsgroups: bionet.molbio.methds-reagnts Subject: Aminopropanol mutant Date: 4 Mar 1996 16:29:27 +0100 Organization: CITI2 - Universite Rene Descartes, Paris Lines: 9 Message-ID: <4hf28n$j59@bisance.citi2.fr> NNTP-Posting-Host: bisance.citi2.fr X-Newsreader: TIN [version 1.2 PL2] Hi netters I am looking for a bacterial mutant blocked in the aminopropanol biosynthetic pathway for a complementation experiment. Has anybody heard of such a thing ? Any comments, ideas, advices are welcome. (It is about to be a question of life or death for me ) Laurent. Email : laurent.naudin@rp.fr From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!eworld.com!Microplate From: Microplate@eworld.com Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Microplate technology Date: 4 Mar 1996 06:52:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <960304064904_26583168@hp1.online.apple.com> NNTP-Posting-Host: net.bio.net The Miptec 96 meeting is in Basle Switzerland June 24-27 you can contact local organiser John Devlin. ARRTIntl@aol.com for further details. From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!newsserver.jvnc.net!crick.bms.com!usenet From: Doughert@synapse.bms.com (Tom Dougherty) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Plasmid Drawing Programme Date: 4 Mar 1996 13:53:27 GMT Organization: Bristol-Myers Squibb Lines: 21 Message-ID: <4heskn$1nd@crick.bms.com> References: <4h4o49$g3d@is.bbsrc.ac.uk> NNTP-Posting-Host: doughertyt.wf.bms.com In article <4h4o49$g3d@is.bbsrc.ac.uk>, Your EMail address (HARVEY@BBSRC.AC.UK) (HARVEY) says: > >I am looking to buy a plasmid drawing programme for use on a PC, something >that will take a list of features and their location and produce a nice >map. I would like to know what programmes are available and any advice >or opinions as to which might be the best option. >Many thanks >Alison Harvey >HARVEY@BBSRC.AC.UK Alison- We have been using a PC plasmid program called "Enhance", and are very happy with the results. It produces a very high quality plasmid (or linear) map on a laser printer. Our version is DOS, but a Windows version is due shortly. It is very easy to learn and use. You can view some of the output in J. Bacteriol. 175, pages 113 and 114. The program is put out by Scientific & Educational Software, PO Box 440, State Line, Pa, USA. Phone is (717)597-5307. Tom Dougherty Dept. Microbiology Bristol-Myers Squibb ***Opinions strictly my own*** From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!tank.news.pipex.net!pipex!warwick!yama.mcc.ac.uk!news.salford.ac.uk!aber!not-for-mail From: jms93@aber.ac.uk (jms93) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA sequence for rat Date: 4 Mar 1996 13:21:19 GMT Organization: University of Wales, Aberystwyth Lines: 31 Message-ID: <4heqof$p5h@osfb.aber.ac.uk> References: NNTP-Posting-Host: pccbgj.ccu.aber.ac.uk Mime-Version: 1.0 X-Disclaimer: Warning - Sender not verified X-Newsreader: WinVN 0.93.11 In article , lcwilliams@medusa.unm.edu says... > >Hello fellow scientists! >Does anyone know where I can get the rat DNA sequence of EGF (Epidermal >Growth Factor) and EGF-R? Thank you in advance for your help. > >Laurie Williams >University of New Mexico Hospital Have you tried the Genbank website? If it has been sequenced, it will be there. The address is as follows.... http://www.ncbi.nih.gov/Search/index.html Another good source of sequence information is the SEQNET Website and that can be found here... http://www.dl.ac.uk/SEQNET/home.html Goof luck! Jonathan Shillingford jms93@aber.ac.uk > From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!swrinde!tank.news.pipex.net!pipex!warwick!yama.mcc.ac.uk!news.salford.ac.uk!aber!not-for-mail From: jms93@aber.ac.uk (jms93) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RNA isolation Date: 4 Mar 1996 13:16:19 GMT Organization: University of Wales, Aberystwyth Lines: 29 Message-ID: <4heqf3$p5h@osfb.aber.ac.uk> References: <4h2ph8$cvv@mark.ucdavis.edu> NNTP-Posting-Host: pccbgj.ccu.aber.ac.uk Mime-Version: 1.0 X-Disclaimer: Warning - Sender not verified X-Newsreader: WinVN 0.93.11 In article <4h2ph8$cvv@mark.ucdavis.edu>, ez022056@rocky.ucdavis.edu says... > >I am looking for a 'quick and easy" RNA isolation protocol. I plan to do >RT-PCR on >20 different cell lines. If anyone knows of such protocol, >please let me know... >Thanks in advance > >-- >******************************** >Edward H. Wang * >Staff Resch. Assc. * >Dept. Internal Med. -Nephrology* >UC Davis * >******************************** The commercial kit that we use is the RNAeasy kit from Qiagen. It allows the isolation of RNA from both whole tissue and cell lines and is very quick, effecint, and the RNA is of very good quality. In total, to isolate RNA takes about 40 minutes and you can do 12 samples at once. We use the kit on a routine basis ands find it works very well. You have to be careful of DNA contamination and I would recoomend an extra wash in the wash buffer as suggested in the protocol. Good luck. Jonathan Shillingford jms93@aber.ac.uk From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!plug.news.pipex.net!pipex!weld.news.pipex.net!pipex!tank.news.pipex.net!pipex!warwick!news.nott.ac.uk!griffin.nott.ac.uk!usenet From: Nigel Kenward Newsgroups: bionet.molbio.methds-reagnts Subject: Re: freeze-squeeze Date: Mon, 04 Mar 1996 11:24:31 +0000 Organization: Dept. Biochemistry, University of Nottingham, U.K. Lines: 21 Message-ID: <313AD2EF.F54@vax.nott.ac.uk> References: Reply-To: mbxnjk@vax.nott.ac.uk NNTP-Posting-Host: macjm2.biochem.nottingham.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) Alternatively, Cut out band. Freeze band (-20) Cut the conical part off an eppendorf and place a small needle hole in it. Pack the end of the conical bit with a small wad od siliconised glass wool. Put the conical bit into a new eppendorf and put the frozen slice into the conical part. Spin 5 mins The buffer in the outer tube will contain the DNA ! Try different spin times etc to get it perfected. Always use good quality agarose ! Good luck -- ____ ___ |\ | | / \ / | NIGEL KENWARD, | \ | | | ___ |__ | Dept. Biochemistry, | \ | | | | | | Nottingham University Medical School. | \| | \____/ \___ |___ Clifton Boulevard. ------------------------------- Nottingham. NG7 2UH. Tel (0115 924 9924) ext 44789 Fax (0115 942 2225) email:mbxnjk@vax.nott.ac.uk "You can tell me.......I'm a doctor" From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!nntp.coast.net!fu-berlin.de!news.belwue.de!news.uni-stuttgart.de!news.ruhr-uni-bochum.de!news.rhrz.uni-bonn.de!ibm.rhrz.uni-bonn.de!UZS13B From: UZS13B@ibm.rhrz.uni-bonn.de (Stefan Kahlert) Newsgroups: bionet.molbio.methds-reagnts Subject: Transfection of proteins into cells: HOW? Date: Mon, 04 Mar 96 10:41:13 MEZ Organization: RHRZ Uni-Bonn Lines: 26 Message-ID: <17740964ES85.UZS13B@ibm.rhrz.uni-bonn.de> References: <4gseqa$qec@oravannahka.Helsinki.FI> <1773CE1A3S85.UZS13B@ibm.rhrz.uni-bonn.de> NNTP-Posting-Host: ibm.rhrz.uni-bonn.de I want to block proteins in cells with specific antibodies, and I'm looking for possible methods to get this antibodies into these cells. Problems: - microinjection is not possible because I work with suspension cells (hard to catch and even harder to find the injected ones to check the result) - electroporation is *not* the way to (please don't ask why. The story is to long to be told here I think). What other methods are possible to get antibodies or other proteins into suspension cells? Thanks in advance Stefan -- Stefan Kahlert, Medizinische Universitaets-Poliklinik Bonn UZS13B@ibm.rhrz.uni-bonn.de or UZS13B@dbnrhrz1.bitnet From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!chi-news.cic.net!nntp.coast.net!fu-berlin.de!news.belwue.de!news.uni-stuttgart.de!news.ruhr-uni-bochum.de!news.rhrz.uni-bonn.de!ibm.rhrz.uni-bonn.de!UZS13B From: UZS13B@ibm.rhrz.uni-bonn.de (Stefan Kahlert) Newsgroups: bionet.molbio.methds-reagnts Subject: Microinjection: where to find info about? Date: Mon, 04 Mar 96 10:36:44 MEZ Organization: RHRZ Uni-Bonn Lines: 23 Message-ID: <177409548S85.UZS13B@ibm.rhrz.uni-bonn.de> References: <4gseqa$qec@oravannahka.Helsinki.FI> <1773CE1A3S85.UZS13B@ibm.rhrz.uni-bonn.de> NNTP-Posting-Host: ibm.rhrz.uni-bonn.de In article <1773CE1A3S85.UZS13B@ibm.rhrz.uni-bonn.de> UZS13B@ibm.rhrz.uni-bonn.de writes: dear colleagues can someone please point me to a god source of informatin about methods and applications of microinjection of proteins and antibodies into adherent (or nonadherent, but I'm not interested in information dealing with the special problems of eg. micromanipulation of xenopus oocytes) mammalian cells? thanks in advance Stefan -- Stefan Kahlert, Medizinische Universitaets-Poliklinik Bonn UZS13B@ibm.rhrz.uni-bonn.de or UZS13B@dbnrhrz1.bitnet From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!newsfeed.internetmci.com!in1.uu.net!csn!nntp-xfer-2.csn.net!news-2.csn.net!csn!news-1.csn.net!rutgers!mcrcr6!vilimf01 From: vilimf01@mcrcr6.med.nyu.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Anyone successfully doing RNAse protection Assays? Message-ID: <1996Mar2.174223.8216@mcrcr6> Date: 2 Mar 96 17:42:23 EDT References: <3135F21D.DA0@unixg.ubc.ca> Organization: NYU Medical Center, New York, NY 10016, USA Lines: 31 In article <3135F21D.DA0@unixg.ubc.ca>, genes@unixg.ubc.ca writes: > I have started doing RNAse protection assays using an Ambion kit(RNAse > II). I can't seem to get it to work. The probe is 600 bp and is made > from a pGEM plasmid containing an insert. The target is made from a > longer sequence that is in another plasmid (probe was actually > subcloned from this longer sequnce). I am trying to titre the reaction > so as not to use up my total RNA sample that is limited. The control > for the kit works fine. Can anyone give me some pointers on making the > probe or the target sample and getting this to work. > Any help will be greatly appreciated. > Craig I too have used the RPAII kit for doing protections. I had no sucess in detecting my message with the kit. I even had problems detecting the sense RNA control that I ran off my gene of interest. (I highly recommend this control for troubleshooting RPA). Other lab members also reported problems with the RPA II kit and recommended the old RPAI kit which is basically the procerure from current protocols in molecular biology. The control sense RNA now gave a HUGE signal and, after gel purifying the probe (also highly recommended), I managed to detect my message in 20ug of total RNA. In speaking to the Ambion people, they confirmed that the new RPA kit is less sensitive than the old procedure. The phenol step is somewhat cumbersome, but getting the result is worth it. For rare messages, or if RPAII isn't working, I recommend the old procedure. By the by, I also modified the hybridization step as per Mironov et al NAR 23(16): 3359 using a buffer devoid of formamide and hybridizing at 85 C for 2-4hr. Faster hyb with better results in my hands, and my probes were 650 and 300bp. Hope my experiences are helpful, good luck. Usual Disclaimers SVEN From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!swrinde!howland.reston.ans.net!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!usenet From: Keld Sorensen Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Background in Southern Blots Date: 4 Mar 1996 18:38:57 GMT Organization: Univ. Illinois / College of Med. Lines: 9 Message-ID: <4hfdc1$od0@piglet.cc.uic.edu> References: <4h8516$n1h@alpha.pcix.com> NNTP-Posting-Host: sliprock-01.dialin.uic.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: jlitt@capecod.net X-URL: news:4h8516$n1h@alpha.pcix.com [mail and post] On the Q on BG in Southerns - does anybody have particularly nice blocking buffers and Hyb buffers?? Keld. From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!usenet From: Keld Sorensen Newsgroups: bionet.immunology,sci.med.immunology,bionet.molbio.methds-reagnts Subject: Re: ELISA reagent supplier help needed! Date: 4 Mar 1996 18:44:31 GMT Organization: Univ. Illinois / College of Med. Lines: 8 Message-ID: <4hfdmf$od0@piglet.cc.uic.edu> References: NNTP-Posting-Host: sliprock-01.dialin.uic.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: jchristn@umich.edu X-URL: news:jchristn.45.000FB026@umich.edu Xref: biosci bionet.immunology:8077 sci.med.immunology:5380 bionet.molbio.methds-reagnts:41318 [Snipped Q about sources of TMB and BCIP.NBT substrates] - Try Pierce Chemical Co E-mail: PierceChem@mcimail.com Phone: 1-800-874-3723 or 1-815-968-0747 Fax: 1-815-968-7316 From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!in1.uu.net!news.tacom.army.mil!news2.acs.oakland.edu!cwis-20.wayne.edu!usenet From: Anton Scott Goustin Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Sequence of Lambda DASH II polylinker Date: 4 Mar 1996 18:17:44 GMT Organization: Wayne State University, Detroit, MI USA Lines: 17 Message-ID: <4hfc48$8dj@cwis-20.wayne.edu> References: NNTP-Posting-Host: asgoff.biosci.wayne.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: Schenker@MPIMG-Berlin-Dahlem.MPG.DE X-URL: news:Schenker.27.00AFC219@MPIMG-Berlin-Dahlem.MPG.DE Schenker@MPIMG-Berlin-Dahlem.MPG.DE (Martin Schenker) wrote: >Hi to everyone! > >I'm looking for the sequence of the polylinker of DASH II. I need to generate primers with a length of >30 bases. These primers sho= uld >include the T3 and T7 promoter sites and extend to the left and right arms of the phage (Not I, Sal I and Xba I -sites). I haven't = found any sequence data, so if someone has some information.... > >P.S.: Is there a Stratagene web page??? A. S. Goustin answers: Stratagene does not have a WWW page, but will respond to inquires via the Internet at: techform@stratagene.= com. I believe I once generated some primers that meet your description. I used them for Long PCR on a Lambda DASH II isolate, with a ~1= 8 kb insert. They seemed to work. I am not sure where I got the sequence info I needed, but it is available. From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!mtuvin From: mtuvin@bcm.tmc.edu (Michael J. Tuvim) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Protein gel Date: 4 Mar 1996 18:38:48 GMT Organization: Baylor College of Medicine, Houston, Tx Lines: 18 Sender: Michael Tuvim Distribution: world Message-ID: <4hfdbo$88h@gazette.bcm.tmc.edu> References: NNTP-Posting-Host: condor.mbcr.bcm.tmc.edu NNTP-Posting-User: mtuvin In article ferlandl@ERE.UMontreal.CA ("Ferland Louis H.") writes: >Most people I know try to not load more than 50 micrograms. We tried >loading 200 ug to detect a low abundance protein we couldn't purify and >got only a mess. We finally had to find a different way to improve the >limit of detection. >On Fri, 23 Feb 1996, lmrc. wrote: >> What is the maximum amount of total protein one can load on a PAGE gel >> for Western analysis?? We routinely load 100-150 ug of total protein on our gels. If you make your gels thicker (3 mm instead of 1,5) you don't have any smears and smiles and generally end up with good enough resolution. The problem is with the antibody: it should be VERY specific to ignore the plethora of accompanying proteins. Secondary AB will also pick nonspecific bands, so it might be worth trying several. Good luck Michael From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!ns1.faseb.org!lamarck.sura.net!newsfeed.internetmci.com!tank.news.pipex.net!pipex!warwick!bradford.ac.uk!leeds.ac.uk!news From: futers@biovax.leeds.ac.uk Subject: Re: Cutting PCR-created restriction sites Message-ID: <1996Mar4.173946.25257@leeds.ac.uk> Reply-To: futers@biovax.leeds.ac.uk NNTP-Posting-Host: bmbvax.leeds.ac.uk Organization: The University of Leeds, UK Date: Mon, 4 Mar 1996 17:39:45 +0000 (GMT) References: Lines: 37 In article , watson_j@bms.com (A. John Watson) writes: >> Marc J. Mass, Ph.D. >> (mass@am.herl.epa.gov) >> wrote: : We have used PCR to create a BclI restriction site not >> : normally present. The fragment is about 500 bp long, and >> : after digestion it should be about 420 bp. We have found >> : that the PCR product is not cutting to completion with Bcl (about 90% of it >> : is cutting). According to our calculations we are certainly >> : using enough enzyme. Does anyone have experience with >> : getting PCR-created restriction sites to cut. >> >> >> : MJ Mass, Ph.D. > >Well, I'd be satisifed with 90% ;^) Is there some reason you need to >digest to completion? BTW, if you check the back of the New England >Biolabs catalog you'll find a list of oligo cleavage results for a number >of different enzymes -- some work well, some don't. In general, it's all >in how much flanking DNA the endonuclease likes to have surroiunding the >recognition site. > >Cheers, > >AJW > >---------------- >John Watson >Bristol-Myers Squibb Co. >watson_j@bms.com >--------------------------------------------------------------------- >"If you're not part of the solution, you're part of the precipitate." >--------------------------------------------------------------------- I am also looking at a Bcl I polymorphism and also only get about 90% digestion. I just ignore the faint upper band in the homozygote. Simon. From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!data-transport.com!scott_jokerst From: scott_jokerst@data-transport.com (R. Scott Jokerst) Newsgroups: bionet.molbio.methds-reagnts Subject: Plasmid Drawing Programs - where to find... Date: 4 Mar 1996 09:38:58 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 44 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hello Alison, In response to your question, I searched Biological Data Transport's information resources for: plasmid OR restriction OR mapping Among the other links returned was one for DNASTAR's LASERGENE software, which you can download to try out and see if it meets your needs. TransportLinks Matches: Computer Software LASERGENE DNA & Protein Sequence Analysis Software by DNASTAR Inc. Download & Testdrive Software! Visit LASERGENE's Software Library If you visit BDT's web site at http://www.data-transport.com and reissue the query, or simply look up DNASTAR directly, you'll be given this hyperlink which will take you directly to the information you are looking for. It's a snap. Hope this helps, Scott **************** In article <4h4o49$g3d@is.bbsrc.ac.uk>, Your EMail address (HARVEY@BBSRC.AC.UK) (HARVEY) says: > >I am looking to buy a plasmid drawing programme for use on a PC, something >that will take a list of features and their location and produce a nice >map. I would like to know what programmes are available and any advice >or opinions as to which might be the best option. >Many thanks >Alison Harvey >HARVEY@BBSRC.AC.UK ---> R. Scott Jokerst scott_jokerst@data-transport.com ---> ---> Biological Data Transport http://www.data-transport.com ---> ---> 510-648-8229 510-648-8279 (FAX) ---> From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.tamu.edu!news From: davesmith@bioch.tamu.edu Newsgroups: bionet.molbio.methds-reagnts Subject: fluorescently labelled protein Date: Mon, 04 Mar 1996 19:10:39 GMT Organization: Texas A&M University, College Station, TX Lines: 7 Message-ID: <4hf86d$i7t@news.tamu.edu> NNTP-Posting-Host: 486young1.tamu.edu X-Newsreader: Forte Free Agent 1.0.82 Anyone out there have a reliable protocol for or experience with labelling purified proteins with fluorescent tags? I'm hoping to follow a fluorescently labelled protein as it escapes from vesicles/liposomes--a sort of marker release experiment. Thanks in advance. dave smith From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!uwm.edu!msunews!netnews.upenn.edu!news.tju.edu!joseph2.tjh.tju.edu!user From: boehnin1@jeflin.tju.edu (Darren Boehning) Newsgroups: bionet.molbio.methds-reagnts Subject: Peptides Date: Mon, 04 Mar 1996 11:39:28 -0500 Organization: Thomas Jefferson University Lines: 6 Message-ID: NNTP-Posting-Host: joseph2.tjh.tju.edu Greetings, Does anyone know of a moderately priced/moderate quality peptide manufacturing company? Please e-mail me with info. Thanks in advance, Darren Boehning boehnin1@jeflin.tju.edu From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!sb-roscoff.fr!vogel From: vogel@sb-roscoff.fr (Lee Vogel) Newsgroups: bionet.molbio.methds-reagnts Subject: Unltracompentent cells Date: 4 Mar 1996 09:01:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <199603041656.RAA17180@sbr.sb-roscoff.fr> NNTP-Posting-Host: net.bio.net Have recently been mucking about with competent cell methods in order to improve yields of a plasmid library. I started with the method of Inoue (1990), growing XLI-blue cells to OD.600=0.6 at room temperature. After the Mg++/Ca++ washing step I suspended the cells either in the glycerin/PEG solution of Nishimura et al, NAR 18 (1990), or in the TB plus DMSO solution of Inoue. Cells were then frozen in a dry-ice bath an left at -80 O.N. The following day transformations were performed with cells preincubated with 25mM beta-Mercapto. or 25mM DTT or nothing. These transformations were compared with supercomptent cells purchased from Stratagene which are "guaranteed" to have a competency greater than 10-9. Cells in the glycerin/PEG gave at least 100 fold less colonies than the Statagene and Inoue cells. In all cases a reducing agent improved transformation, but beta-mercapto. worked beter than DTT. Interestingly , the Inoue preparation with beta-mercapto. gave marginally beter results than the Stratagene cells (approx. 30% more colonies : 130 as opposed to 100 for Stratagene). Unfortunately, I can not provide transformation efficiencies as this was a "real" experiment involving the transformation of a ligation reaction for which the concentration of ligated vector is unknown. From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!BORCIM.WUSTL.EDU!brett From: brett@BORCIM.WUSTL.EDU (brett) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Transfection of proteins into cells: HOW? Date: 4 Mar 1996 17:15:15 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 38 Sender: daemon@net.bio.net Distribution: world Message-ID: <199603050105.TAA14934@borcim.wustl.edu> NNTP-Posting-Host: net.bio.net > >I want to block proteins in cells with specific antibodies, >and I'm looking for possible methods to get this antibodies >into these cells. >Problems: > >- microinjection is not possible because I work with suspension cells > (hard to catch and even harder to find the injected ones to check > the result) > >- electroporation is *not* the way to (please don't ask why. The story > is to long to be told here I think). > >What other methods are possible to get antibodies or other proteins into >suspension cells? > >Thanks in advance > >Stefan > > > > >-- >Stefan Kahlert, Medizinische Universitaets-Poliklinik Bonn >UZS13B@ibm.rhrz.uni-bonn.de or UZS13B@dbnrhrz1.bitnet You can get T7RNAP into cells using liposomes Brett Lindenbach Program in Immunology Washington University - St Louis brett@borcim.wustl.edu "I own my own pet virus. I get to pet and name her." - Cobain From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.uwa.edu.au!karenmk From: karenmk@uniwa.uwa.edu.au (Karen Kroeger) Newsgroups: bionet.molbio.methds-reagnts Subject: Transcription factors and transfections Date: 5 Mar 1996 00:39:16 GMT Organization: The University of Western Australia Lines: 24 Message-ID: <4hg2fk$vjk@styx.uwa.edu.au> NNTP-Posting-Host: uniwa.uwa.edu.au X-Newsreader: TIN [version 1.2 PL2] Does anyone have or know where I can obtain mammalian expression vectors for AP-2 and Ets for use in co-transfection experiments, to study transcription factors binding to promoter DNA in hmuan T cells. I was also looking for an antibody to Ets transcription factor ? Again for transfection experiments, I was wondering if anyone has or knows or a commercially available source of a reporter gene vector which contains a weak promoter, that has low/or no inducibility by PMA. We have previously used the pGL3 and pGL2 luciferase vectors from Promega, but find the SV40 promoter too strong and too inducible for our purpose. Any advice would be helpful ! On a different note, I am thinking of using the One-hybrid system from Clontech, and was wondering if any one that has used it has any advice on the pitfalls etc, whether it is difficult etc. Thankyou. Please relp to email address. Thankyou Karen Kroeger Department of Biochemistry University of Western Australia Perth, W.A., Australia E-mail : karenmk@uniwa.uwa.edu.au From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!PLUTO.CIS.FPU.AC.JP!fk01090 From: fk01090@PLUTO.CIS.FPU.AC.JP (Atsushi Ishikawa) Newsgroups: bionet.molbio.methds-reagnts Subject: anti-myristoylated glycine Ab Date: 4 Mar 1996 22:58:46 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <199603050655.PAA16055@fpujm.cis.fpu.ac.jp> NNTP-Posting-Host: net.bio.net Hi all: I am looking for the anti-myristoylated glycine Ab. Could anyone give me some info? Thanks in advance. Regards, Atsushi Ishikawa ---------------------------------------------------------------------------- Atsushi Ishikawa Fukui Prefectural University e-mail fk01090@pluto.cis.fpu.ac.jp TEL 81-776-61-6000 (ext. 3516) FAX 81-776-61-6015 ---------------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!SMTPGWY.AGRIC.NSW.GOV.AU!Michelle.Gleeson From: Michelle.Gleeson@SMTPGWY.AGRIC.NSW.GOV.AU Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Can dessicants be dried in microwave? Date: 4 Mar 1996 21:13:54 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <9602058260.AA826071118@smtpgwy.agric.nsw.gov.au> NNTP-Posting-Host: net.bio.net Dear Donald, I have never tried the microwave method, but as you can dry herbs and flowers in a microwave it may well work. There is only one way to find out whether or not it wrecks the microwave ;) Anyway, several hours in a lab oven at 80C is hot enough - when the silica gel crystals are bright blue again they are dry. Michelle gleesom@agric.nsw.gov.au From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!SMTPGWY.AGRIC.NSW.GOV.AU!Michelle.Gleeson From: Michelle.Gleeson@SMTPGWY.AGRIC.NSW.GOV.AU Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Can dessicants be dried in microwave? Date: 4 Mar 1996 21:13:48 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <9602058260.AA826071120@smtpgwy.agric.nsw.gov.au> NNTP-Posting-Host: net.bio.net Dear Donald, I have never tried the microwave method, but as you can dry herbs and flowers in a microwave it may well work. There is only one way to find out whether or not it wrecks the microwave ;) Anyway, several hours in a lab oven at 80C is hot enough - when the silica gel crystals are bright blue again they are dry. Michelle gleesom@agric.nsw.gov.au From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!daresbury!bioftp.unibas.ch!infobiogen.fr!jussieu.fr!univ-lyon1.fr!dsi.unimi.it!news.IT.net!dish.news.pipex.net!pipex!tank.news.pipex.net!pipex!iol!imci2!imci3!imci4!newsfeed.internetmci.com!in2.uu.net!news.epix.net!clsmppp40.epix.net!user From: bgreuel@epix.net (Brian T. Greuel) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Transfection of proteins into cells: HOW? Date: Mon, 04 Mar 1996 21:41:26 -0500 Organization: epix.net Lines: 12 Message-ID: References: <4gseqa$qec@oravannahka.Helsinki.FI> <1773CE1A3S85.UZS13B@ibm.rhrz.uni-bonn.de> <17740964ES85.UZS13B@ibm.rhrz.uni-bonn.de> NNTP-Posting-Host: clsmppp40.epix.net In article <17740964ES85.UZS13B@ibm.rhrz.uni-bonn.de>, UZS13B@ibm.rhrz.uni-bonn.de (Stefan Kahlert) wrote: > > I want to block proteins in cells with specific antibodies, > and I'm looking for possible methods to get this antibodies > into these cells. Lipofection should work to get proteins into cells. --Brian Greuel From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!rutgers!csn!news-1.csn.net!ub!dsinc!newsfeed.pitt.edu!scramble.lm.com!news.math.psu.edu!chi-news.cic.net!nntp.coast.net!howland.reston.ans.net!newsfeed.internetmci.com!news.connectnet.com!max-lj-71.connectnet.com!user From: boxboy@connectnet.com (Kelly J. Thibault) Newsgroups: bionet.molbio.methds-reagnts Subject: electroporation of Streptomyces sp. Date: Mon, 04 Mar 1996 17:47:56 -0800 Organization: Boxboy Express Lines: 5 Message-ID: NNTP-Posting-Host: max-lj-140.connectnet.com X-Newsreader: Yet Another NewsWatcher 2.0.6b4 I am looking for any information on the electroporation of Streptomyces species. Any help you can give would be greatly appreciated. Please e-mail me directly. Thanks! Kelly From owner-methds-reagnts@net.bio.net Sun Mar 03 22:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!newsfeed.sunet.se!news00.sunet.se!sunic!mn6.swip.net!plug.news.pipex.net!pipex!weld.news.pipex.net!pipex!tank.news.pipex.net!pipex!iol!imci2!news.internetMCI.com!newsfeed.internetmci.com!chi-news.cic.net!ddsw1!news.mcs.net!van-bc!unixg.ubc.ca!dcwong From: dcwong@unixg.ubc.ca (Donald Chun Kit Wong) Newsgroups: bionet.molbio.methds-reagnts Subject: Can dessicants be dried in microwave? Date: 5 Mar 1996 02:09:53 GMT Organization: University of British Columbia, Vancouver, B.C., Canada Lines: 8 Message-ID: <4hg7ph$28q@nntp.ucs.ubc.ca> NNTP-Posting-Host: interchg.ubc.ca X-Newsreader: TIN [version 1.2 PL2] We have been using "DriRite" and silica gel as dessicant. Some bottles are sitting around with pink crystals since people never bothered to dry them again. Does anyone know if it's possible to dry / rejuvenate these in a microwave? Our laboratory ovens don't go up far beyond 100 C. Thanks. Donald Wong Pathology, U of B.C. From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!in1.uu.net!news.ner.bbnplanet.net!news3.near.net!yale!news.ycc.yale.edu!BIOMED!twilliam From: Tori Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Amersham Hybond N problem Date: Tue, 5 Mar 1996 15:42:05 EST Organization: Yale University Lines: 25 Message-ID: References: NNTP-Posting-Host: biomed.med.yale.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: I also used the Hybond-N recently and had a horrible time. According to the et br staining I had run the perfect gel with genomic dna but after transfering and probing and exposing I had a solid black rectangle. I called the company and they said that I need to soak the membrane in 5x SSC before probing. They also gave me some suggestions for stripping the filter but nothing worked. I was using unpurified 32P random hexanucleotide labeled DNA fragments. I soaked the membrane the second time around and reprobed but all I got was high background and hybridization to my mol wt markers. I am going to try to reprobe and see what happens. *************************************************************************** Tori D. Williams twilliam@biomed.med.yale.edu DST SK DK PHD2B SCI 0(+> LIVELONG... AGGIEPRIDE MAC = ME!!!! :) *************************************************************************** On Fri, 1 Mar 1996, Paul N Hengen wrote: > BRITTA SCHULZ (schulzb@pz350.ipsp.uni-hohenheim.de) wrote: > > : Who can help me to find out, which company produces the uncharged membrane > : formerly sold by Amersham as Hybond N ? > From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!daresbury!bioftp.unibas.ch!news.vub.ac.be!news.belnet.be!news.rediris.es!news.uoregon.edu!tank.news.pipex.net!pipex!swrinde!cssun.mathcs.emory.edu!hobbes.cc.uga.edu!news From: phillip buckhaults Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Chemiluminescence, western blotting, problems... Date: Tue, 05 Mar 1996 14:34:15 +0800 Organization: University of Georgia, Athens Lines: 24 Message-ID: <313BE067.2813@bscr.uga.edu> References: <4hhrse$ium@sage.cc.purdue.edu> NNTP-Posting-Host: l-pha.biochem.uga.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) Giampaolo Minetti wrote: > > Hello, > I am experiencing problems with Enhanced Chemiluminescence Detection > (Amersham ECL) and western blotting. > What happens is that after adding the ragent, incubating for 1 min and > taking the nitrocellulose into the cassette for exposure (I use to > cover the membrane with Saran Wrap), everything is OK. > After 2 or 3 minutes, however, the entire membrane is luminescent. The > bands I'm interested in are there, but you cannot take a good > exposure because of the extremely high background. (much ascii deleted) Giampaolo, do you drain your blot well after soaking in the ECL reagent? try letting all the liquid drain that will, touching the edge of the blot to the container to get the last bit off. anyway, this helped us. good luck. -phil From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!newsfeed.internetmci.com!in2.uu.net!brighton.openmarket.com!decwrl!news-server.ncren.net!mac218.ciit.org!user From: Sprankle@ciit.org (Cathy Sprankle) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Amersham Hybond N problem Date: Tue, 05 Mar 1996 15:24:32 +1000 Organization: Chemical Industry Institute for Toxicology Lines: 41 Message-ID: References: <1996Mar2.165022.8215@mcrcr6> NNTP-Posting-Host: mac218.ciit.org In article <1996Mar2.165022.8215@mcrcr6>, vilimf01@mcrcr6.med.nyu.edu wrote: > In article , schulzb@pz350.ipsp.uni-hohenheim.de (BRITTA SCHULZ) writes: > > Who can help me to find out, which company produces the uncharged membrane > > formerly sold by Amersham as Hybond N ? > > The problem is , they are now selling a new membrane, calling it Hybond N as > > well, but it is very different. However, since we have the new Hybond N all > > we get is black autoradiographs ( we are using the Dig-system) The new > > membrane is very thick and stiff, it doesn't look like nylon > > at all, and it is produced by another manufacturer. (snip) > > Britta > > I just recieved something from MSI (http://www.msifilters.com) > saying that Amersham had replaced its supplier and implied that that > its MAGNA membrane would give the same results as the the old Amersham > Hybond-N membranes. (snip) > Usual Disclaimers, SVEN Does anyone know about when these changes took place? We started having problems with our northerns last summer. We have always used Hybond N, and if there was a change in the membranes it would explain a great deal. (Of course, we weren't smart enough at the time to record stuff like lot numbers, etc.! Live and learn...) Thanks, Cathy -- Catherine Sprankle CIIT e-mail: sprankle@ciit.org Opinions expressed (such as they are!) are strictly mine and do not reflect those of CIIT. From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!surfnet.nl!swsbe6.switch.ch!swidir.switch.ch!in2p3.fr!univ-lyon1.fr!cri.ens-lyon.fr!news From: Olivier Gandrillon Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Touchdown PCR protocoll? Date: Tue, 05 Mar 1996 23:33:14 +0000 Organization: Ecole Normale Superieure de Lyon Lines: 54 Message-ID: <313CCF3A.6183@ens-lyon.fr> References: <4h198b$e88@gwdu19.gwdg.de> Reply-To: Olivier.Gandrillon@ens-lyon.fr NNTP-Posting-Host: mac-lr5-143b.ens-lyon.fr Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) Hi I have been using the following protocol with some (i.e. not always) success to get rid of contaminating bands in my PCR reactions: 95°C for 5 mn, then 72°C for 2 cycles (95°C, 30"; 72°C, 30"; 72°C, 45") then 70°C for 2 cycles 68°C for 2 cycles 66°C for 2 cycles 64°C for 2 cycles 62°C for 3 cycles 60°C for 3 cycles 58°C for 24 cycles 72°C for 10mn Goto 4°C Of course the final temperature has to be determined first: you want to use a temperature where you know you amplify your fragment of interest and the contaminating bands. Hope this helps. Olivier Volker Blaschke wrote: > > Dear netters, > > I am looking for advice on how to do touchdown PCR. My current protocoll > is 30 s @ 95 C, 1 min @ 63 C and 2 min @ 70 C. > I should get a product of 1550 bp, which is unfortunately only faint. The > main product is 500 bp with a huge band. Now, I was thinking that > touchdown PCR might increase specificity and rid me of the 500 bp > product. I am not sure if further increasing annealing temp would make > that much of a difference. > > What would you do for touchdown? > > I was thinking of starting at 70 C, but I am not sure what the decrease > in annealing temp should be per cycle ? Should I go down 1 C / cycle for > seven cycles and continue for another 28 cycles at 63 C, or should I > decrease at say 0.5 C for 14 cycles? > > Any input is most welcome! > > Volker > > -- > Dr. Volker Blaschke > Depts. of Dermatology, Biochemistry > Georg-August-University, Goettingen, Germany From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!chi-news.cic.net!nntp.coast.net!howland.reston.ans.net!torn!resunix.ri.sickkids.on.ca!news From: Marc Visconti Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PMSF timing Date: Tue, 05 Mar 1996 11:40:22 -0400 Organization: The Hospital For Sick Children Lines: 6 Message-ID: <313C6066.3E1@resunix.ri.sickkids.on.ca> References: <924F353101B938D9@office.mmc.org> NNTP-Posting-Host: resunix.ri.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) To: varyc.mmcri@mmc.org How are you preparing PMSF? You should call Sigma and get the specs on it, but PMSF works best when solubilized in anhydrous ethanol and added to buffers dropwise with slight agitation. I add PMSF prior to cell disruption and have no problems with degradation. marc From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!tank.news.pipex.net!pipex!peer-news.britain.eu.net!lyra.csx.cam.ac.uk!daresbury!not-for-mail From: "Michail P. Sahsamanoglou" Newsgroups: bionet.molbio.methds-reagnts Subject: Membrane properties Date: 5 Mar 1996 16:33:01 -0000 Lines: 7 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <4hhqbt$sn4@mserv1.dl.ac.uk> Comments: Authenticated sender is X-mailer: Pegasus Mail for Windows (v2.23) MIME-Version: 1.0 Original-To: methods@dl.ac.uk Hi Netters. These are my questions: Are PVDF membranes exclusively used for protein immunoblots and microsequencing or can they be used also for nucleic acid blotting? What are the differences regarding blot applications between NC, nylon and PVDF and what is the consensus for their use? I will appreciate all your answers. TIA Mike From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!swrinde!cssun.mathcs.emory.edu!news.cc.emory.edu!usenet From: Carol Stugard Newsgroups: bionet.molbio.methds-reagnts Subject: PCR-SSCP ON ABI 373 WITH GENESCAN 672 Date: 5 Mar 1996 18:01:41 GMT Organization: Emory University Lines: 14 Message-ID: <4hhvi5$fgp@moe.cc.emory.edu> NNTP-Posting-Host: alvin.gen.emory.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.methds-reagnts I am amplifying a PCR fragment that is 187 base pairs with dye amidite FAM on the foreward primer and HEX on the reverse. The protocol I am following for loading samples (after centricon 100) is 1 ul sample + 1ul 100mM naOH + 2.5 ul deionized formamide (with blue dextran) + 0.5 ul Genescan standard heated to 95 C for 5 minutes, put on ice 5 minutes. Native gel is 6.5% acrylamide+ 10% glycerol (in 1 X TBE). Gel runs 18 hours (not cooled) but temp does not exceed 25 C. There are 2 nuclear targets for this PCR. I am getting only 2 peaks which are both blue comigrating with green. There are no sinsle-stranded peaks that are either blue or green like you would expect from single strands. What can I try to make this gel system run true single strands? Is it the heating step? From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!ACD.TUSK.EDU!Prakash From: Prakash@ACD.TUSK.EDU (C. S. Prakash) Newsgroups: bionet.molbio.methds-reagnts Subject: Biotech NewsReport Date: 5 Mar 1996 12:45:04 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 32 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net A monthly newsletter "NBIAP News Report" that describes briefly the news and research items of interest to agricultural biotech community is available both in print and electronic version. For internet access to the News Report, textfiles, and databases use one of the following procedures. 1. Through WWW: http://www.nbiap.vt.edu/ 2. Use telnet or gopher to connect to ftp.nbiap.vt.edu 3. Use ftp to connect to ftp.nbiap.vt.edu. Use "anonymous" as your user-id, your email address as your password. Type "cd pub/nbiap". To have the News Report automatically emailed, send an email message to news@nbiap.biochem.vt.edu and type SUBSCRIBE NEWSREPORT in the message section. National Biological Impact Assessment Program (NBIAP) runs this Information Systems for Biotechnology, which is a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. It does not necessarily reflect the views of the U.S. Department of Agriculture or of Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge. . For Additional Information: P.L. Traynor, Editor, Information Systems for Biotechnology, 120 Engel Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308, tel: 540-231-2620, fax: 540-231-2614, email: nbiap@vt.edu From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!news.internetMCI.com!darwin.sura.net!nih-csl!postman From: bernard@elsie.nci.nih.gov (Bernard Murray) Subject: Re: Can dessicants be dried in microwave? Message-ID: <1996Mar5.191128.17821@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: <4hg7ph$28q@nntp.ucs.ubc.ca> Date: Tue, 5 Mar 1996 19:11:28 GMT Lines: 26 In article <4hg7ph$28q@nntp.ucs.ubc.ca>, dcwong@unixg.ubc.ca says... > >We have been using "DriRite" and silica gel as dessicant. Some bottles >are sitting around with pink crystals since people never bothered to dry >them again. Does anyone know if it's possible to dry / rejuvenate these >in a microwave? Our laboratory ovens don't go up far beyond 100 C. Thanks. > >Donald Wong >Pathology, U of B.C. > I don't think you have to use an oven "beyond 100 C" as you are drying the water out rather than boiling it. I leave a pot of silica gel in an oven kept at 60degC and this dries okay (eventually). If you are in a hurry and want to use the microwave then spread the crystals out thinly on a paper towel and microwave in brief bursts. If they start very damp you may have to change the towel. Be careful as the crystals can get very hot. In all cases avoid breathing the dust. Happy dessicatin' ! Bernard Bernard Murray, Ph.D. bernard@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA) From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!newsfeed.internetmci.com!in2.uu.net!news.tufts.edu!diamond.tufts.edu!skrishna From: Sanjay Krishnaswamy Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Can one gene be used to select for OR against.... Date: Tue, 5 Mar 1996 12:57:29 -0500 Organization: Tufts University Lines: 14 Message-ID: References: <4h5f48$cb5@hydra.cc.umb.edu> NNTP-Posting-Host: diamond.tufts.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: Actually, while the Tet resistance gene from Tn10 _can_ be selected against based on the conferred sensitivity to fusaric acid (or quinaldic acid, or some other reagents) I've found those results not clean. There is another method, I have to think about how it works but I think it involves exposing cells to tet (which stops tet sensitive cells from growing), then penicillin (which kills growing cells), then washing off the antibiotic. I think I've got a protocol for this somewhere if you're interested. _______________________________________________________________________________ Sanjay Krishnaswamy sanjay@bur.visidyne.com _______________________________________________________________________________ From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!daresbury!bioftp.unibas.ch!infobiogen.fr!jussieu.fr!oleane!tank.news.pipex.net!pipex!swrinde!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!martinlab From: klenchin@macc.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: electroporation of Streptomyces sp. Date: Tue, 05 Mar 96 15:53:29 GMT Organization: UW-Madison Lines: 14 Message-ID: <4hho1p$8rs_001@biochem.wisc.edu> References: NNTP-Posting-Host: martinlab.biochem.wisc.edu X-Newsreader: News Xpress Version 1.0 Beta #4 In article , boxboy@connectnet.com (Kelly J. Thibault) wrote: ->I am looking for any information on the electroporation of Streptomyces ->species. Any help you can give would be greatly appreciated. Please ->e-mail me directly. ->Thanks! ->Kelly Just do a medline (or something similar) search for the last ~ 5 years. I recall many reports. Also you can try to get from Bio-Rad (is it for free?) a comprehensive (or so they claim) brochure on electroporation techniques. - Dima From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!newsfeed.internetmci.com!in2.uu.net!newsflash.concordia.ca!news.nstn.ca!news.cs.indiana.edu!purdue!mozo.cc.purdue.edu!not-for-mail From: minetti@sage.cc.purdue.edu (Giampaolo Minetti) Newsgroups: bionet.molbio.methds-reagnts Subject: Chemiluminescence, western blotting, problems... Date: 5 Mar 1996 11:58:54 -0500 Organization: Purdue University Lines: 62 Message-ID: <4hhrse$ium@sage.cc.purdue.edu> NNTP-Posting-Host: sage.cc.purdue.edu Hello, I am experiencing problems with Enhanced Chemiluminescence Detection (Amersham ECL) and western blotting. What happens is that after adding the ragent, incubating for 1 min and taking the nitrocellulose into the cassette for exposure (I use to cover the membrane with Saran Wrap), everything is OK. After 2 or 3 minutes, however, the entire membrane is luminescent. The bands I'm interested in are there, but you cannot take a good exposure because of the extremely high background. I thought that I was using a too high concentration of secondary antibody (goat anti-rabbit, horseradish peroxidase-conjugated BioRad) so I switched from a 1:10000 dilution to 1:30000. The only effect was a longer delay before the background luminescence showed up. The problem is the same, no matter what the primary antibody is, and no matter what solution (5% BSA or 5% non fat milk) I use for blocking the membrane before the primary. Also, I'm using a brand new Amersham ECL reagent. Since it looked like the secondary antibody was damaged, I tried another Goat-anti-rabbit-HRP (Pierce), with exactly the same result. There is some problem related to the secondary antibody. I've tried to soak a piece of plain nitrocellulose into the reagent, and nothing happened. The washing buffer is a Tris/HCl ph 7.5, 150 mM NaCl, 0.05% Tween 20. What I know for sure is that the horseradish-luminol reaction is prone to artifactual results. The presence of heme or hemoglobin affects the result, because of a peroxidase-like activity of hemoglobin. (but in this case I don't have any hemoglobin in my samples!). Another amazing observation of mine is that if you have carbonic anhydrase in your gel (as often happens because C.A. is commonly used as a molecular weight marker in most of the commercial preparations) and you transfer the protein on nitrocellulose and you soak your nitrocellulose in the Chemiluminescence reagent (WITHOUT any primary or secondary antibody) and you expose the film, you'll end up with a wonderful band of 29-30 kDa. I feel that my problem is a contamination in one of the following: buffers, electrophoresis apparatus, blotting apparatus, BSA and, last but not least, DEIONIZED WATER As for the nature of this contaminant... no idea. (actually I'm strongly tempted to consider Fe contamination in my deionized H2O ?!?) If anyone else ran into the same problem, please help. Thank you very much for you attention Giampaolo . . ~..~. ~.~.~.~ ~.~.~:~ Giampaolo Minetti ~.:~ Purdue University \:/ Department of Chemistry /:\ 327 Wetherill Building /:.~\ West Lafayette IN-47906 /-~_~_\ Tel. 317-494-5275 (_______) ****************************^*^*^*^ minetti@sage.cc.purdue.edu From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!surfnet.nl!swsbe6.switch.ch!scsing.switch.ch!news.belwue.de!news.uni-stuttgart.de!news From: ifao@po.uni-stuttgart.de (Wayne Coco) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Hydropathy plot program Date: 5 Mar 1996 14:48:48 GMT Organization: Microbiology, Univ. Stuttgart, Germany Lines: 11 Message-ID: <4hhk8g$3bp2@info4.rus.uni-stuttgart.de> References: <1996Feb11.133100@vaxc.cc.monash.edu.au> <4g25a4$inb@erika.cica.es> NNTP-Posting-Host: imbpc7.biologie.uni-stuttgart.de X-Newsreader: WinVN 0.92.6+ > >| Was wondering if anyone can direct me to a program that will create >| hydropathy plots from amino acid seqeunces. > I believe older versions of DNA Stryder (for the Mac) are shareware. This small, but very handy program whips out RE sites, orfs, codon usage, hydropathy plots and more. MUCH simpler than PCgene. Wayne From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!howland.reston.ans.net!tank.news.pipex.net!pipex!peer-news.britain.eu.net!lyra.csx.cam.ac.uk!mole.bio.cam.ac.uk!cdw1000 From: cdw1000@mole.bio.cam.ac.uk (Christopher Walentas (Bioc)) Newsgroups: bionet.molbio.methds-reagnts Subject: Native PAGE @ pH < 6.5 Date: 5 Mar 1996 13:59:48 GMT Organization: University of Cambridge, England Lines: 16 Message-ID: <4hhhck$ae8@lyra.csx.cam.ac.uk> NNTP-Posting-Host: mole.bio.cam.ac.uk Keywords: Native PAGE X-Newsreader: NN version 6.5.0 #3 (NOV) I was wondering if anyone had a buffering system that they could recommend for the resolution of two proteins using native PAGE at a pH at or below 6.5? I've got a mixture of two species which differ by a series of histidines, and need to utilise their charge differences (which are only relevant below their pKa), but cannot go too acidic due their pI is around 4. I've tried citric acid at pH 5.5 (100mM) but with not much luck-- the proteins are 100kDa and hardly made it into the resolving gel by the time the BPBlue had scooted off the bottom. I've looked into a number of Practical Approach-type reference manuals, but for the most part they are too general and of little or no help. Any advice in this matter would be most appreciated. Cheers, tripp From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!agate!howland.reston.ans.net!tank.news.pipex.net!pipex!oleane!in2p3.fr!swidir.switch.ch!swsbe6.switch.ch!scsing.switch.ch!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!uni-erlangen.de!lrz-muenchen.de!win_ibelgaufts.lmb.uni-muenchen.de!user From: Ibelgauf@lmb.uni-muenchen.de (Horst Ibelgaufts) Newsgroups: bionet.molbio.methds-reagnts Subject: Why use 3´to5´exo-negative DNA-Polymerases? Date: Tue, 05 Mar 1996 10:51:43 +0100 Organization: Leibniz-Rechenzentrum, Muenchen (Germany) Lines: 20 Distribution: world Message-ID: NNTP-Posting-Host: win_ibelgaufts.lmb.uni-muenchen.de Dear fellow netters, maybe it is a stupid question, but I have been wondering about the rationale behind using DNA polymerases specifically designed to be devoid of a 3´ to 5´ exonuclease activity (such as Sequenase or Taquenase). Is it that these enzymes have a higher processivity and polymerize faster? thanks for any enlightenment :-) and greetings from sunny bright (but cold) Munich Horst -- H IBELGAUFTS http://www.lmb.uni-muenchen.de/groups/ibelgaufts/cytokines.html _______________________________________________________________ Ultra posse nemo obligatur _______________________________________________________________ From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!FYS.RULIMBURG.NL!F.Schaap From: F.Schaap@FYS.RULIMBURG.NL (Frank Schaap) Newsgroups: bionet.molbio.methds-reagnts Subject: re: rat cardiomyocyte cell line Date: 5 Mar 1996 04:19:59 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <01I1ZBJNU8O2000941@RL0002.RULIMBURG.NL> NNTP-Posting-Host: net.bio.net Ed Castro wrote: Hello everyone, I am wondering whether anyone has information concerning the availability of a rat cardiomyocyte cell line. Any information is greatly appreciated. Thanks in advance, Edmundo Edmundo, A clonal cell line derived from embryonic rat heart tissue has been described by Kimes and Brandt (ExpCellRes 98:367 1976). The cell line, H9c2, has both cardiac muscle as well as skeletal muscle properties. This cell line is employed by many researchers and is available through ATCC. Caviedes et al. described a cell line, RCVC, derived from rat ventricular cells (JMolCellCardiol 25:829 1993.) Kind Regards, Frank From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!tank.news.pipex.net!pipex!warwick!bsmail!usenet From: Harry.Witchel@bristol.ac.uk (Harry J. Witchel) Subject: Re: Can dessicants be dried in microwave? Content-Type: Text/Plain; charset=US-ASCII Message-ID: Sender: usenet@uns.bris.ac.uk (Usenet news owner) Nntp-Posting-Host: rm2.pys.bris.ac.uk Reply-To: harry.witchel@bristol.ac.uk Organization: Physiology / Med. School / Bristol X-Newsreader: WinVN 0.99.6 References: <9602058260.AA826071120@smtpgwy.agric.nsw.gov.au> Mime-Version: 1.0 Date: Tue, 5 Mar 1996 11:38:48 GMT Lines: 23 Dear Michelle -- This is really interesting! Can you really dry flowers and herbs in a microwave (rather than cooking them)? How does that work; do you just use a very low setting? Harry In article <9602058260.AA826071120@smtpgwy.agric.nsw.gov.au>, Michelle.Gleeson@SMTPGWY.AGRIC.NSW.GOV.AU says... > > Dear Donald, > > I have never tried the microwave method, but as you can dry herbs > and flowers in a microwave it may well work. There is only one way to > find out whether or not it wrecks the microwave ;) Anyway, several > hours in a lab oven at 80C is hot enough - when the silica gel > crystals are bright blue again they are dry. > > > Michelle > gleesom@agric.nsw.gov.au > From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!agate!newsxfer2.itd.umich.edu!gatech!gt-news!cc.gatech.edu!cssun.mathcs.emory.edu!swrinde!howland.reston.ans.net!nntp.coast.net!harbinger.cc.monash.edu.au!bunyip.cc.uq.oz.au!munnari.OZ.AU!metro!metro!wabbit.its.uow.edu.au!mac599c.uow.edu.au!Mark Dowton From: mark_dowton@uow.edu.au (Mark Dowton) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Can dessicants be dried in microwave? Followup-To: bionet.molbio.methds-reagnts Date: Wed, 06 Mar 1996 14:42:03 +1000 Organization: University of Wollongong Lines: 22 Distribution: world Message-ID: References: <9602058260.AA826071120@smtpgwy.agric.nsw.gov.au> NNTP-Posting-Host: mac599c.uow.edu.au I have dried dessicant in small lots (1-2 g) in a microwave. It works well, but obviously best if it the dessicant is well spread out. Otherwise, the evaporating water gets reabsorbed by surrounding dessicant. It is quite nice to watch it rapidly turn bright blue. One caution, use a glass container, not plastic, as the latter sometimes melts. Mark_Dowton@uow.edu.au In article <9602058260.AA826071120@smtpgwy.agric.nsw.gov.au>, Michelle.Gleeson@SMTPGWY.AGRIC.NSW.GOV.AU wrote: > Dear Donald, > > I have never tried the microwave method, but as you can dry herbs > and flowers in a microwave it may well work. There is only one way to > find out whether or not it wrecks the microwave ;) Anyway, several > hours in a lab oven at 80C is hot enough - when the silica gel > crystals are bright blue again they are dry. > > > Michelle > gleesom@agric.nsw.gov.au From owner-methds-reagnts@net.bio.net Mon Mar 04 22:00:00 1996 Path: biosci!daresbury!