From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: microplate@aol.com (MICROPLATE) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: San Diego Molecular Biology Conf Date: 2 Jul 1995 14:48:55 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 3 Sender: root@newsbf02.news.aol.com Message-ID: <3t6pmn$3g2@newsbf02.news.aol.com> References: Reply-To: microplate@aol.com (MICROPLATE) NNTP-Posting-Host: newsbf02.mail.aol.com Please send me details of the Conf and call for papers. Roy L. Manns Polyfiltronics Inc 100 weymouth street Rockland Ma 02370 USA TKS. Roy Manns Polyfiltronics home From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!rutgers!gatech!news.sprintlink.net!news1.channel1.com!news.channel1.com!channel1!harold.drabkin Distribution: world Newsgroups: bionet.molbio.methds-reagnts Subject: glycerol From: harold.drabkin@channel1.com (Harold Drabkin) Message-ID: <40.14530.3148@channel1.com> References: <3stjlc$f13@neuro.usc.edu> Date: Sun, 02 Jul 1995 00:10:00 -0500 Organization: Channel 1(R) 617-864-0100 Info Lines: 17 Subject: glycerol W>t of glycerol in most of the commercial restriction W>enzymes? I want to add glycerol to my protein samples to prevent W>freezing at -20 C. Thanks for the info. W>-- W>--------------------------------------------------------------------- W>William Sun, Ph.D. Phone: (213)740-3406 W>Hedco Neuroscience Building Fax: (213)740-5687 W>University of Southern California Pager: (310)499-8670 The info is usually supplied in the catalogs; mostly 50% --- * OLXWin 1.00 * Back Up My Hard Drive? I Can't Find The Reverse Switch! From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!rutgers!gatech!news.sprintlink.net!news1.channel1.com!news.channel1.com!channel1!harold.drabkin Distribution: world Newsgroups: bionet.molbio.methds-reagnts Subject: CAT assay question From: harold.drabkin@channel1.com (Harold Drabkin) Message-ID: <40.14529.3148@channel1.com> References: Date: Sun, 02 Jul 1995 00:10:00 -0500 Organization: Channel 1(R) 617-864-0100 Info Lines: 22 Subject: CAT assay question E>Following the TLC of a CAT reaction, four (or a subset of four) spots might E>be visualzed. These correspond to unacetylated chloramphemicol, two E>different species of mono-acetylated chloramphenicol, and di-acetylated E>chloramphenicol. The presence of di-acetylated chloramphenicol probably E>indicates that the reaction has preceeded too far, and is not useful for E>quantitation. E>My question is: When calculating the "percent conversion" for a particular E>reaction, do you take into account both mono-acetylated species, or just E>the top most form (the more abundant form)? Is the issue actually just a E>matter of consistency in your number crunching from assay to assay? I always include the two monospecies together. One of them (the faint one) is due to (as I remember) a non-enzymatic rearrangement. Thus, it is valid to do so. If the reacton gives diacetyl, it's off linear range anyway. --- * OLXWin 1.00 * All wiyht. Rho sritched mg kegtops awound? From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!agate!spool.mu.edu!uwm.edu!msunews!harbinger.cc.monash.edu.au!news.cs.su.oz.au!inferno.mpx.com.au!news.unimelb.EDU.AU!ucsvc.ucs.unimelb.edu.au!wehi.edu.au!woozle.mel.dbe.CSIRO.AU!mel.dit.csiro.au!its.csiro.au!news Newsgroups: bionet.molbio.methds-reagnts Subject: cytomegalovirus promoter Message-ID: From: D.Adelson@prospect.anprod.csiro.au (David Adelson) Date: Fri, 30 Jun 1995 04:54:39 GMT Sender: news@its.csiro.au (News Manager) Organization: CSIRO Division of Animal Production Nntp-Posting-Host: medea.prospect.anprod.csiro.au X-Newsreader: NetSuite News for OS/2 [version: 3.3j] Lines: 17 G'day netters, Is there anyone out there who can provide me with a plasmid containing the cytomegalovirus promoter? I need it to drive expression of green fluorescent protein in sheep cells. I know that Clontech sells something I might be able to adapt to my purpose called pCMVbeta, but they want A$793 for 25 micrograms which I would rather not have to fork out. thanks in advance, ****************************************************** *Dave Adelson CSIRO Divison of Animal Production* *"What, me worry?" Sydney Lab, Sydney, AUSTRALIA * *ALL OPINONS ARE MY OWN, USUAL DISCLAIMER APPLIES * ****************************************************** From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!agate!news.duke.edu!news-feed-1.peachnet.edu!hobbes.cc.uga.edu!gnti.biochem.uga.edu!user From: buckhaults@bscr.cc.uga.edu (phillip buckhaults) Newsgroups: bionet.molbio.methds-reagnts Subject: Promoter Finder Kit from Clontech Date: Sat, 01 Jul 1995 17:47:27 -0500 Organization: uga Lines: 13 Distribution: world Message-ID: Reply-To: Buckhaults@bscr.uga.edu NNTP-Posting-Host: gnti.biochem.uga.edu Has anyone experience with Clontech's Promoter-Finder kit for PCR walking along the human genome? We have been working with it for the past few months, and have never gotten their positive control to work. Phillip Buckhaults Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center Life Science Building University of Georgia Athens Georgia 30602 (706) 542-1701 ________________________________________________________________________________ "Let us not confuse effort with achievement" From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!rutgers!gatech!swrinde!news.uh.edu!mac-918.sr2-building.uh.edu!user From: biolc4@jetson.uh.edu (Wenfu) Newsgroups: bionet.molbio.methds-reagnts Subject: Why probe hybridizes to vector? Followup-To: bionet.molbio.methds-reagnts Date: 2 Jul 1995 22:37:53 GMT Organization: UH Dept of Biol Lines: 8 Distribution: world Message-ID: NNTP-Posting-Host: mac-918.sr2-building.uh.edu When I was using isolated DNA fragment( I am sure no any vector sequence in it) to probe digested plasmids, I found my probe hybridizes to the vector. This kind of thing happen to me several times when I was doing the similar thing. Although it did not matter for my results, I am really curious about the reasons for. Any idea would be appreciated. From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!agate!news.duke.edu!news-feed-1.peachnet.edu!gatech!news.uoregon.edu!vixen.cso.uiuc.edu!usenet.ucs.indiana.edu!bronze.ucs.indiana.edu!ellenmq From: ellenmq@ucs.indiana.edu ( ) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR cloning Date: 3 Jul 1995 02:51:18 GMT Organization: Indiana University Lines: 23 Message-ID: <3t7lv6$17a@usenet.ucs.indiana.edu> References: <3scee1$cqs@news.ycc.yale.edu> NNTP-Posting-Host: bronze.ucs.indiana.edu X-Newsreader: TIN [version 1.2 PL2] Junhyong (junhyong_kim@quickmail.cis.yale.edu) wrote: : Hi, : I heard somewhere that there are now other commercial T-tailed vectors : available for PCR product cloning than TA-cloning kit from Invitrogen. : Has anybody tried others? Is it cheaper? : We tried making some but the results are rather variable... We've used Novagen's T7-Blue TA cloning vector with good success. You don't need the "kit" just buy the vector which makes it less expensive. They recommend using NovaBlue E.coli which has the same genotype as Stratagene's XL1-Blue cells. It's not necessary to use that particular E.coli strain, in fact we used a regular cloning strain of E.coli called DH11SF' available commercially from Gibco/BRL Life technologies. Alternatively standard DH5alpha cells should also be fine for propagation. You can check out Novagen's www site at this address: http://www.novagen.com/ for more information on this vector if you have a Web browser like Netscape available to you; they have both their catalog, vector sequences and plasmid maps available. -Ellen Quardokus, Indiana University, Dept. of Biology From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!agate!spool.mu.edu!uwm.edu!msunews!harbinger.cc.monash.edu.au!yoyo.aarnet.edu.au!goliath.camtech.com.au!news From: Rick Tearle Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Storage temperatures Date: 1 Jul 1995 13:02:28 GMT Organization: CAMTECH (SA) Pty Ltd Lines: 6 Message-ID: <3t3h14$bhu@goliath.camtech.com.au> References: NNTP-Posting-Host: ppp54.camtech.com.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: nbai@teleport.com X-URL: news:nbai.12.00088C53@teleport.com Dear Ted, I know about the problems of repeated temperature shifts between 0 and -20C, but what happens at -70C? Rick From owner-methds-reagnts@net.bio.net Sat Jul 01 23:00:00 1995 Path: biosci!CAMD2.KKPCR.RE.KR!yskim From: yskim@CAMD2.KKPCR.RE.KR Newsgroups: bionet.molbio.methds-reagnts Subject: (none) Date: 2 Jul 1995 22:45:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <9507030536.AA27978@camd2.kkpcr.re.kr> NNTP-Posting-Host: net.bio.net Dear Netters : I'm planning to collect or clone only 'promoter region' from plant. In order to detour many tackles and to save time, I wanna use PCR tools. Some consensus sequences were known in plant promoter region. So I intend to use that sequence as a primer. But I have problems. 1) Those sequences are too short to be used as a primer( < 8 bases). 2) Only one-side primer ( consensus region ) will be constructed, how can I construct the other side primer? And inform me how to construct 'promoter library' from plant. Thank in advances. Kim, Yong-Sig Internet : yskim@camd2.kkpcr.re.kr From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!cs.utexas.edu!news.sprintlink.net!cam.news.pipex.net!pipex!edi.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!yama.mcc.ac.uk!liv!flynna From: flynna@liverpool.ac.uk (Mr A.M. Flynn) Subject: myosin purification Message-ID: Sender: news@liverpool.ac.uk (News System) Nntp-Posting-Host: fir-12.liv.ac.uk Organization: The University of Liverpool X-Newsreader: TIN [version 1.2 PL2] Date: Mon, 3 Jul 1995 16:45:56 GMT Lines: 13 Hello, I was wondering whether anyone has any experience in purifying type II myosin, (non-muscle). I have already attempted this but, aparently, got a very poor recovery. I used liver as my starting material, so perhaps this isn't the best source. Is this protein prone to protease degradation? If someone could send me a reference containing a purification protocol, with any pertinent points such as preferred choice of starting material, or whether all classes of myosin will come through the purification method I would be extremely grateful. Thanks very very much! From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!swrinde!hookup!news.mcgill.ca!bio182 From: bg2z@musicb.mcgill.ca (Dino DeAngelis) Newsgroups: bionet.molbio.methds-reagnts Subject: biological/biomedical journal rankings: available on the net? Date: 3 Jul 1995 16:29:31 GMT Organization: McGill University Lines: 5 Message-ID: <3t95tb$gau@sifon.cc.mcgill.ca> NNTP-Posting-Host: 132.206.101.182 X-Newsreader: News Xpress Version 1.0 Beta #3 Is there a site on the net where I can find the updated ranking of biological publications ? If not on the net, where and how could I find it? Thank you so much in advance! Dino From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!news.uoregon.edu!news.dacom.co.kr!news.kreonet.re.kr!usenet.kornet.nm.kr!ames!waikato!news.massey.ac.nz!sysadmin From: "R.D.Johnson" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: laying membranes on bacteria plates Date: 4 Jul 1995 05:21:00 GMT Organization: Massey University, New Zealand Lines: 17 Message-ID: <3taj3s$a08@cc-server9.massey.ac.nz> References: <9506301903.AA22337@compbio2.med.wayne.edu> NNTP-Posting-Host: cc-cs-mg2-dip2-6.massey.ac.nz Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: jeff@COMPBIO2.MED.WAYNE.EDU X-URL: news:9506301903.AA22337@compbio2.med.wayne.edu A quick (not dirty) method is as follows. Place nylon membrane on the agar plate surface. plate the bacteria directly on the membrane at dilution of choice. Grow overnight or as usual until good colonies are clear. Lift filters from plate and place (bacteria side up) on a few Mls denaturation soln on a bench for 2 minute. Transfer filter to a few Mls of neutralisation soln and leave 5 min. remove filter allow to dry and then back in the microwave at high power for one minute to fix DNA. Filter is ready to probe. Good luck Rich From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!news.sprintlink.net!europa.chnt.gtegsc.com!newsxfer.itd.umich.edu!news.cic.net!infoserv.illinois.net!brutus.bright.net!usenet.eel.ufl.edu!usenet.cis.ufl.edu!usenet.ufl.edu!NewsWatcher!user From: smerage@cmg.health.ufl.edu (Jeffrey Smerage) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Stripping Hybond membranes Date: Mon, 03 Jul 1995 12:43:51 -0500 Organization: University of Florida Lines: 7 Message-ID: References: NNTP-Posting-Host: 128.227.142.163 I regularly strip Hybond membranes, and yes you should loose a little of your DNA. I have never tried to quantitate my losses although they don't make a significant different difference in my subsequent exposure times. I uses a 0.1X SSC, 0.1% SDS solution at 95°C twice to strip my membranes. This came from a Biotechniques article titled "Evaluation of DNA Probe Removal from Nylon Membrane", Biotechniques, 1992, 13:572-575. From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!news.uoregon.edu!news.dacom.co.kr!news.kreonet.re.kr!usenet.kornet.nm.kr!ames!waikato!news.massey.ac.nz!sysadmin From: "R.D.Johnson" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Why probe hybridizes to vector? Date: 4 Jul 1995 05:32:53 GMT Organization: Massey University, New Zealand Lines: 8 Message-ID: <3tajq5$a08@cc-server9.massey.ac.nz> References: NNTP-Posting-Host: cc-cs-mg2-dip2-6.massey.ac.nz Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: biolc4@jetson.uh.edu X-URL: news:biolc4-020795165223@mac-918.sr2-building.uh.edu Often, when gel purifying a fragment from a vector such a pUC a residual amount of pUC is carried over which although not detectable ona gel is enough to be labelled and show up on an autorad. Rich From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!news.uoregon.edu!news.dacom.co.kr!news.kreonet.re.kr!usenet.kornet.nm.kr!ames!waikato!news.massey.ac.nz!sysadmin From: "R.D.Johnson" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Why probe hybridizes to vector? Date: 4 Jul 1995 05:32:42 GMT Organization: Massey University, New Zealand Lines: 8 Message-ID: <3tajpq$a08@cc-server9.massey.ac.nz> References: NNTP-Posting-Host: cc-cs-mg2-dip2-6.massey.ac.nz Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: biolc4@jetson.uh.edu X-URL: news:biolc4-020795165223@mac-918.sr2-building.uh.edu Often, when gel purifying a fragment from a vector such a pUC a residual amount of pUC is carried over which although not detectable ona gel is enough to be labelled and show up on an autorad. Rich From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!gatech!usenet.eel.ufl.edu!usenet.cis.ufl.edu!usenet.ufl.edu!gnv.ifas.ufl.edu!tanli From: tanli@gnv.ifas.ufl.edu (LI, TANG) Newsgroups: bionet.molbio.methds-reagnts Subject: P1 cloning Message-ID: <1995Jul3.112841.6737@tower> Date: 3 Jul 95 11:28:41 -0500 Lines: 14 Hi, Does anyone know Dr. Nat sternberg's email address? I would like to contact with him and ask for some help in P1 cloning system.Some people told me that Dupont Com. sells P1 vector . But when I caled them , they said' no' now. They did sold before. I try to use P1 to create a library. However I don't know where I can get or buy the P1 vector and packing matterial. Any help will be appreciated. Tang Li Dept of Pathobiology College of Vet. Med. Sci. From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!gatech!news-feed-1.peachnet.edu!news.netins.net!news.dacom.co.kr!news.kreonet.re.kr!worak.kaist.ac.kr!biochem.kaist.ac.kr!hbkim From: hbkim@biochem.kaist.ac.kr (Hanbok Kim) Newsgroups: bionet.molbio.methds-reagnts Subject: protease isolation? Date: 3 Jul 1995 23:55:51 GMT Organization: Biochemistry Lab, KAIST Lines: 8 Message-ID: <3ta027$od5@worak.kaist.ac.kr> NNTP-Posting-Host: biochem.kaist.ac.kr X-Newsreader: TIN [version 1.2 PL2] I try to clone protease gene from the environment. It seems to be important how selevtive conditions were chosen, such as pH of media and culture temperature. Effective methods to clone it or references will be greatly appreciated. Han, hbkim@biochem.kaist.ac.kr From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!lll-winken.llnl.gov!venus.sun.com!nntp-hub2.barrnet.net!news.Stanford.EDU!amy9.Stanford.EDU!rruss From: Ray Russ Newsgroups: bionet.virology,bionet.molbio.proteins,bionet.immunology,bionet.molbio.methds-reagnts,bionet.cellbiol Subject: gp-120 participant has questions... Date: Mon, 3 Jul 1995 13:44:55 -0700 Organization: Stanford University Lines: 29 Message-ID: References: NNTP-Posting-Host: amy9.stanford.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: Xref: biosci bionet.virology:3765 bionet.molbio.proteins:4982 bionet.immunology:4716 bionet.molbio.methds-reagnts:30398 bionet.cellbiol:2508 I was one of a group of participants in a phase II gp-120 trial which was administered by San Francisco General Hospital/UCSF several years ago. I am curious if anyone out there knows how long I will continue to test "indeterminate" on Western Blot, Elysa (sp?) and PCR. Any answers/suggestions/input would be greatly appreciated. Please keep in mind that I am for all practical pourposes a neophyte as far as immunology goes and was just a volunteer so please take it easy on the technical aspects - thanks. ______________________________________________________________________________ Ray Russ Stanford Linear Accelerator Center Operational Health Physics Department ______________________________________________________________________________ From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: wzhao80850@aol.com (WZhao80850) Newsgroups: bionet.molbio.methds-reagnts Subject: P24/CD9 western antibody! Date: 3 Jul 1995 16:19:14 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 6 Sender: root@newsbf02.news.aol.com Message-ID: <3t9jc2$ngv@newsbf02.news.aol.com> Reply-To: wzhao80850@aol.com (WZhao80850) NNTP-Posting-Host: newsbf02.mail.aol.com Hi there, I am looking for a western blotting antibody for P24/CD9 protein, which is a member of membrane protein family. Any information is grateful. Thanx in advance! Wei From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!nntp.gmd.de!news.rwth-aachen.de!news.rhrz.uni-bonn.de!news.uni-stuttgart.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!fauern!lrz-muenchen.de!ipp-garching.mpg.de!genmac8.biochem.mpg.de!user From: spang@vms.biochem.mpg.de (Anne Spang) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Western help needed! Date: 3 Jul 1995 18:18:00 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 13 Message-ID: References: <3t2kio$7td@newsbf02.news.aol.com> NNTP-Posting-Host: genmac8.biochem.mpg.de Hi Wei! If your bands are curved on the western blot, they should also be curved on the SDS-Page. So, your first problem might be the SDS-page itselfs. Do you have high salt concentrations or detergents in your sample? What about your blotting efficiency? Is your protein an abundant protein? Can you overexpress or enrich it as control of your Westerns? What about other antibodies? Do they give a simliar faint signals? I often use antibodies in a very high concentration and I am working with proteins which are not very abundant in the cell... I do not think that your problem primarily is related to the ECL-Kit. .. but please give more informations about your problem! Anne From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!emory!darwin.sura.net!fconvx.ncifcrf.gov!fcsparc6!pnh From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) Subject: Boomerang DNA Amplification Images Message-ID: Summary: Where to get the information and images Sender: Paul N. Hengen Nntp-Posting-Host: fcsparc6.ncifcrf.gov Organization: Frederick Cancer Research and Development Center Distribution: bionet Date: Mon, 3 Jul 1995 19:16:51 GMT Lines: 44 Some people were trying to contact me about getting the Boomerang DNA amplification images which originally came from Kevin Ahern (ahernk@bcc.orst.edu). However, since he sometimes does not reply to personal e-mail, you can now get the information from his form letter (boomerang.txt) explaining the history of the boomerang technique and the self-explanatory picture (either bda.gif or bda.tif) from my ftp site at ftp.ncifcrf.gov in the directory pub/methods/boomerang. Interestingly, the method only requires one primer and is therefore NOT covered by the existing U.S. patent on DNA amplification using a set of two primers (PCR). For additional information on similar techniques, see the references below. Happy Boomeranging!!! @article{Arnold1991, author = "C. Arnold and I. J. Hodgson", title = "Vectorette {PCR}: a novel approach to genomic walking", journal = "PCR Methods and Applications", volume = "1", pages = "39-42", comment = "vectorette PCR", year = "1991"} @article{Devon1995, author = "R. S. Devon and D. J. Porteous and A. J. Brookes", title = "Splinkerettes -- improved vectorettes for greater efficiency in {PCR} walking", journal = "Nucleic Acids Res.", volume = "23", number = "9", pages = "1644-1645", comment = "vectorette PCR", year = "1995"} ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - - - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/ - - - - - - * * - - - Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - * ******************************************************************************* From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!rockyd!notes From: Sean Stevens Subject: Re: Isol. of Transcription factors X-Nntp-Posting-Host: roeder_mac3.rockefeller.edu Content-Type: text/plain; charset=iso-8859-1 Message-ID: To: schraute@uni-bonn.de Sender: notes@rockyd.rockefeller.edu (News Administrator) Content-Transfer-Encoding: 8bit Organization: The Rockefeller University References: Mime-Version: 1.0 Date: Tue, 4 Jul 1995 04:14:41 GMT X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-Url: news:schraute-0307951848380001@rhrz-ts1-p9.rhrz.uni-bonn.de Lines: 49 schraute@uni-bonn.de (Bernhard Schrautemeier) wrote: >I am looking for a method for positive selection of (cyano)bacterial >transcription factors expressed in (E. coli) gene libraries to isolate the >corresponding gene. > >Also I heard that it is possible to bind promoter-containing DNA sequences >to certain matrices (e. g. latex; J. Biomater. Sci. Polym. Ed. 5(4), >293-302, 1994; I don´t have access to this journal) and thus to >selectively purify binding proteins, e.g. for use in footprinting. > >I would be glad to get some help! > >-- >____________________________________________________________ >Bernhard Schrautemeier Tel: +49-228-73-5277 or 5536 >Botanisches Institut Fax: +49-228-73-5513 >der Universitaet Bonn E-mail: schraute@uni-bonn.de >Kirschallee 1 x________________________________ >53115 Bonn x >Germany x >___________________________x Bernhard- There is a much easier method that should work for you. It uses matrix attachment, but reverses it to be the bacterial proteins bound to the nitrocellulose and then you probe this with the radiolabelled DNA of interest. Thus you would express the library proteins, stick them to the filters, denature, renature, and expose to DNA, wash and use the spots on the blots to pick the bacteria bearing the desired gene. A few rounds of purification and you should have it. The technique is referred to as -southwestern screening- and an explanation and protocol is found in 'Current Protocols' or 'The Red Book' as it is sometimes called. I can supply you with references besides. To biochemically isolate factors I would strongly suggest columns work- you may have to fractionate initially using phosphocellulose or a similar matrix, then use a DNA affinity method ie oligos covalently linked to agarose. Descriptions can be found in papers out of the Roeder or Tjian labs. I can also look them up for you if you so desire. Many standard methods books such as the 'Practical Approach' series also contain these methods in detail. Good luck and e-mail me with any more comments or queries. Sean Stevens The Rockefeller University stevens@rockvax.rockefeller.edu From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!swrinde!emory!gatech!darwin.sura.net!wvnvms!xunfang From: xunfang@wvnvms.wvnet.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA purification from agarose gels Message-ID: <1995Jul3.223558.14926@wvnvms> Date: 3 Jul 95 22:35:58 EDT References: <3sp9rk$1tp@lyra.csx.cam.ac.uk> Organization: West Virginia Network for Educational Telecomputing Lines: 17 In article <3sp9rk$1tp@lyra.csx.cam.ac.uk>, rw200@cus.cam.ac.uk (R. Woodward) writes: > Dear all > I would like to canvas opinion on the best kit or perhaps method to use > when purifying DNA fragments from agarose gels, at the moment we use the > 'EluQuik' kit from S&S the yield is good and so is the purity but its > alittle fiddly especially if you perform alot of isolations at one time > . I want to use the purified fragments in ligation reactions. > Thanks to all Robert > > R.Woodward > Dept Pharmacology > Tennis Court Rd > University of Cambridge > Email rw200@cus.cam.ac.uk Try Qiaquick Gel Extraction Kit from Qiagen. It takes only 20 minutes for one sample. The recovery is around 50% or more. From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-stuttgart.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!fauern!gs.dfn.de!news.gwdg.de!news From: pwolff@willow.mpiz-koeln.mpg.de (Patricia) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Glass Beads Date: 3 Jul 1995 13:16:47 GMT Organization: MPI fuer Zuechtungsforschung Lines: 13 Message-ID: <3t8qjv$p34@gwdu19.gwdg.de> References: <1995Jun15.160450.1@hkucc.hku.hk> NNTP-Posting-Host: mac38.mpiz-koeln.mpg.de X-Posted-From: InterNews 1.0.7@mac38.mpiz-koeln.mpg.de X-Authenticated: pwolff on FTP host willow.mpiz-koeln.mpg.de Hi Shannon, Working on yeast I used acid-washed Glass Beads (by Sigma-Aldrich) to break up the cells...Is it that, what you wanted to know? Hope I could help you... Patricia Patricia Wolff MPI fuer Zuechtungsforschung koeln E-mail: pwolff@mpiz-koeln.mpg.de From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!nntp.gmd.de!news.rwth-aachen.de!news.rhrz.uni-bonn.de!rhrz-ts1-p9.rhrz.uni-bonn.de!user From: schraute@uni-bonn.de (Bernhard Schrautemeier) Newsgroups: bionet.molbio.methds-reagnts Subject: Isol. of Transcription factors Date: Mon, 03 Jul 1995 18:48:38 +0200 Organization: Bot. Inst. Univ. Bonn Lines: 20 Message-ID: NNTP-Posting-Host: rhrz-ts1-p9.rhrz.uni-bonn.de I am looking for a method for positive selection of (cyano)bacterial transcription factors expressed in (E. coli) gene libraries to isolate the corresponding gene. Also I heard that it is possible to bind promoter-containing DNA sequences to certain matrices (e. g. latex; J. Biomater. Sci. Polym. Ed. 5(4), 293-302, 1994; I don´t have access to this journal) and thus to selectively purify binding proteins, e.g. for use in footprinting. I would be glad to get some help! -- ____________________________________________________________ Bernhard Schrautemeier Tel: +49-228-73-5277 or 5536 Botanisches Institut Fax: +49-228-73-5513 der Universitaet Bonn E-mail: schraute@uni-bonn.de Kirschallee 1 x________________________________ 53115 Bonn x Germany x ___________________________x From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!ieunet!maths.tcd.ie!news.tcd.ie!acer.gen.tcd.ie!dbarton From: dbarton@acer.gen.tcd.ie (Dr David E Barton) Subject: Primer Sets for Linkage Analysis Message-ID: Sender: dbarton@acer.gen.tcd.ie (Dr David E Barton) Organization: Irish National Centre for Medical Genetics Date: Mon, 3 Jul 1995 12:28:57 GMT Lines: 12 I am looking for sources of primer sets for genetic linkage searches. I am aware of the Research Genetics set (and would welcome comments on it) but I want to know if there are other sources. They need to be available with fluorescent labels, as I am using an ALF. Thanks for your help, David. | David Barton | National Centre for Medical Genetics | Our Lady's Hospital for Sick Children | Crumlin, Dublin 12, Ireland. | Tel 455 0515 Fax 455 8873 From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!spool.mu.edu!bloom-beacon.mit.edu!news.starnet.net!wupost!news.utdallas.edu!corpgate!bcarh189.bnr.ca!nott!cunews!freenet.carleton.ca!FreeNet.Carleton.CA!ax041 From: ax041@FreeNet.Carleton.CA (Rachelle Leger) Subject: Stripping Hybond membranes Message-ID: Sender: ax041@freenet.carleton.ca (Rachelle Leger) Reply-To: ax041@FreeNet.Carleton.CA (Rachelle Leger) Organization: The National Capital FreeNet Date: Mon, 3 Jul 1995 11:07:11 GMT Lines: 15 I seem to loose material from my blot when I strip Hybond N Plus membranes Is is difficult to know for sure that this is really happening but when I reuse it the next time I need to expose much longer. Is this your experience? I strip by pouring boiling .1% SDS and .1% SSC on the membrane and skaking for 10 minutes. Thanks for any response Rachelle Leger McGill Cancer Centre McGill University Leger@medcor.mcgill.ca ax401@freenet.carleton.ca From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!BGUMAIL.BGU.AC.IL!raveh From: raveh@BGUMAIL.BGU.AC.IL (Dina Raveh BGU) Newsgroups: bionet.molbio.methds-reagnts Subject: Call for LacZ fragment for terminal bash Date: 3 Jul 1995 06:21:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <3t7lv6$17a@usenet.ucs.indiana.edu> We have a protein whose DNA sequence ends at +2. Does anyone have a lacZ gene which we could blunt onto this and get readthru? Thanks, Dina From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!news.sprintlink.net!cam.news.pipex.net!pipex!edi.news.pipex.net!pipex!warwick!griffin.nott.ac.uk!ppdirg.nottingham.ac.uk!user From: pdzirg@vme.nott.ac.uk (Ian Graham) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: help: cloning oligos Date: 3 Jul 1995 16:52:19 GMT Organization: Nottingham University Lines: 28 Message-ID: References: <3t59u9$gva@ari.net> NNTP-Posting-Host: ppdirg.nottingham.ac.uk In article <3t59u9$gva@ari.net>, Soon Paik wrote: > Dear Netters, > > In order to generate a ribozyme expression construct, > 1) I have generated two complemetary oligos (49mers) with EcoRI and XhoI > overhangs on each end. So that when hybridized they will be like the > following. > 5'-aattcc----------------------tc-3' > 3'-gg----------------------agagct-5' > 6) checked many colonies and cannot find the right inserts. > > What have I done wrong? > Any suggestions? Well, Soon, I would consider the most likely option to be that you have a strong secondary structure in your oligos, which is preventing them from annealing to each other, hence not clonable. This is especially likely if you are trying to generate an RNA with a "hammerhead" type structure. The way to get around this _may_ be to put them into ice-water after the 70 deg treatment, then allow them to warm gently to room temp. Why not mix the two oligos together before kinasing them, so that the heat inactivation of the enzyme and the denaturation of the oligos is combined in a single step? An alternative suggestion is that any complex secondary structure may need to be removed before the kinase treatment, since the enzyme works well only on free 5' ends. Five mins at 70 deg followed by 3 mins on ice-water should do the trick. If none of these things work, you may have to clone the sequence in two parts, if that is feasible. That should overcome secondary structure effects, but will obviously take longer. Hope this helps - good luck!! Ian Graham PhD Dept of Genetics, University of Nottingham ian.graham@vme.nott.ac.uk From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!cam.news.pipex.net!pipex!edi.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!dundee.ac.uk!rdcsaunders.anatphys.dundee.ac.uk!rdcsaunders From: rdcsaunders@anatphys.dundee.ac.uk (Robert D. C. Saunders) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Stripping Hybond membranes Date: Mon, 3 Jul 1995 16:58:11 Organization: University of Dundee Lines: 17 Message-ID: References: NNTP-Posting-Host: rdcsaunders.anatphys.dundee.ac.uk X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] In article ax041@FreeNet.Carleton.CA (Rachelle Leger) writes: >From: ax041@FreeNet.Carleton.CA (Rachelle Leger) >Subject: Stripping Hybond membranes >Date: Mon, 3 Jul 1995 11:07:11 GMT >I seem to loose material from my blot when I strip Hybond N Plus membranes >Is is difficult to know for sure that this is really happening but when I >reuse it the next time I need to expose much longer. >Is this your experience? >I strip by pouring boiling .1% SDS and .1% SSC on the membrane and skaking >for 10 minutes. This has been my experience too. Both with N and N+. Furthermore I have suffered this with other nylon membranes. I use nitrocellulose wherever possible. Robert From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!nntp.gmd.de!news.rwth-aachen.de!news.rhrz.uni-bonn.de!news.uni-stuttgart.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!fauern!lrz-muenchen.de!usenet From: Juan MacFarlane Newsgroups: bionet.molbio.methds-reagnts Subject: Seeking for a source of Nitrocefin Date: 3 Jul 1995 13:21:57 GMT Organization: Institute for Physical Biochemistry, Uni-Muenchen Lines: 13 Distribution: world Message-ID: <3t8qtl$560@sunserver.lrz-muenchen.de> NNTP-Posting-Host: pc6.pbm.med.uni-muenchen.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Dear Netters I'm doing beta-lactamase assays and I would like to try it with the colourimetric substrate known as Nitrocefin. If anyone knows a source for this compound, preferably in Germany, I would be extremely grateful if they could inform me of the company name, address, fax no. etc. Juan Macfarlane Institut fur Physikalische Biochemie Schillerstrasse 44 80336 muenchen, Germany Fax: 089-5996 483 Email: juanmac@pbm.med.uni-muenchen.de From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Newsgroups: bionet.virology,bionet.molbio.proteins,bionet.immunology,bionet.molbio.methds-reagnts,bionet.cellbiol Path: biosci!bcm!cs.utexas.edu!swrinde!emory!news-feed-1.peachnet.edu!darwin.sura.net!nih-csl!dbrml69.niaid.nih.gov!user From: Seth_Pincus@NIH.gov (Seth Pincus) Subject: Re: Epitope Mapping Kits: Chiron Mimotopes vs. Genosys Spots Kit? Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: dbrml69.niaid.nih.gov Organization: NIAID References: Date: Sun, 2 Jul 1995 15:14:07 GMT Lines: 19 Xref: biosci bionet.virology:3761 bionet.molbio.proteins:4978 bionet.immunology:4715 bionet.molbio.methds-reagnts:30384 bionet.cellbiol:2505 In article , "Ed Seijo (BIO)" wrote: > Does anyone have any experience with either of these kits or epitope > mapping in general. I would appreciate any information. > Thanks, > Ed Seijo We have published three papers describing the use of these techniques to map the human anti-HIV antibody response in fine detail. They have all been published in the Journal of Clinical Investigation 1993-4. The citations are: 91: 1987-96 93: 140-46 93: 2505-13 If these contain information germaine to your question and you want more details, then feel free to get in touch by E-mail or phone (I'm in the FASEB directory). From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!nntp.gmd.de!news.rwth-aachen.de!news.rhrz.uni-bonn.de!news.uni-stuttgart.de!news.belwue.de!news.uni-konstanz.de!marvin.biologie.uni-konstanz.de!user From: stephan.witte@uni-konstanz.de (Stephan Witte) Newsgroups: bionet.molbio.methds-reagnts Subject: 2HybridSystem: Gal4 or LexA  Followup-To: bionet.molbio.methds-reagnts Date: 3 Jul 1995 13:42:52 GMT Organization: University of Constance Lines: 15 Distribution: world Message-ID: NNTP-Posting-Host: marvin.biologie.uni-konstanz.de Hi, there is a question regarding the two hybrid system again: There are LexA and Gal4 based systems available. What is the difference between those two systems? What are the advantages / disadvantages of both systems? Thanks a lot, Stephan  Stephan Witte Inst. of Immunology University of Constance stephan.witte@uni-konstanz.de From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!cam.news.pipex.net!pipex!edi.news.pipex.net!pipex!sunic!sunic.sunet.se!news.funet.fi!ousrvr.oulu.fi!jkortes From: jkortes@paju.oulu.fi (Jarkko Kortesmaa) Newsgroups: bionet.molbio.methds-reagnts Subject: How to import MD PhosphorImager images? Date: 3 Jul 1995 14:11:49 GMT Organization: University of Oulu, Department of Biochemistry Lines: 13 Message-ID: <3t8tr5$8qc@ousrvr.oulu.fi> NNTP-Posting-Host: raita.oulu.fi Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: TIN [version 1.2 PL2] Dear netters, Is is possible to export images from the ImageQuaNT program of Molecular Dynamics PhosphorImager? I would like to paste the images into a WinWord document. -- ------------------------------------------------------------------------------ Jarkko Kortesmaa Dept. of Biochemistry and Biocenter Oulu jkortes@paju.oulu.fi University of Oulu Finland ------------------------------------------------------------------------------ From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-ulm.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!fauern!lrz-muenchen.de!ipp-garching.mpg.de!usenet From: Peter Zwickl Newsgroups: bionet.molbio.methds-reagnts Subject: E. coli B834 cells Date: 3 Jul 1995 06:35:38 GMT Organization: Max-Planck-Institute for Biochemistry Lines: 4 Message-ID: <3t833q$54em@sat.ipp-garching.mpg.de> NNTP-Posting-Host: bmmac26.biochem.mpg.de 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 Which company sells the E. coli B-strain B834(DE3), suitable for high- level expression of recombinant proteins with T7-polymerase? From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!ihnp4.ucsd.edu!swrinde!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!msunews!harbinger.cc.monash.edu.au!microb12.med.monash.edu.au!CDMENU From: CDMENU@ccs1.cc.monash.edu.au Newsgroups: bionet.molbio.methds-reagnts Subject: electroporation of BL21(DE3)pLysS Date: Mon, 3 Jul 1995 07:22:11 GMT Organization: Monash University Lines: 7 Message-ID: NNTP-Posting-Host: microb12.med.monash.edu.au X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Hi! Has anyone tried to electroporate the E.coli strain BL21(DE3)pLysS? If so, could you please send me the protocol for the preparartion of the cells and also the protocol for electroporation. My Email address is: jackiec@ccs1.cc.monash.edu.au Thanks very much! Jackie From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!daresbury!trane.uninett.no!Norway.EU.net!EU.net!howland.reston.ans.net!swrinde!emory!darwin.sura.net!fconvx.ncifcrf.gov!fcsparc6!pnh From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) Subject: Re: electroporation of BL21(DE3)pLysS Message-ID: Summary: E. coli BL21(DE3)pLysS Sender: Paul N. Hengen Nntp-Posting-Host: fcsparc6.ncifcrf.gov Organization: Frederick Cancer Research and Development Center References: http://www-lmmb.ncifcrf.gov/~pnh/ - - - - - - * * - - - Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - * ******************************************************************************* From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-ulm.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!fauern!lrz-muenchen.de!ipp-garching.mpg.de!usenet From: Peter Zwickl Newsgroups: bionet.molbio.methds-reagnts Subject: (no subject) Date: 3 Jul 1995 06:52:52 GMT Organization: Max-Planck-Institute for Biochemistry Lines: 4 Message-ID: <3t8444$54bg@sat.ipp-garching.mpg.de> NNTP-Posting-Host: bmmac26.biochem.mpg.de 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?ALL Which company sells the E. coli strain B834, suitable for high-level expression of recombinant proteins with the T7 polymeras? From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!emory!cssun.mathcs.emory.edu!hobbes.cc.uga.edu!gnti.biochem.uga.edu!user From: buckhaults@bscr.cc.uga.edu (phillip buckhaults) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: How to import MD PhosphorImager images? Date: Sun, 02 Jul 1995 13:09:14 -0500 Organization: uga Lines: 33 Distribution: world Message-ID: References: <3t8tr5$8qc@ousrvr.oulu.fi> NNTP-Posting-Host: gnti.biochem.uga.edu In article <3t8tr5$8qc@ousrvr.oulu.fi>, jkortes@paju.oulu.fi (Jarkko Kortesmaa) wrote: > Dear netters, > > Is is possible to export images from the ImageQuaNT program of Molecular > Dynamics PhosphorImager? > > I would like to paste the images into a WinWord document. > > -- > ------------------------------------------------------------------------------ > Jarkko Kortesmaa Dept. of Biochemistry and Biocenter Oulu > jkortes@paju.oulu.fi University of Oulu > Finland > ------------------------------------------------------------------------------ The Molecular Dynamics Software has a "Convert" option (under the file menu, i think) which converts the file from 16 bit to 8 bit data. The output file still has the .gel extension, but is actually a .tiff file that most word programs will read. The file somehow has less information after the conversion, so you should do any quantitation on the origional file, but the screen display and printouts look the same (at least to me). Phillip Buckhaults Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center Life Science Building University of Georgia Athens Georgia 30602 (706) 542-1701 ________________________________________________________________________________ "Let us not confuse effort with achievement" From owner-methds-reagnts@net.bio.net Sun Jul 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!psuvax1!news.pop.psu.edu!news.cac.psu.edu!howland.reston.ans.net!swrinde!hookup!nstn.ns.ca!dragon.acadiau.ca!pat426.acadiau.ca!002233w From: 002233w@axe.acadiau.ca (ANTHONY WILSON) Subject: RAPD Gel Analysis Message-ID: <002233w.103.2FF8278E@axe.acadiau.ca> Lines: 16 Sender: news@relay.acadiau.ca Nntp-Posting-Host: pat426.acadiau.ca Organization: Acadia University Date: Mon, 3 Jul 1995 17:23:26 GMT Lines: 16 I was wondering if anyone has had any success with contacting the Spanish group that authored the Gel Manager software package that was advertised in the RAPD newsgroup a few weeks back... Our lab is interested in finding a simple gel analysis program that takes digitized gel images and provides interpretation of the data. Gel Manager is the first program we've come across that appears to fit the bill, but we haven't had any luck with contacting the programming group in Spain... Any help or any other suggestions of possibly useful packages?? Thanks... Tony Wilson Acadia University From owner-methds-reagnts@net.bio.net Mon Jul 03 23:00:00 1995 Path: biosci!daresbury!is.bbsrc.ac.uk!pc0519.ri.bbsrc.ac.uk!user From: Andy.Law@bbsrc.ac.uk (Andy Law (Big Nose)) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: How to import MD PhosphorImager images? Date: Tue, 04 Jul 1995 09:37:37 +0000 Organization: Roslin Institute Lines: 33 Distribution: world Message-ID: References: <3t8tr5$8qc@ousrvr.oulu.fi> NNTP-Posting-Host: pc0519.ri.bbsrc.ac.uk X-Newsreader: Value-Added NewsWatcher 2.0b24.0+ In article , buckhaults@bscr.cc.uga.edu (phillip buckhaults) wrote: > In article <3t8tr5$8qc@ousrvr.oulu.fi>, jkortes@paju.oulu.fi (Jarkko > Kortesmaa) wrote: > > > Dear netters, > > > > Is is possible to export images from the ImageQuaNT program of Molecular > > Dynamics PhosphorImager? > > > > I would like to paste the images into a WinWord document. > > > > -- > > > > > The Molecular Dynamics Software has a "Convert" option (under the file > menu, i think) which converts the file from 16 bit to 8 bit data. The > output file still has the .gel extension, but is actually a .tiff file > that most word programs will read. The file somehow has less information > after the conversion, so you should do any quantitation on the origional > file, but the screen display and printouts look the same (at least to me). > Phillip Buckhaults I'm pretty sure that NIH image can read the 16 bit files, so if you can get the file across to that you should be able to play around with it directly. We'll be able to spot it if you start painting in bands and spots though ;-} -- Andy Law ( Andy.Law @ bbsrc.ac.uk ) ( Big Nose in Edinburgh ) From owner-methds-reagnts@net.bio.net Mon Jul 03 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-ulm.de!news.belwue.de!News.Uni-Marburg.DE!news.th-darmstadt.de!news.uni-mainz.de!GATERMAN@mzdmza.zdv.uni-mainz.de From: gaterman@mzdmza.zdv.uni-mainz.de Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Why probe hybridizes to vector? Date: 4 Jul 1995 09:28:05 GMT Organization: Johannes Gutenberg-Univ. Mainz Lines: 17 Distribution: world Message-ID: <3tb1j5$m4i@kralle.zdv.Uni-Mainz.DE> References: Reply-To: gaterman@mzdmza.zdv.uni-mainz.de NNTP-Posting-Host: vzdmzy.zdv.uni-mainz.de In article , biolc4@jetson.uh.edu (Wenfu) writes: > >When I was using isolated DNA fragment( I am sure no any vector sequence in >it) >to probe digested plasmids, I found my probe hybridizes to the vector. >This kind of thing happen to me several times when I was doing the similar >thing. Although it did not matter for my results, I am really curious >about the reasons for. >Any idea would be appreciated. 1 if the probe is too long, almost any hibridization will cause a signal probe size should be small (< 200bp). Normally , this can be achieved by using some oligo-priming kit or by sonicating the probe. 2 check your hibridizytion conditions sgg From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!usenet.ins.cwru.edu!path22304.path.cwru.edu!user From: sbv@pop.cwru.edu (Scott Vande Pol) Newsgroups: bionet.molbio.methds-reagnts Subject: 5-FOA solubility Followup-To: bionet.molbio.methds-reagnts Date: 5 Jul 1995 17:46:13 GMT Organization: Case Western Reserve University Lines: 11 Message-ID: NNTP-Posting-Host: path22304.path.cwru.edu We are using FOA (5-floroorotic acid) for selection against URA-3 plasmids in yeast. FOA is expensive to buy and difficult to get into solution. Does anyone know of a non-toxic solvent (such as DMSO) in which a stable 100X FOA stock solution can be made? -- Scott Vande Pol Case Western Reserve University Department of Pathology Cleveland Oh. 44106 sbv@pop.cwru.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!mg690769 From: mg690769@bcm.tmc.edu (Michael Gerdes) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA purification from agarose gels Date: 5 Jul 1995 17:49:04 GMT Organization: Baylor College of Medicine, Houston, Tx Lines: 15 Message-ID: <3tejag$lnc@gazette.bcm.tmc.edu> References: NNTP-Posting-Host: condor.mbcr.bcm.tmc.edu In article bio.tamu.edu writes: >I am interested in what other people think about Qiagens new Qiaquick >columns compared to Promega's wizard PCR preps. Has anybody done a direct >comparison of these two kits for purification of DNA from gels? I have been using the Qiaquick spin tubes for several months now, and have been very pleased. The cost is fairly low, and they can also be used for hot nucleotide removal after labeling reactions, but with a different binding buffer. I don't know how they compare to Promega's though. MiJoGe Baylor Coll of Medicine Dept Cell Biology From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!news.sprintlink.net!cam.news.pipex.net!pipex!dish.news.pipex.net!pipex!edi.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!hgmp.mrc.ac.uk!news From: Michael Rhodes Newsgroups: bionet.molbio.methds-reagnts Subject: AZLON BAGS Date: 4 Jul 1995 12:26:49 GMT Organization: U. K. Human Genome Mapping Project Resource Centre Lines: 7 Message-ID: <3tbc29$oaj@mercury.hgmp.mrc.ac.uk> NNTP-Posting-Host: dysprosium.hgmp.mrc.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) -- Does anyone know a supplier of AZLON bags in the UK thanks Michael Rhodes From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!europa.chnt.gtegsc.com!news.umbc.edu!haven.umd.edu!umabnet.ab.umd.edu!umabnet.ab.umd.edu!kwalker From: "Dr. Kimberly Walker" Newsgroups: bionet.molbio.methds-reagnts Subject: Who knows the tricks for S1 or primer Ext Date: Wed, 5 Jul 1995 13:32:26 -0400 Organization: University of Maryland at Baltimore Lines: 4 Message-ID: NNTP-Posting-Host: umabnet.ab.umd.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Is ther anyone out there who knows the tricks to the trade for MungBean/S1 mapping v. Primer extension? I gotta do it quick and I want to know if there are pitfalls I should watch out for. Kim From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!dish.news.pipex.net!pipex!oleane!jussieu.fr!univ-lr.fr!cribx1.u-bordeaux.fr!orcades.u-bordeaux2.fr!user From: vekris@u-bordeaux2.fr (Antoine VEKRIS) Newsgroups: bionet.molbio.methds-reagnts Subject: human rDNA's request Date: Mon, 03 Jul 1995 18:08:35 +0000 Organization: Universite Bordeaux II Lines: 14 Message-ID: NNTP-Posting-Host: orcades.u-bordeaux2.fr Hello, I am searching cloned, human rDNAs. lets say clones pR7.3, pR5.8 ! Can anybody help me thanks Antoine vekris@u-bordeaux2.fr -- Antoine VEKRIS University of Bordeaux II Medical Biochemistry and Molecular Biology laboratory From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!OSU.EDU!beall.3 From: beall.3@OSU.EDU (Clifford Beall) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: pCDNAI and pCDM8 problems in stable transfection ? Date: 5 Jul 1995 09:53:33 -0700 Organization: Ohio State University Lines: 34 Sender: daemon@net.bio.net Distribution: world Message-ID: <199507051649.MAA25986@beauty.magnus.acs.ohio-state.edu> References: NNTP-Posting-Host: net.bio.net In article , marcvdc@lmb1.rug.ac.be wrote: > Dear Netters, > > > It seems that there are problems with the stable transfections of genes > cloned in pCDM8. Although the transient transfections of the gene gave > high expression levels, no protein was detected in the stable transfectans. > The same was done with an biological inactive form of the protein and this > gave the same results. So, it is not because of the toxicity of the gene > expressed. > > Now I wonder wether someone had the same problems with pCDM8. Are the same > problems seen in the pCDNAI (in vitrogen)? Maybe, I will use this vector in > the future but then I want to be sure not to encounter the same problems. > > Please respond also to me personal. > > Thanks, > > Marc Dear Marc, You need a selectable marker to get stable transformants, because only about 1 in 10~6 cells stably integrates introduced DNA. pCDM8 does not contain such a marker. You can use pcDNA 3, and select with G418 (you can find details in Current Protocols in Molecular Biology or other source). Good luck, Cliff Beall Research Scientist Ohio State Neurobiotechnology beall.3@osu.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!gatech!swrinde!dish.news.pipex.net!pipex!oleane!jussieu.fr!citi2.fr!news From: Laurentiu COCEA Newsgroups: bionet.molbio.methds-reagnts Subject: Re: how to work out curvature of DNA??? Date: 5 Jul 1995 16:47:12 GMT Organization: CITI2 - Universite Rene Descartes, Paris Lines: 36 Message-ID: <3tefmg$6de@bisance.citi2.fr> References: NNTP-Posting-Host: rpc5.citi2.fr gqva12@udcf.gla.ac.uk (G Gallagher) wrote: > > Hello out there > > Does anyone know how to work out the extent to which the secondary structure > and/or curvature of dsDNA is altered by mutations? > > Specifically, I want to guage the effect of differing sizes of a CA repeat. > > There doesn't seem to be an obvious programme in the GCG package. > > Many thanks > > Grant Gallagher Hi- if by "mutations" you mean nucleotide substitutions, then I don't think this can change the "secondary structure" of a dsDNA. Actually, a secondary structure is either a hairpin structure of a dsDNA (does it occur in vivo?) or a Z-DNA. Never heard of a "secondary structure" of a dsDNA and if you actually think of DNA bending then it depends on specific proteins that are able to do it by specific interections with the nucleotides. On the other hand, a dsDNA fgt of less than 250 bp does not bend well, at least it cannot bend enough to have its ends "touch" each other - so there are some limitations. One of the main features of a dsDNA molecule is the fact that its structure (the double helix) does NOT depend on the specific sequence of nucleotides that make it. On the other hand, protein secondary structures do depend on their primary structure (i.e., the sequence of amino acids). If you think different and you have some arguments please let me know. Laurentiu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!newsjunkie.ans.net!inet.d48.lilly.com!40!sk Newsgroups: bionet.molbio.methds-reagnts Subject: GFP expression in mammalian cells Message-ID: From: sk@lilly.com (Stuart Kuhstoss) Date: Wed, 5 Jul 95 16:32:22 GMT Followup-To: bionet.molbio.methds-reagnts Distribution: world Organization: Lilly Research Laboratories Nntp-Posting-Host: sak.d50.lilly.com X-Newsreader: VersaTerm Link v1.1 Lines: 14 We've been trying to express GFP in mammalian cell lines with very little success. We've used both vectors from Clonetech (pGFP-N1) and a vector constructed here with comparable lack of success. The cell lines tried were HeLa, 293, NRKE, all with no success. A different collegue has said that he sees expression in MCF7 cells, but I haven't tried these yet. I've heard scuttlebutt about these sorts of problems elsewhere. Does anyone have any feel for what (if any) are the barriers to expression in mammalian cells? I've heard that codon usage may be a problem. Any experience with this? We haven't done Southerns and Northerns yet, but I have no reason to think that these vectors won't fire in the cell types listed above. Any suggestions would be greatly appreciated. Stu Kuhstoss SK@LILLY.COM From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!howland.reston.ans.net!EU.net!uknet!newsfeed.ed.ac.uk!udcf.gla.ac.uk!gqva12 From: gqva12@udcf.gla.ac.uk (G Gallagher) Subject: how to work out curvature of DNA??? Message-ID: Sender: news@udcf.gla.ac.uk (News) Organization: Glasgow University Computing Service Date: Wed, 5 Jul 1995 15:22:36 GMT Lines: 12 Hello out there Does anyone know how to work out the extent to which the secondary structure and/or curvature of dsDNA is altered by mutations? Specifically, I want to guage the effect of differing sizes of a CA repeat. There doesn't seem to be an obvious programme in the GCG package. Many thanks Grant Gallagher From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!agate!news.duke.edu!godot.cc.duq.edu!newsfeed.pitt.edu!gatech!swrinde!cs.utexas.edu!news.sprintlink.net!dish.news.pipex.net!pipex!demon!btnet!matsu.nis.co.jp!wnoc-tyo-news!wnoc-sfc-news!wnoc-kyo-news!icspub!odins-suita!odins-sci!usenet From: Shin-ichiro HIRAGA Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Sequencing AT rich regions Date: 5 Jul 1995 08:26:13 GMT Organization: Dept. Biol., Fac. Sci., Osaka University Lines: 5 Message-ID: <3tdib5$ph2@ns2.sci.osaka-u.ac.jp> References: <3suf6b$alv@mserv1.dl.ac.uk> <3td6p8$ims@ns2.sci.osaka-u.ac.jp> NNTP-Posting-Host: apple.bio.sci.osaka-u.ac.jp Mime-Version: 1.0 Content-Type: text/plain; charset=iso-2022-jp Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) X-URL: news:3td6p8$ims@ns2.sci.osaka-u.ac.jp I have posted a wrong one. Please ignore. Shin-ichiro Hiraga From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!news.itd.umich.edu!86.13.med.umich.edu!manisha From: manisha@umich.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Sourses for Staphy. Entero. B toxoid ? Date: Wed, 5 Jul 1995 11:12:22 Organization: University of Michigan Lines: 10 Message-ID: NNTP-Posting-Host: 86.13.med.umich.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Hello! I wanted to know all the sourses to buy Staphylococcal Enterotoxin B Toxoid in the U. S. Thanks in advance Please reply to: Manisha@Umich.Edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!newsserver.jvnc.net!synapse.bms.com!news-admin From: patel_p@bms.com (taylor) Newsgroups: bionet.molbio.methds-reagnts Subject: Need lacZ gene to be used as a promoter probe reporter Date: 5 Jul 1995 14:14:06 GMT Organization: Bristol-Myers Squibb Lines: 13 Message-ID: <3te6nf$h7e@synapse.bms.com> NNTP-Posting-Host: patel_p.bms.com Hi: I need a procaryotic promoter less (but has its own bacterial RBS) lacZ gene. Basically, I need to use this gene as a reporter gene in a specific bacterial promoter assay. Any info. on this will be greatly appreciated. I have tried all of the common biotech. companies (viz. Promega, Invitrogen etc.) Thanks in advanve Pramathesh e-mai: Patel_P@BMS.com From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!elroy.jpl.nasa.gov!lll-winken.llnl.gov!venus.sun.com!rutgers!netnews.upenn.edu!parvati3-10.wistar.upenn.edu!user From: waterman_m@a1.mscf.upenn.edu (Matthew J. F. Waterman) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: efficient blunt ligation? Followup-To: bionet.molbio.methds-reagnts Date: 5 Jul 1995 13:46:14 GMT Organization: University of Pennsylvania Lines: 11 Message-ID: References: NNTP-Posting-Host: parvati3-10.wistar.upenn.edu In article , hdang@channel.neusc.bcm.tmc.edu (hong dang) wrote: > > Still wondering why it is so outrageous that this could actually work? > > Hong > Yes, please tell why this is such an incredible scientific accomplishment. I do it all the time, it is really very easy. From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!gatech!EU.net!uknet!daresbury!not-for-mail From: Newsgroups: bionet.molbio.methds-reagnts Subject: 32P-postlabeling Date: 5 Jul 1995 14:52:31 +0100 Lines: 16 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3te5ev$1h0@mserv1.dl.ac.uk> X-Sender: p_hanelt@popserv1.rz.uni-ulm.de Original-To: methods@dl.ac.uk Hi netters, I'm working with the 32P-postlabeling method (intensification of the DNA adducts with P1 nuclease). There are a lot of different methods for determination of the "normals". Who can help me to find a good working method and can send me a fax to let me see how a typical TLC looks like. (I'm not sure how to interprete what I see). I'm looking forward to hearing from you and to get some "good working and easy protocols". Thanks for your help Sabine Sabine Hanelt Uni Klinik Ulm Abt. Medizinische Genetik Fax: 49-731-501-3438 Tel: 49-731-501-3432 From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!nntp.gmd.de!news.rwth-aachen.de!news.rhrz.uni-bonn.de!news.uni-stuttgart.de!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!informatik.uni-bremen.de!nordwest.pop.de!olis.north.de!indrom!unihil!MHH!NewsWatcher!user From: CAH@ifm.mh-hannover.de (Christoph Ahlers) Subject: Re: DDRT-PCR Northerns Message-ID: Sender: news@thor.mhrz.mh-hannover.de (NetNews @ MHRZ) Organization: Inst. Molecular Biology References: <3sil5t$gsq@bubba.NMSU.Edu> <3so6mq$7oe@bubba.NMSU.Edu> Date: Tue, 4 Jul 1995 14:27:27 GMT Lines: 22 In article <3so6mq$7oe@bubba.NMSU.Edu>, smori@nmsu.edu (Shahram Mori) wrote: > that in my display gels I see what looks like differentially regulated > fragments under test but NOT control (and I do > everything in duplicates). When you say duplicates, do you mean using fresh batches of RNA? Only bands which are reproducibly differentially expressed, ie, those showing differential expression in seperate lots of RNA, can be expected to show differential expression on a Northern. Even then, it's still not guaranteed. According to most of the literature, bands should be larger than 200 bp. I'd be interested in receiving a summary via e-mail of responses you receive to your original question- Chris Ahlers Institute for Molecular Biology Hannover Medical School 30625 Hannover, Germany cah@ifm.mh-hannover.de From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!EU.net!uknet!bcc.ac.uk!is.bbsrc.ac.uk!pc0518.ri.bbsrc.ac.uk!user From: peter.wilson@bbsrc.ac.uk (pete wilson) Newsgroups: bionet.molbio.methds-reagnts Subject: ignore...testing Date: Wed, 05 Jul 1995 12:05:10 +0000 Organization: Roslin Institute BBSRC Lines: 2 Message-ID: NNTP-Posting-Host: pc0518.ri.bbsrc.ac.uk ignore testing From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!howland.reston.ans.net!newsserver.jvnc.net!news.caren.net!news.join.ad.jp!wnoc-tyo-news!news.iij.ad.jp!wincgw1!creamy!icspub!odins-suita!odins-sci!usenet From: shiraga@gen-info.osaka-u.ac.jp (Shin-ichiro HIRAGA) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Sequencing AT rich regions Message-ID: <3td6p8$ims@ns2.sci.osaka-u.ac.jp> Date: 5 Jul 95 05:08:56 GMT References: <3suf6b$alv@mserv1.dl.ac.uk> Organization: Dept. Biol., Fac. Sci., Osaka University Lines: 5 NNTP-Posting-Host: apple.bio.sci.osaka-u.ac.jp Mime-Version: 1.0 Content-Type: text/plain; charset=iso-2022-jp Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) X-URL: news:3suf6b$alv@mserv1.dl.ac.uk I think that the optimum temp of Tth pol is 75 degree C. I'm not sure but the local AT rich region may be denatured at that temp. I think you should try T7 pol or Sequanase system for the region. Shin-ichiro Hiraga From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!cam.news.pipex.net!pipex!dish.news.pipex.net!pipex!warwick!lyra.csx.cam.ac.uk!macr1-5.welc.cam.ac.uk!user From: jpcd0@mole.bio.cam.ac.uk (John Dixon) Newsgroups: bionet.molbio.methds-reagnts Subject: Is lambda EMBL 3A suppressed by supE? Date: Wed, 05 Jul 1995 11:55:32 +0000 Organization: Wellcome CRC Institute Lines: 17 Message-ID: NNTP-Posting-Host: macr1-5.welc.cam.ac.uk Can anyone tell me if the amber mutations in lambda EMBL 3A (Aam Bam) are suppressed by supE as well as supF? Are the mutations overcome by prolonged incubation? I was using lambda ZAP (Sam100) and if the lambda are incubated >8hrs all form plaques on sup0 hosts. Can this be a problem for AamBam lambda? Thanks in advance Johnny D -- John Dixon Lab 44 (223) 334131 Wellcome/CRC Institute Fax 44 (223) 334134 Dept Genetics Cambridge University United Kingdom e-m: jpcd0@mole.bio.cam.ac.uk From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!dish.news.pipex.net!pipex!edi.news.pipex.net!pipex!sunic!sunic.sunet.se!trane.uninett.no!daresbury!not-for-mail From: Newsgroups: bionet.molbio.methds-reagnts Subject: test Date: 5 Jul 1995 11:34:47 +0100 Lines: 2 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3tdps7$m5r@mserv1.dl.ac.uk> X-Sender: p_hanelt@popserv1.rz.uni-ulm.de Original-To: methods@dl.ac.uk test From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!EU.net!uknet!daresbury!not-for-mail From: Newsgroups: bionet.molbio.methds-reagnts Subject: test Date: 5 Jul 1995 11:34:17 +0100 Lines: 2 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3tdpr9$m51@mserv1.dl.ac.uk> X-Sender: p_hanelt@popserv1.rz.uni-ulm.de Original-To: methods@dl.ac.uk test From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!gatech!howland.reston.ans.net!Germany.EU.net!news.dfn.de!news.rwth-aachen.de!news.rhrz.uni-bonn.de!rhrz-ts1-p14.rhrz.uni-bonn.de!user From: schraute@uni-bonn.de (Bernhard Schrautemeier) Newsgroups: bionet.molbio.methds-reagnts Subject: Transcription factors with SurfZAP? Date: Wed, 05 Jul 1995 14:26:20 +0200 Organization: Bot. Inst. Univ. Bonn Lines: 16 Message-ID: NNTP-Posting-Host: rhrz-ts1-p14.rhrz.uni-bonn.de Does anybody know if Stratagene´s SurfZAP Kit can also be used for isolating (prokaryotic) transcription factor genes, i. e. does it work for protein-DNA interaction, probing SurfZAP libraries with labeled DNA fragments? I would welcome any experience in this respect. Thanks in advance! -- ____________________________________________________________ Bernhard Schrautemeier Tel: +49-228-73-5277 or 5536 Botanisches Institut Fax: +49-228-73-5513 der Universitaet Bonn E-mail: schraute@uni-bonn.de Kirschallee 1 x________________________________ 53115 Bonn x Germany x ___________________________x From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!nntp.usm.edu!whale.st.usm.edu!jstemple From: jstemple@whale.st.usm.edu (Jeffrey Shane Temple) Newsgroups: bionet.molbio.methds-reagnts Subject: Sequencing PCR products Date: 4 Jul 1995 19:37:13 GMT Organization: University of Southern Mississippi Lines: 12 Message-ID: <3tc599$cs1@server.st.usm.edu> NNTP-Posting-Host: whale.st.usm.edu X-Newsreader: TIN [version 1.2 PL1] I am planning to use a relatively new product from Amersham to try and sequence PCR products. The name of the product is Sequenase PCR product sequencing kit. Before I buy the kit, I would like to have some feedback from others that may have used this kit before. Is it as easy as the catalog describes it and most importantly, is it worth $300? If you haven't used this kit before and have another suggestion, feel free to voice it. All comments will be greatly appreciated. Jeffrey Temple Univ of Southern MS Dept of Chem/Biochem From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!psgrain!iafrica.com!ticsa.com!cstatd.cstat.co.za!ucthpx!ovisun.ovi.ac.za!ovisun.ovi.ac.za!not-for-mail From: news@ovisun.ovi.ac.za (Usenet News) Newsgroups: bionet.molbio.methds-reagnts,bionet.general,bionet.microbiology Subject: Looking for Clostridium McAb Date: 5 Jul 1995 09:23:35 -0000 Organization: Onderstepoort Veterinary Institute Lines: 24 Message-ID: <3tdlmn$425@ovisun.ovi.ac.za> NNTP-Posting-Host: ovisun.ovi.ac.za Xref: biosci bionet.molbio.methds-reagnts:30427 bionet.general:16033 bionet.microbiology:2640 I am looking for monoclonal antibodies to Clostridium botulinum C1 and D neurotoxins, Clostridium tetani tetanus toxin and Clostridium perfringens alpha-, beta- and epsilon toxins. What is the going rate for monoclonal antibodies? I plan to use the reagents in ELISA systems to detect the toxins of interest. Please respond via email. My email address is: charlotte@moon.ovi.ac.za Kind regards Ms Charlotte Ellis Fax no: 27 12 56 56 573 Address: Onderstepoort Private Bag X5 Onderstepoort 0110 South Africa From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!europa.chnt.gtegsc.com!howland.reston.ans.net!vixen.cso.uiuc.edu!usenet.ucs.indiana.edu!sunflower.bio.indiana.edu!gilbertd From: gilbertd@sunflower.bio.indiana.edu (Don Gilbert) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Bionet USENET Articles (Search GOPHER) Date: 5 Jul 1995 02:11:22 GMT Organization: Biology, Indiana University - Bloomington Lines: 17 Message-ID: <3tcsca$7ou@usenet.ucs.indiana.edu> References: NNTP-Posting-Host: sunflower.bio.indiana.edu There are at least two archives of Bionet news that can be searched. One is the BioSci archive at net.bio.net, another is IUBio archive at iubio.bio.indiana.edu. Both offer seaches for gopher and www browsers. If one is unavailable at some point, try the other. Recently the net.bio.net archive has been moving and the computer host has had hardware bugs. For the IUBio archive: gopher to iubio.bio.indiana.edu, search in Network-News/ http://iubio.bio.indiana.edu/ and likewise " In article lonnie writes: >I was wondering if anybody has tried to do a search at the Bionet USENET >Article GOPHER lately. ... The message that I have gotten >for the last week has been "unable to connect". -- -- d.gilbert--biocomputing--indiana u--bloomington--gilbertd@bio.indiana.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!ames!waikato!comp.vuw.ac.nz!auckland.ac.nz!NewsWatcher!user From: tbratt@sbsnov1.auckland.ac.nz (Tomas Bratt) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: How to remove albumin? Date: 5 Jul 1995 00:49:40 GMT Organization: University of Auckland Lines: 23 Message-ID: References: NNTP-Posting-Host: 130.216.20.12 In article , Morty@bchm.unp.ac.za (Rory.Morty) wrote: > Hi there! > > Any ideas about how to COMPLETELY remove trace amounts of albumin from an > otherwise pure preparation of protein. MEC is not feasable because the > molecular weight of the protein of interest is too close to 68 kDa. The > trace amounts of albumin have not been removed by a number of IEC steps, or > chromatography on Cibacron Blue or phenyl sepharose. Any ideas? > > Rory Morty > e-mail: morty@gate2.cc.unp.ac.za Try protein G affinity chromatography, but be sure that the albumin binding part of protein G has not been deleted in the protein G you use. Your protein will pass through and the albumin could be eluted and protein G used again Tomas -- David Franklin, Auckland, New Zealand From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!usc!elroy.jpl.nasa.gov!swrinde!hookup!rover.ucs.ualberta.ca!news From: ksayani@gpu.srv.ualberta.ca (Karim Sayani) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: San Diego Molecular Biology Conf Date: 4 Jul 1995 22:03:46 GMT Organization: University of Alberta Lines: 3 Message-ID: <3tcds2$11dq@rover.ucs.ualberta.ca> References: Reply-To: ksayani@gpu.srv.ualberta.ca NNTP-Posting-Host: pgs.surg.med.ualberta.ca X-Newsreader: WinVN 0.92.4 Pls send me more info on The San Diego Molecular Biology Conference, 1995 Thank you, Karim From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!NewsWatcher!user From: hdang@channel.neusc.bcm.tmc.edu (hong dang) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: efficient blunt ligation? Date: Tue, 04 Jul 1995 17:17:32 -0500 Organization: BCM, Neuroscience Lines: 32 Message-ID: NNTP-Posting-Host: d.neusc.bcm.tmc.edu X-Newsreader: Yet Another NewsWatcher 2.0b27.2 >hdang@channel.neusc.bcm.tmc.edu (hong dang): >> Just did this with EcoRV digested vector and PCR fragment: gel purify the vector >> fragment with QIAquick kit, SAP (shrimp alkaline phosphatase, USB) 1 unit >> for 40 min at 37C then heat inactivate at 65C for 15-20 mins; ligate at >> 16C overnight with 3:1 of I:V, 400 units T4 ligase, left on the bench >> (r.t.) in the morning, transform in the afternoon; next day: vector alone >> 9 colonies, with insert, about 1000. >> >> Hong >You were a very lucky man Hong, but do you think you should tell >such lies on the internet. Maybe I should clarify a bit: 1) I did start with 2-3 ug of plasmid (about 5.6 kb) and 2-3 molar fold excess of EcoRV fragment (300 bp) from 700 bp PCR product obtained using Pfu polymerase. So this may not qualify as "EFFICIENT" blunt ligation. 2) T4 DNA ligase was concentrated version from NEB. All reactions were done in a total volume of 20-40 microliters. 3) Final transformation rxn should contain about 200 ng DNA per tube (did not check but I used half of the products obtained from the previous step in each stage, e.g. 10 ul of 20 ul ligation mix to transform). 4) I don't work for any of the companies mentioned in any of my posts, and I am responsible for all contents of my messages. Still wondering why it is so outrageous that this could actually work? Hong -- Under construction! From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!cam.news.pipex.net!pipex!dish.news.pipex.net!pipex!uknet!daresbury!not-for-mail From: Andrei Popov Newsgroups: bionet.molbio.methds-reagnts Subject: RE: how to remove albumin Date: 4 Jul 1995 21:27:38 +0100 Lines: 13 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3tc87q$hfu@mserv1.dl.ac.uk> Importance: High Sensitivity: Company-Confidential Original-To: methods@dl.ac.uk (Receipt Notification Requested) (Non Receipt Notification Requested) (IPM Return Requested) Hi I used for this purpose a small column with anti-albumin mAB coupled to Sepharose 4B CL. worked fine regards Andrei From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!hookup!rover.ucs.ualberta.ca!gpu1!mdeyholo From: mdeyholo@gpu1.srv.ualberta.ca (Michael Deyholos) Newsgroups: bionet.molbio.methds-reagnts Subject: Purifying Bacto-Agar: Suggestions? Date: 4 Jul 1995 20:07:11 GMT Organization: University of Alberta Lines: 15 Message-ID: <3tc71f$ip8@rover.ucs.ualberta.ca> NNTP-Posting-Host: gpu1.srv.ualberta.ca X-Newsreader: TIN [version 1.2 PL2] To save money, I'd like to use bacto-agar (e.g. DIFCO) in place of noble agar as part of a solid growth medium for screeing seedling-lethal mutations in Arabidopsis. I've heard of washing bacto-agar in cold TE or water for a few days in a cold room. Apparently, the agar turns pure white after a few washes. Does anyone else have experience with this? Anyone know of a supplier of "PHYT-AGAR" Thanks, Mike From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!hookup!rover.ucs.ualberta.ca!gpu1!mdeyholo From: mdeyholo@gpu1.srv.ualberta.ca (Michael Deyholos) Newsgroups: bionet.molbio.methds-reagnts Subject: SOB Medium: Why add Mg late? Date: 4 Jul 1995 20:01:36 GMT Organization: University of Alberta Lines: 7 Message-ID: <3tc6n0$ip8@rover.ucs.ualberta.ca> NNTP-Posting-Host: gpu1.srv.ualberta.ca X-Newsreader: TIN [version 1.2 PL2] Just curious why Sanbrook/Maniatis reccommend autoclaving SOB, then adding MgCl2, then re-autoclaving. Why not add the MgCl2 to the original soltuion prior to autoclaving? Thanks, Mike Deyholos McGill Dept. of Biology From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!gatech!news.uoregon.edu!psgrain!iafrica.com!ticsa.com!cstatd.cstat.co.za!ucthpx!quagga.ru.ac.za!nntp.und.ac.za!pc048.ag.unp.ac.za!Morty From: Morty@bchm.unp.ac.za (Rory.Morty) Newsgroups: bionet.molbio.methds-reagnts Subject: How to remove albumin? Date: Mon, 3 Jul 1995 22:46:00 GMT Organization: University of Natal , Pietermaritzburg, South Africa Lines: 10 Message-ID: NNTP-Posting-Host: pc048.ag.unp.ac.za Keywords: Albumin; Protein purification; contamninants Hi there! Any ideas about how to COMPLETELY remove trace amounts of albumin from an otherwise pure preparation of protein. MEC is not feasable because the molecular weight of the protein of interest is too close to 68 kDa. The trace amounts of albumin have not been removed by a number of IEC steps, or chromatography on Cibacron Blue or phenyl sepharose. Any ideas? Rory Morty e-mail: morty@gate2.cc.unp.ac.za From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: davidm7643@aol.com (DavidM7643) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Alternative to Zeocin? Date: 4 Jul 1995 15:30:16 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 1 Sender: root@newsbf02.news.aol.com Message-ID: <3tc4s8$cff@newsbf02.news.aol.com> References: Reply-To: davidm7643@aol.com (DavidM7643) NNTP-Posting-Host: newsbf02.mail.aol.com Zeocin is bleomycin From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news2.near.net!news.delphi.com!usenet From: lonnie Newsgroups: bionet.molbio.methds-reagnts Subject: Bionet USENET Articles (Search GOPHER) Date: Tue, 4 Jul 95 14:49:47 -0500 Organization: Delphi (info@delphi.com email, 800-695-4005 voice) Lines: 8 Message-ID: NNTP-Posting-Host: bos1f.delphi.com I was wondering if anybody has tried to do a search at the Bionet USENET Article GOPHER lately. Several months ago I successfully did a search and was able to read the articles located, but I have been unsuccessful at reading the articles found during recent searches. The message that I have gotten for the last week has been "unable to connect". I would appreciate any feedback. Lonnie From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!gatech!bloom-beacon.mit.edu!world!rrohan From: rrohan@world.std.com (Richard M Rohan) Subject: Re: PCR problem - please help Message-ID: Organization: The World, Public Access Internet, Brookline, MA X-Newsreader: TIN [version 1.2 PL2] References: <3sea7s$1kb@mserv1.dl.ac.uk> <3sikq8$gsq@bubba.NMSU.Edu> Distribution: bionet Date: Tue, 4 Jul 1995 18:41:41 GMT Lines: 32 Shahram Mori (smori@nmsu.edu) wrote: : Vince Mulholland (mulholl@sasa.gov.uk) wrote: : : I've got a contaminating band in my PCR reactions. I'm using : : allele-specific amplification to differentiate between species of : : nematodes. The set-up is as follows; : : 'A'-specific 'B'-specific Universal : : --> --> <-- : : 390 bp 240 bp : : So, the PCR reaction contains three primers. I get a particular band : : for each species or both bands when there is a mixture. This has been : : working fine up until a few weeks ago. I still get a single band when : : I amplify a single species. However, when there is a mixture of the : : two species I get a contaminating 800 bp band. This is the odd part - : : it is ONLY present when there is a mixture. : : Anyone had similar problems? Any ideas gratefully accepted. Souds like heteroduplex formation to me. If the allele-specific products are sufficiently similar they could be hybridizing in later cycles to form an A:B heteroduplex. Since this heteroduplex is partially single-stranded it will migrate slower in the gel, thus giving the appearance of a larger fragment. Acrylamide gels are more susceptible to this problem than agarose gels. If you can detect the two allele-specific bands, why worry about the larger fragment? Rich Rohan rrohan@world.std.com From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!hookup!nstn.ns.ca!newsflash.concordia.ca!sunqbc.risq.net!athena.ulaval.ca!www.crhdq.ulaval.ca!user From: rioux@rsvs.ulaval.ca (Stéphane Rioux) Newsgroups: bionet.molbio.methds-reagnts Subject: E. coli transformation Date: 4 Jul 1995 16:52:33 GMT Organization: Centre de recherche de L'Hôtel-Dieu de Québec Lines: 12 Message-ID: NNTP-Posting-Host: www.crhdq.ulaval.ca I have a question which I didn't yet obtain a satisfactory answer. When E. coli is chemically transformed what happen? Do competents cells uptake many plasmids molecules or in the average only one plasmid molecule? Thank you for any help Stephane Rioux Centre de Recherche de l'Hotel-Dieu de Quebec Quebec, Canada G1R 2J6 From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!gatech!EU.net!Belgium.EU.net!chaos.kulnet.kuleuven.ac.be!idefix.CS.kuleuven.ac.be!infoserv.rug.ac.be!lmb32!marcvdc From: marcvdc@lmb1.rug.ac.be Newsgroups: bionet.molbio.methds-reagnts Subject: pCDNAI and pCDM8 problems in stable transfection ? Date: Tue, 4 Jul 1995 14:28:31 GMT Organization: Laboratory of Molecular Biology - University of Gent, Belgium Lines: 19 Message-ID: NNTP-Posting-Host: lmb32.rug.ac.be X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #1] Dear Netters, It seems that there are problems with the stable transfections of genes cloned in pCDM8. Although the transient transfections of the gene gave high expression levels, no protein was detected in the stable transfectans. The same was done with an biological inactive form of the protein and this gave the same results. So, it is not because of the toxicity of the gene expressed. Now I wonder wether someone had the same problems with pCDM8. Are the same problems seen in the pCDNAI (in vitrogen)? Maybe, I will use this vector in the future but then I want to be sure not to encounter the same problems. Please respond also to me personal. Thanks, Marc From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.acns.nwu.edu!news From: ctebeau@melre.acns.nwu.edu (Christopher M. Tebeau) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR Question Date: 4 Jul 1995 14:52:51 GMT Organization: Northwestern University Lines: 27 Message-ID: <3tbkk3$mqi@news.acns.nwu.edu> References: NNTP-Posting-Host: 165.124.225.219 Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 In article , kucukhu@mail.auburn.edu says... > > > Hello everybody, > > I am trying to amplify a 4.3 kb fragment from a virus to subclone >into a common vector..... I had a similar problem recently trying to subclone a 1.5 kb phage fragment using primers with pstI on each end. I got it to clone as follows: gel purify pcr reaction, digest entire elution, phenol chloroform extract and etoh precipitate the digest, ligate 1/10 and 9/10 of digest O.N. etc... When I did this w/o the phenol:chloroform step it didn't work. I am not sure why?? I have also subcloned smaller pcr fragments 150-500 bp with BamH1/EcoR1 primers with the same steps. Here I thought it would be important to inactivate BamH1 by P:C before the ligation because it doesn't heat inactivate according to the Gibco catalog. Anyway, I hope this helps, and if anyone knows of a good discussion in the literature about cloning PCR products I would appreciate hearing about it. Christopher M. Tebeau Northwestern University ctebeau@merle.acns.nwu.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: crousej@aol.com (CrouseJ) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: HepG2 nuclear extract Date: 4 Jul 1995 11:00:24 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 4 Sender: root@newsbf02.news.aol.com Message-ID: <3tbl28$7vp@newsbf02.news.aol.com> References: <3tbj9r$1a2@mserv1.dl.ac.uk> Reply-To: crousej@aol.com (CrouseJ) NNTP-Posting-Host: newsbf02.mail.aol.com I'm away from the office, but its a shot in the dark. Check out BioTechniques from volume 18 number 6 page 984. There is a protocol there see if it helps. Regards. From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!cam.news.pipex.net!pipex!dish.news.pipex.net!pipex!uknet!daresbury!not-for-mail From: Rainer Lemke Newsgroups: bionet.molbio.methds-reagnts Subject: HepG2 nuclear extract Date: 4 Jul 1995 15:30:19 +0100 Lines: 10 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3tbj9r$1a2@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk Hello, I am urgently in need of a protocoll for preparing a nuclear extract from HepG2 cells (or related). I already looked up the chapters in Current protocolls and Maniatis, but they only give a general procedure. I would be very happy, if you could email or fax a protocoll, which worked in your hands. Thanks a lot! Rainer lemke@server1.rz.uni-leipzig.de Fax (Rainer Lemke) +49 341/2114492 (University of Leipzig) From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!THORIN.UTHSCSA.EDU!HARDIES From: HARDIES@THORIN.UTHSCSA.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: B. megaterium rec. DNA guidlines? Date: 5 Jul 1995 01:05:58 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <950703102944.20211d13@thorin.uthscsa.edu> Can anyone please point me to recombinant DNA guidlines for working in Bacillus megaterium? Steve Hardies Hardies@uthscsa.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!uwm.edu!fnnews.fnal.gov!gw1.att.com!news.bu.edu!acs2.bu.edu!leach From: leach@bu.edu (martin LEACH) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Need lacZ gene to be used as a promoter probe reporter Date: 5 Jul 1995 17:45:48 GMT Organization: Boston University Lines: 20 Message-ID: <3tej4c$lfh@news.bu.edu> References: <3te6nf$h7e@synapse.bms.com> NNTP-Posting-Host: acs2.bu.edu X-Newsreader: TIN [version 1.2 PL2] Try clonetech...http://www.clonetech.com/ i've used their pnassbeta construct....seems ok... Martin taylor (patel_p@bms.com) wrote: : Hi: : I need a procaryotic promoter less (but has its own bacterial RBS) : lacZ gene. Basically, I need to use this gene as a reporter gene in a : specific bacterial promoter assay. Any info. on this will be greatly appreciated. : I have tried all of the common biotech. companies (viz. Promega, : Invitrogen etc.) : Thanks in advanve : Pramathesh : e-mai: Patel_P@BMS.com From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!ihnp4.ucsd.edu!news.service.uci.edu!miledi1 From: user@darwin.bio.uci.edu (User) Newsgroups: bionet.molbio.methds-reagnts Subject: monoclonal Abs in bacteria Date: Thu, 06 Jul 95 01:00:51 GMT Organization: University of California Irvine Lines: 21 Message-ID: <3tfck3$9ms_001@service.uci.edu> NNTP-Posting-Host: miledi1.bio.uci.edu X-Newsreader: News Xpress Version 1.0 Beta #3 Hi netters! Does anybody know any protocolo for the expression of immunoglobulin H an L genes in E coli. Advice on plasmids and cloning of the H and L cDNAs will be appreciated. ataulfo email mataulfo@darwin.bio.uci.edu UC Irvine Charlie "El puto amo de la barraca" ''The fucking lord of the barracks'' ["CRIO@DARWIN.BIO.UCI.EDU" or "onbriboc@lg.ehu.es"] From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!usc!news.cerf.net!newsserver.sdsc.edu!nic-nac.CSU.net!charnel.ecst.csuchico.edu!csusac!csus.edu!news.ucdavis.edu!chip!ez015890 From: ez015890@chip.ucdavis.edu (Traci Roth) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Who knows the tricks for S1 or primer Ext Date: 5 Jul 1995 23:16:24 GMT Organization: University of California, Davis Lines: 14 Message-ID: <3tf6g8$5lh@mark.ucdavis.edu> References: NNTP-Posting-Host: chip.ucdavis.edu X-Newsreader: TIN [version 1.2 PL2] Dr. Kimberly Walker (kwalker@umabnet.ab.umd.edu) wrote: : Is ther anyone out there who knows the tricks to the trade for : MungBean/S1 mapping v. Primer extension? I gotta do it quick and I want : to know if there are pitfalls I should watch out for. : Kim Kim, If you want to get S1 done quickly, I would suggest the Ambion S1 kit which worked well for me (no I am not a rep). Also, make sure you have reasonably good RNA for either procedure. For primer ex. I would suggest purifying primers before use. Feel free to email me if you want more details From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Newsgroups: bionet.virology,bionet.molbio.proteins,bionet.immunology,bionet.molbio.methds-reagnts,bionet.cellbiol Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!dbrml69.niaid.nih.gov!user From: Seth_Pincus@NIH.gov (Seth Pincus) Subject: Re: gp-120 participant has questions... Message-ID: Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: dbrml69.niaid.nih.gov Organization: NIAID References: Date: Wed, 5 Jul 1995 00:59:53 GMT Lines: 40 Xref: biosci bionet.virology:3791 bionet.molbio.proteins:4990 bionet.immunology:4736 bionet.molbio.methds-reagnts:30465 bionet.cellbiol:2514 You probably participated in one of several blinded studies. UCSF should have a study coordinator who can give you that information, plus others such as published articles resulting from the studies. But to be fair, it is highly unlikely that anyone without a detailed knowledge of your medical records can answer your questions. If your physician is not explaining these to you, get another opinion. In article , Ray Russ wrote: > I was one of a group of participants in a phase II gp-120 trial which was > administered by San Francisco General Hospital/UCSF several years ago. > > I am curious if anyone out there knows how long I will continue to test > "indeterminate" on Western Blot, Elysa (sp?) and PCR. > > Any answers/suggestions/input would be greatly appreciated. > > Please keep in mind that I am for all practical pourposes a neophyte as > far as immunology goes and was just a volunteer so please take it easy on > the technical aspects - thanks. > > > > ______________________________________________________________________________ > > Ray Russ > > Stanford Linear Accelerator Center > Operational Health Physics Department > > > > > > ______________________________________________________________________________ From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!lll-winken.llnl.gov!osi-east2.es.net!oracle.pnl.gov!mica.inel.gov!cwis.isu.edu!news.cc.utah.edu!mac64_14.med.utah.edu!user From: brad@corona.med.utah.edu (Brad Nicholson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Call for LacZ fragment for terminal bash Followup-To: bionet.molbio.methds-reagnts Date: Wed, 05 Jul 1995 14:46:06 +0000 Organization: Department of Pathology Lines: 20 Distribution: world Message-ID: References: <3t7lv6$17a@usenet.ucs.indiana.edu> NNTP-Posting-Host: mac64_14.med.utah.edu In article , raveh@BGUMAIL.BGU.AC.IL (Dina Raveh BGU) wrote: > We have a protein whose DNA sequence ends at +2. Does anyone have a lacZ > gene which we could blunt onto this and get readthru? > > Thanks, Dina Hello Dina, You might look for someone who has one of the vectors from Robert Simons, pRS415, pRS551 etc. He has both protein and operon fusions to lacZ, with a multi-cloning site with EcoRI-SmaI-BamHI (both orientations), it also has a transcriptional terminator in front of the cloning sites to prevent read- through. This may work for you. The reference is: Simons, R., Houman, F. & Kleckner, N.. 1987, Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene, 53, 85-96. Good luck, Brad From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!NMU.EDU!MKNEPPER From: MKNEPPER@NMU.EDU ("Knepper, Marc A") Newsgroups: bionet.molbio.methds-reagnts Subject: CTAB: How to crystallize? Date: 5 Jul 1995 16:03:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <05JUL95.20493267.0014.MUSIC@NMU.EDU> NNTP-Posting-Host: net.bio.net Greetings! Does anyone have a protocol for crystallizing CTAB (cetylmethylammonium bromide)? Any help would be greatly appreciated. Thanks. Marc A. Knepper mknepper@nmu.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!gatech!newsfeed.pitt.edu!godot.cc.duq.edu!hudson.lm.com!news.pop.psu.edu!psuvax1!uwm.edu!vixen.cso.uiuc.edu!usenet.ucs.indiana.edu!bronze.ucs.indiana.edu!ellenmq From: ellenmq@ucs.indiana.edu ( ) Newsgroups: bionet.molbio.methds-reagnts Subject: Req: Info. on 2D-SDS-PAGE Date: 5 Jul 1995 22:39:10 GMT Organization: Indiana University Lines: 20 Message-ID: <3tf4ae$fsk@usenet.ucs.indiana.edu> NNTP-Posting-Host: bronze.ucs.indiana.edu X-Newsreader: TIN [version 1.2 PL2] Hi, I posted earlier re: finding a company which might do 2D SDS-PAGE. I only heard of one lab called Kendrick Labs. Are there others? Has anyone used Kendrick Labs for 2D SDS-PAGE? Would people recommend sending off for 2D-SDSPAGE or setting up in their own lab for it based on the infrequency of need to do this protocol? Thanks in advance. Ellen M. Quardokus, Dept. of Biology Indiana University-Bloomington ellenmq@bronze.ucs.indiana.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!gatech!swrinde!dish.news.pipex.net!pipex!oleane!jussieu.fr!math.ohio-state.edu!newsfeed.acns.nwu.edu!news.acns.nwu.edu!news From: tar205@nwu.edu (TRACEY ANN RISTOW) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Gel printer/photodoc system Date: 5 Jul 1995 22:12:19 GMT Organization: Northwestern University, Evanston, IL. USA Lines: 17 Message-ID: <3tf2o3$qq5@news.acns.nwu.edu> Reply-To: tar205@nwu.edu NNTP-Posting-Host: lucky125.acns.nwu.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 We use the GelPrint 2000i by Biophotonics Corp (1-800-848-5235). It allows you to save approx. 500 images on its hard drive, but also has disk capabilites. The system prints out approx. 4" x 6" thermal prints of the captured images. Overall, we like this system except we have had a few problems with camara (Note: the company has since corrected the problems). The system costs between $12,000 - 14,000, but this also includes a PC. Hope this was of some help. Tracey Ristow Molecular Epidemiology Northwestern Memorial Hospital Chicago, IL 60657 tar205@nwu.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!ACD.TUSK.EDU!Prakash From: Prakash@ACD.TUSK.EDU (C. S. Prakash) Newsgroups: bionet.molbio.methds-reagnts Subject: RE: silicon carbide whisker Date: 5 Jul 1995 15:26:51 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 42 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HSIP56FTXE000435@Acd.Tusk.Edu> NNTP-Posting-Host: net.bio.net I do not know about the patent, but I am positive that the first report of use of silicon-carbide whisker mediated transformation was by Dr. Andrew Cockburn of USDA/ARS who transformed mosquito eggs initially and then many plants. Group led by Dr. Martin Wilson of ICI Seeds first reported the development of fertile transgenic plants (corn) using this approach in 1994. _C. S. Prakash >On 5 Jul 1995 lees@SASK.USASK.CA wrote: > >> >> Who is patent asignee for the silicon carbide whisker-mediated >> transformation of plants? And from when do they keep this patent? >> >> Thanks for any related information. >> >> >> Lee> >I think it's Cornell Univ. that has the patent. > >Charles F. (Chuck) Austerberry, Ph.D. >Assistant Professor of Biology >Creighton University >Omaha, NE 68178-0103 >e-mail: cfauster@creighton.edu > >> >> >> > > C. S. Prakash Voice (334) 727 - 8023 Tuskegee University Fax (334) 727 - 8067 School of Agriculture,Tuskegee Email: Prakash@Acd.Tusk.Edu Alabama 36088 USA From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!gatech!bloom-beacon.mit.edu!boulder!sprint!aplummer From: aplummer@sprint.uccs.edu (AL R. PLUMMER) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: 5-FOA solubility Date: 5 Jul 1995 20:34:54 GMT Organization: University of Colorado at Boulder Lines: 25 Message-ID: <3tet1e$sok@lace.Colorado.EDU> References: NNTP-Posting-Host: sprint.uccs.edu X-Newsreader: TIN [version 1.2 PL2] Scott Vande Pol (sbv@pop.cwru.edu) wrote: : We are using FOA (5-floroorotic acid) for selection against URA-3 plasmids : in yeast. FOA is expensive to buy and difficult to get into solution. : Does anyone know of a non-toxic solvent (such as DMSO) in which a stable : 100X FOA stock solution can be made? : -- : Scott Vande Pol : Case Western Reserve University : Department of Pathology : Cleveland Oh. 44106 : sbv@pop.cwru.edu I know this will sound bad, but I have two methods for getting 5-FOA into solution: 1)Apply a slight-not high!-amount of heat to your 5-FOA solution. I know they say that it is heat labile, but in my hand it underegoes very little degredation. 2)Raise the pH of your solution with NaOH until it goes into solution (Don't use too much NaOH). I have used both methods depending on the medium I am preparing: SD(Glucose) or SG(Glycerol) and have been able to select ura3- mutants or 'cure' plasmids. Hope this helps, -Al(aplummer@sprint.uccs.edu) From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!gatech!europa.chnt.gtegsc.com!howland.reston.ans.net!swrinde!cs.utexas.edu!news.tamu.edu!sewild1.tamu.edu!user From: Yasha@bioch.tamu.edu (Yasha Hartberg) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: how to work out curvature of DNA??? Date: Wed, 05 Jul 1995 16:17:44 +0300 Organization: Texas A&M University Lines: 24 Message-ID: References: NNTP-Posting-Host: sewild1.tamu.edu In article , gqva12@udcf.gla.ac.uk (G Gallagher) wrote: > Hello out there > > Does anyone know how to work out the extent to which the secondary structure > and/or curvature of dsDNA is altered by mutations? > > Specifically, I want to guage the effect of differing sizes of a CA repeat. > > There doesn't seem to be an obvious programme in the GCG package. Secondary structure prediction of DNA is not as well established as for RNA, but you may be able to use the same algorithms as RNA secondary structure prediction programs by substituting in the free energy values determined recently for DNA base stacking. As for DNA bending, I don't think it is well enough understood to develop prediction algorithms. There are experimental techniques available to determine the extent of DNA bending, however. You might begin looking for papers by Richard Sinden or Jan Klysik. I think they have done some work in this area. Yasha Hartberg Texas A&M University "The most beautiful thing in Tokyo is McDonald's." Andy Warhol From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.starnet.net!wupost!waikato!comp.vuw.ac.nz!matai.vuw.ac.nz!johnnyjw From: johnnyjw@matai.vuw.ac.nz (Johnny Wharton) Newsgroups: bionet.molbio.methds-reagnts Subject: Mycoplasma Detection Date: 6 Jul 95 09:18:46 +1200 Organization: Victoria University of Wellington, NZ Lines: 14 Distribution: world Message-ID: <1995Jul6.091846@matai.vuw.ac.nz> NNTP-Posting-Host: matai.vuw.ac.nz Howdy Does anybody out there know the sequences of the primers supplied in the Stratagene mycoplasma detection primer set or any other useful information regarding PCR detection of mycoplasmic contamination of cell-culture? Any information would be gratefully recieved by mail at the address: MIMRCH@wnmeds.ac.nz thanks, Craig Hilton From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!agate!news.Stanford.EDU!not-for-mail From: ladasky@leland.Stanford.EDU (John Ladasky) Newsgroups: bionet.molbio.methds-reagnts Subject: I lost the inverse PCR article! Date: 5 Jul 1995 22:05:03 -0700 Organization: Stanford University, CA 94305, USA Lines: 14 Message-ID: <3tfqtv$s8c@elaine15.Stanford.EDU> NNTP-Posting-Host: elaine15.stanford.edu Greetings, everyone, A few weeks ago, someone posted an article giving a detailed inverse PCR procedure. Well, I thought I saved this article, but it seems that I did not. The Triglia and Hartl papers are both very sketchy, and I've been attempting to fill in the blanks without success. If anyone saved that article with the detailed protocol, I would appreciate it greatly if you would send it to me. Thanks in advance! -- Unique ID : Ladasky, John Joseph Jr. Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???) Location : Stanford University, Dept. of Structural Biology, Fairchild D-105 Keywords : immunology, music, running, Green From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!gatech!news.uoregon.edu!news.bc.net!unixg.ubc.ca!gstuart From: gstuart@unixg.ubc.ca (Gregory Stuart) Newsgroups: bionet.molbio.methds-reagnts Subject: Help - isolation of DNA from snake cross-sections Date: 6 Jul 1995 04:51:06 GMT Organization: University of British Columbia, Vancouver, B.C., Canada Lines: 18 Message-ID: <3tfq3q$2uk@nntp.ucs.ubc.ca> NNTP-Posting-Host: unixg.ubc.ca Hello - I am posting this for a friend. He is isolating DNA from cross-sections of snakes (snake slices), as follows : grinding the frozen tissue in liquid nitrogen; overnight proteinase K incubation; phenol-CHCl3 extractions, CHCl3 extractions. His A280/A260 ratio is >1, indicating a lot of protein. He does not see much proteinaceous material at the interface. Apparently, the proteinase K digests are working, as the tissue suspensions clarify during the incubations. Also, there appears to be some problem with carry-over of (colored) contaminants during the DNA isolations. The intended use for the DNA is PCR and multiplex PCR. Any hints, ideas, and suggestions would be welcome. A working protocol would be most appreciated! :-) I will post a summary if sufficient useful material is received. -- -- Greg Stuart gstuart2@sol.uvic.ca Fax : 604-656-8148 Centre for Environmental Health, University of Victoria, 9865 West Saanich Rd., Sidney, British Columbia, CANADA. V8L 3S1. From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!news.moneng.mei.com!sol.ctr.columbia.edu!news.mtu.edu!msunews!harbinger.cc.monash.edu.au!news.cs.su.oz.au!metro!news From: cjackson@blackburn.med.su.oz.au (cjackson) Newsgroups: bionet.molbio.methds-reagnts Subject: Merck Index on the internet? Date: 5 Jul 1995 02:51:35 GMT Organization: Your Organization Lines: 3 Distribution: inet Message-ID: <3tcunn$5kp@metro.ucc.su.OZ.AU> NNTP-Posting-Host: ts-h08-15-30.ucc.su.oz.au X-Newsreader: WinVN 0.92.6+ Does anybody know whether the Merck Index is available on the internet? Or any other similar database? From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!torn!news.unb.ca!coranto.ucs.mun.ca!leif!vdrover From: vdrover@kean.ucs.mun.ca Newsgroups: bionet.molbio.methds-reagnts Subject: klenow Date: 5 Jul 95 14:28:19 -0230 Organization: Memorial University. St.John's Nfld, Canada Lines: 10 Message-ID: <1995Jul5.142819.1@leif> NNTP-Posting-Host: leif.ucs.mun.ca just a quick question...i want to end-fill restriction fragments (ie:lamda DNA cut with HindIII) to incorporate 32P-dNTPs. I've got Klenow from pharmacia but the buffers for the reaction vary from place to place. Does anyone have a reliable buffer (component concentrations as well) to us with Klenow? please mail responses to vdrover@kean.ucs.mun.ca vic drover memorial university of newfoundland st. john's, newfoundland canada From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!news.starnet.net!wupost!howland.reston.ans.net!news.sprintlink.net!newsfeed.internetmci.com!news.compuserve.com!news.production.compuserve.com!news From: John M. Garrison <74222.3137@CompuServe.COM> Newsgroups: bionet.molbio.methds-reagnts Subject: Re: How to import MD PhosphorImager images? Date: 5 Jul 1995 13:32:07 GMT Organization: via CompuServe Information Service Lines: 31 Message-ID: <3te48n$j88$1@mhade.production.compuserve.com> References: <3t8tr5$8qc@ousrvr.oulu.fi> jkortes@paju.oulu.fi (Jarkko Kortesmaa) writes: >>Is is possible to export images from the ImageQuaNT program of >>Molecular >>Dynamics PhosphorImager? The Molecular Dynamics PhosphorImager (like all Molecular Dynamics scanners) uses a standard 16-bit TIFF file. Even though this is an "industry-standard" file, it is not supported by many other software programs. Most programs will only accept 8 bit and 24 bit TIFF's. (A 24 bit TIFF is really 8 bit TIFF x 3 color channels, while a 16 bit TIFF is 16 bits in one greyscale channel). Molecular Dynamics provides a utility called Convert 16 to 8 which allows you to make an 8 bit representation of a 16 bit TIFF and save it as a new file. Since your monitor is an 8 bit greyscale display anyway, you will not see a visual difference. However, the new file will be 1/2 the file size and will not be as accurate for quantitation. This 8 bit TIFF can be opened by Word, Excel, Photostyler, and Corel draw, to name a few. Depending on your software version, the utility will be built into ImageQuant or bundled with it. Please e-mail me with more specifics if you need more help. specifics if you need more help. specifics if you need more help. -- John M. Garrison jgarrison@mdyn.com Molecular Dynamics (my opinions are solely my own - but they're correct) From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!agate!ames!lll-winken.llnl.gov!noc.near.net!usenet.uchc.edu!TimHusky From: tkuwada@neuron.uchc.edu (TimHusky) Newsgroups: bionet.molbio.methds-reagnts Subject: Ligations-HELP Date: 6 Jul 1995 02:52:28 GMT Organization: Univ of CT Health Center Lines: 23 Sender: -Not-Authenticated-[4752] Message-ID: <3tfj5c$pa5@threed.uchc.edu> NNTP-Posting-Host: tanzlab.uchc.edu X-Posted-From: InterNews 1.0@threed.uchc.edu. Xdisclaimer: No attempt was made to authenticate the sender's name. Iam having trouble with a seemingly straight forward sticky-end ligation. I have used up to 800U/20ul rxn with Insert:plasmid ratio's of 3:1 - 13:1, total DNA of up to 300ng. These rxn have been overnight @ 16C.I have several questions: 1) Am I using too much or too little DNA? 2) Should I clean my DNA from LMP gels? I have tried using DNA in LMP and cleaning it w/ a promega kit (bad yield) 3) I am using 400U/ul T4 ligase (NE Biolabs), has anyone else had any problems w/ their ligase or buffer? 4) Will spiking my buffer w/extra ATP help & what is the deal w/ PEG, does it increase ligation efficiency 5) How can I increase the competency of my DH5-alpha cells,Iam currently using a 50mM CaCl2 2X wash of cells at .3-.4OD @ 590nm I would greatly appreciate any tips. Tim Kuwada Med Student Univ Conn Health Center tkuwada@neuron.uchc.edu From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!agate!ames!lll-winken.llnl.gov!noc.near.net!usenet.uchc.edu!TimHusky From: tkuwada@neuron.uchc.edu (TimHusky) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA Sequencing-HELP Date: 6 Jul 1995 02:32:25 GMT Organization: Univ of CT Health Center Lines: 22 Sender: -Not-Authenticated-[4752] Message-ID: <3tfhvp$oq6@threed.uchc.edu> References: NNTP-Posting-Host: tanzlab.uchc.edu X-Posted-From: InterNews 1.0@threed.uchc.edu. Xdisclaimer: No attempt was made to authenticate the sender's name. The temp of the gel can affect the resolution of your bands. When you load your second run the gel may be "hotter" than your first load, thus giving you better resolution. The optimum temp should be above 55C, dont go too hot as this may crack your plates. I dont know if you are doing this, but pre-warming your gel (70W 1hr) will greatly improve your resolution; try placing a piece of styrofoam over the plates to facilitate warming. Also the top - mid portions of the gel are easier to read. If you run your first load for 3hrs and then develop it (no second load), you may be able to visualize your blurred range. You may have to run mulitple gels but it may be shorter and easier in the long run. Good luck Manitoba (I was born in Winnipeg) Tim Kuwada Medical Student Univ. Connecticut From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!news.uoregon.edu!europa.chnt.gtegsc.com!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: squirenige@aol.com (SquireNige) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: HELP with glcyerol-tolerant sequencing gels Date: 5 Jul 1995 21:27:55 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 15 Sender: root@newsbf02.news.aol.com Message-ID: <3tfe6r$73t@newsbf02.news.aol.com> References: Reply-To: squirenige@aol.com (SquireNige) NNTP-Posting-Host: newsbf02.mail.aol.com Hey, I think this deal with the glyceroo tolerant buffer is somewaht of a misnomer. I routinely have run 5% gels using TBE having diluted sequenase in the glycerol buffer and i dont seem to have any problems. The artifact caused by the glycerol runs up in the 400 bp region so for shorter runs than this I wouldnt worry about it. It doesnt seem as pronounced using the 5% gesl either so that may be a way out for you? Good luck Nigel nwalker@welchlink.welch.jhu.edu Johns Hopkins University From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!rutgers!uwm.edu!news.moneng.mei.com!news.ecn.bgu.edu!vixen.cso.uiuc.edu!news.ksu.ksu.edu!news.mid.net!news.creighton.edu!bluejay.creighton.edu!cfauster From: Charles F Austerberry Newsgroups: bionet.molbio.methds-reagnts Subject: Re: silicon carbide whisker Date: Wed, 5 Jul 1995 15:13:29 -0500 Organization: Creighton University, Omaha Nebraska USA Lines: 22 Message-ID: References: NNTP-Posting-Host: bluejay.creighton.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: I think it's Cornell Univ. that has the patent. Charles F. (Chuck) Austerberry, Ph.D. Assistant Professor of Biology Creighton University Omaha, NE 68178-0103 e-mail: cfauster@creighton.edu On 5 Jul 1995 lees@SASK.USASK.CA wrote: > > Who is patent asignee for the silicon carbide whisker-mediated > transformation of plants? And from when do they keep this patent? > > Thanks for any related information. > > > Lee > > > From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!arabidopsis.botany.wisc.edu!user From: trichmon@students.wisc.edu (Todd Richmond) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Purifying Bacto-Agar: Suggestions? Date: Wed, 05 Jul 1995 14:43:29 -0500 Organization: UW-Madison Dept of Botany Lines: 33 Message-ID: References: <3tc71f$ip8@rover.ucs.ualberta.ca> NNTP-Posting-Host: arabidopsis.botany.wisc.edu X-Newsreader: Yet Another NewsWatcher 2.0b27.4 In article <3tc71f$ip8@rover.ucs.ualberta.ca>, mdeyholo@gpu1.srv.ualberta.ca (Michael Deyholos) wrote: > To save money, I'd like to use bacto-agar (e.g. DIFCO) in place of noble > agar as part of > a solid growth medium for screeing seedling-lethal mutations in Arabidopsis. > > I've heard of washing bacto-agar in cold TE or water for a few days in a > cold room. Apparently, the agar turns pure white after a few washes. > > Does anyone else have experience with this? > > Anyone know of a supplier of "PHYT-AGAR" > > Thanks, > Mike We've washed "plant tissue culture certified" agar with 95% EtOH a couple of times and then strained it through cheesecloth or Miracloth and let it dry. We needed really clean agar for some transformation experiments and this took care of some of the problems we were having. It might work for bacto-agar as well. Todd ********************************************************** Todd Richmond trichmon@students.wisc.edu B129 Birge Hall UW-Madison Dept of Botany Knowledge=power=energy=matter=mass; a good bookshop is just a genteel Black Hole that knows how to read - "Guards! Guards!", Terry Pratchett From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!EU.net!uknet!daresbury!not-for-mail From: Andrei Popov Newsgroups: bionet.molbio.methds-reagnts Subject: RE: 5-FOA solubility Date: 5 Jul 1995 21:59:17 +0100 Lines: 17 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3teuf5$mfu@mserv1.dl.ac.uk> Importance: High Sensitivity: Company-Confidential Original-To: methods@dl.ac.uk (Receipt Notification Requested) (Non Receipt Notification Requested) (IPM Return Requested) >We are using FOA (5-floroorotic acid) for selection against URA-3 plasmids >in yeast. FOA is expensive to buy and difficult to get into solution. >Does anyone know of a non-toxic solvent (such as DMSO) in which a stable >100X FOA stock solution can be made? I remember once dissolving 5-FOA in water titrated with NaOH. The stuff dissolved and I used to spread it on agar plates. By the way, you might have a look at Yeast (1991) vol7:607. Proline causes hypersensitivity to 5-FOA, and you can save a bit of money on it. There was also a posting (bionet.molbio.yeast) a couple of weeks ago by one canadian company which offered very cheap 5-FOA. regards Andrei From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!NMSU.EDU!hroychow From: hroychow@NMSU.EDU (Hiranya Roychowdhury) Newsgroups: bionet.molbio.methds-reagnts Subject: KODAK LPD4 Date: 5 Jul 1995 13:59:25 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hello Netters, I have a roll of LPD4 film (KODAK; B/W) that I bought about 13mo back and used once. Consequently I forgot the speed of the film. Does anybody know the ASA/DIN number of this film. The local Camera Shop guy could not help me (yes, there is only one in this town). Thank you in advance, Hiranya. >>>>>>>>>>>>>>>>>>>>>>>>>>> Hiranya S. Roychowdhury Plant Genetic Engineering Lab. Box 3GL, NM State Univ. Las Cruces, NM 88003 Phone: (505) 646-5785 hroychow@nmsu.edu <<<<<<<<<<<<<<<<<<<<<<<<<<< From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!biosci!not-for-mail From: BIOSCI Administrator Newsgroups: bionet.molbio.methds-reagnts Subject: New net.bio.net IP address still not propagated correctly! Date: 5 Jul 1995 11:34:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: kristoff@net.bio.net Distribution: world Message-ID: <3telvn$fsh@net.bio.net> NNTP-Posting-Host: net.bio.net Although we sent in a change request to the InterNIC registration service some time back, as of this morning, 5 July, it had still not made it through their work queue. I called InterNIC this morning and have been assured that they will have the new number, 204.31.212.2, for net.bio.net in their system by this evening. The consequence of this delay is that every time our system broadcasts the correct number, it often gets reset at other sites by other servers such as InterNIC which continue to hold the old number. This means service disruptions, etc. If the InterNIC does indeed process this request this evening, we should finally get the IP address problem resolved at most sites by the weekend. On the hardware side, we ran successfully from Friday to Monday on a minimal system configuration, then added another CPU board on Monday and experienced two crashes later that day but none on Tuesday. More board swapping will go on later today as we continue to attempt to diagnose the problem. Since we are running wth less CPUs than usual, the system is a bit bogged down, but functional. Nonetheless, avoidance of unimportant demands on the CPUs would be appreciated. Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Path: biosci!SASK.USASK.CA!lees From: lees@SASK.USASK.CA Newsgroups: bionet.molbio.methds-reagnts Subject: silicon carbide whisker Date: 5 Jul 1995 11:55:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Who is patent asignee for the silicon carbide whisker-mediated transformation of plants? And from when do they keep this patent? Thanks for any related information. Lee From owner-methds-reagnts@net.bio.net Tue Jul 04 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!ns1.faseb.org!darwin.sura.net!nih-csl!bernard From: bernard@elsie.nci.nih.gov (Bernard Murray) Subject: Re: Alternative to Zeocin? Message-ID: <1995Jul5.190617.18581@alw.nih.gov> Summary: No, Zeocin is apparently phleomycin D1 Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: elsie.nci.nih.gov Organization: National Institutes of Health References: <3tc4s8$cff@newsbf02.news.aol.com> Date: Wed, 5 Jul 1995 19:06:17 GMT Lines: 22 In article <3tc4s8$cff@newsbf02.news.aol.com>, davidm7643@aol.com (DavidM7643) writes: > Zeocin is bleomycin NOT SO! I have the Zeocin data sheet (from Invitrogen) in front of me and this states that it is "not as toxic as bleomycin on fungi". The compound is apparently phleomycin D1. For those of you that are using it, forget about the need for "low salt LB" medium and use 1x YT. This is much richer and the antibiotic works just fine on plates and in culture (no cross-resistance to amp, tet or kan). My main worry about the pZeoSV vector is that it requires CMV as the promoter for the Zeocin resistance gene. So; a) Why is such a very strong promoter needed? Is the gene product inefficient? b) Do the SV40 and CMV promoters interfere with each other? c) What happens in COS cells (where the SV40 and CMV promoters will be strongly modulated by the T-antigen) compared to "normal" cells (basal promoter activity)? Bernard Murray, Ph.D. bernard@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA) From owner-methds-reagnts@net.bi