From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!BORCIM.WUSTL.EDU!brett From: brett@BORCIM.WUSTL.EDU (brett) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: packaging protocol Date: 2 Feb 1998 06:56:01 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802021451.IAA16499@borcim.wustl.edu> NNTP-Posting-Host: net.bio.net > Hello! I am using the stratagen packaging kit GIGApack. At the end of >the protocol, it is recommended to add chloroform after adding SM medium. >Does anybody know the aim and the mechanism of this step. > Thanks. > Christophe and Sylvain. Your phage will be stable and remain free of other organisms this way. Brett Lindenbach brett@borcim.wustl.edu Dept Molecular Microbiology Washington University School of Medicine Box 8230 660 S. Euclid Ave St Louis, MO 63110 From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!fu-berlin.de!zrz.TU-Berlin.DE!news-ber1.dfn.de!news-ham1.dfn.de!news.mu-luebeck.de!not-for-mail From: "Lars Komorowski" Newsgroups: bionet.molbio.methds-reagnts Subject: HPLC-Manager-Software Date: Mon, 2 Feb 1998 20:48:43 +0100 Organization: Med. Universitaet zu Luebeck Lines: 6 Message-ID: <6b586e$9d9$1@gwsun.medinf.mu-luebeck.de> NNTP-Posting-Host: 141.83.167.168 X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 Who can help me ? I lost data from HPLCManager 3.0 for DOS from Pharmacia LKB. And I don't have the disks anymore (they were from 1991). Who can send me the installation files or maybe newer ones if they work ? Lars From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!ato.dlo.nl!M.D.vanderhee From: M.D.vanderhee@ato.dlo.nl (Miranda v/d Rhee) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Bio-Rad's gene gun tubing coating Date: 2 Feb 1998 06:52:38 -0800 Organization: ATO-DLO Lines: 33 Sender: daemon@net.bio.net Distribution: world Message-ID: <34D65B91.7070@ato.dlo.nl> NNTP-Posting-Host: net.bio.net Bezstarosti (bezstarosti@bc1.fgg.eur.nl) wrote: > We have problems with the coating of the tubing, the distribution of > the gold particles is uneven. The gold sticks to one side of the tube > even after rotation of the tubing prep station. Does somebody have a > solution or a few tips to improve the coating. > > Thanking you in advance > K. Bezstarosti > EUR > Rotterdam > Netherlands > ---------------------------------------------------------------------- We have the same problem. The gold is most dense on one side of the tubing. The gold also smears to other places, but is not evenly distributed there. We get a marble-like pattern of gold on the tubing. Also, the gold clots after coating with DNA, which is not solved by sonication. We use the Helios Gene Gun for plant leaf tissue. There is severe tissue damage in the centre of the shot and only in the periphery we get GUS expressing spots. Does anyone have a solution for these problems? Does anyone have experience with the Helios Gene Gun on plant (leaf) tissue? Thanking you in advance, M. van de Rhee Agrotechnological Research Insitute (ATO-DLO) P.O. Box 17 6700 AA Wageningen The Netherlands m.d.vanderhee@ato.dlo.nl fax: 31-317475347 From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!WWW.BRIC.POSTECH.AC.KR!mhlee From: mhlee@WWW.BRIC.POSTECH.AC.KR (Min-Ho Lee) Newsgroups: bionet.molbio.methds-reagnts Subject: Database of the Korean biological journals (Abstracts) Date: 2 Feb 1998 18:03:29 -0800 Organization: BRIC Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <34D5612A.52880E1C@bric.postech.ac.kr> NNTP-Posting-Host: net.bio.net Dear Nettiers, I'm constructing a database of the Korean biological journals. You can find freely more about 2,000 abstracts ('90 - current issues) and many full texts+MAA- (PDF file format) in this site: http://bric.postech.ac.kr/e_index.html http://bric.postech.ac.kr/publication/bric_journal.html Abstracts of 8 major Korean biological journals, Experimental and Molecular Medicine, Journal of Biochemistry and Molecular Biology, Journal of Microbiology, Journal of Microbiology and Biotechnology, Journal of Plant Biology, Korean Journal of Zoology, Korean Journal of Biological Science, and Molecules and Cells were contained in this database. Min-Ho Lee mhlee@bric.postech.ac.kr BRIC, POSTECH http://bric.postech.ac.kr/+AH4-mhlee/ From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!ais.net!uunet!in2.uu.net!hearst.acc.Virginia.EDU!murdoch.acc.Virginia.EDU!not-for-mail From: Rajesh Kumar Newsgroups: bionet.molbio.methds-reagnts Subject: Preparation of nuclei Date: Mon, 02 Feb 1998 18:45:20 -0400 Organization: University of Virginia Lines: 9 Message-ID: <34D64C7F.66D3@virginia.edu> NNTP-Posting-Host: ppp-32-47.itc.virginia.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.02Gold (Macintosh; I; PPC) Hi, I am looking for a protocol for preparation of nuclei from cultured cells. I will highly appreciate any input. Thanks Rajesh rk6n@virginia.edu From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!newsfeed.direct.ca!torn!nott!uottawa!aix1.uottawa.ca!s535290 From: Robot-Boy Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Better chelator of zinc than EDTA? Date: Mon, 2 Feb 1998 16:58:47 -0500 Organization: University d'/of Ottawa Lines: 12 Message-ID: References: <6b03p1$8ln@bgtnsc02.worldnet.att.net> NNTP-Posting-Host: aix1.uottawa.ca Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII To: Damaged Goods In-Reply-To: <6b03p1$8ln@bgtnsc02.worldnet.att.net> > Hi, > > Im wondering if anyone knows and could post about a better chelator of > zinc than EDTA? > > Thanks. I think CDTA is supposed to be better than EDTA. ed From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!204.156.128.20!news1.best.com!noos.hooked.net!news.well.com!not-for-mail From: "Todd Martinsky" Newsgroups: bionet.molbio.methds-reagnts Subject: scanarray Date: Mon, 2 Feb 1998 14:10:49 -0800 Organization: Technology Mentors, Inc. Lines: 5 Message-ID: <6b5gfd$7cm$1@was.hooked.net> NNTP-Posting-Host: italy-35.ppp.hooked.net X-Newsreader: Microsoft Outlook Express 4.72.2106.4 X-MimeOLE: Produced By Microsoft MimeOLE V4.72.2106.4 for those who want to scan microarrays, but can't do it yet go to http://www.hooked.net/~telechem/scan/ for more information. Good luck. From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.14!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!fu-berlin.de!news.belwue.de!news.uni-hohenheim.de!news From: Ralf Weigel Newsgroups: bionet.molbio.methds-reagnts Subject: pBI101 vectors Date: Tue, 03 Feb 1998 00:14:21 -0500 Organization: Allg. Virologie, Uni Hohenheim Lines: 18 Message-ID: <34D6A7AD.63F2@uni-hohenheim.de> NNTP-Posting-Host: tomate.genetik.uni-hohenheim.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02E (OS/2; I) Dear netters, could anyone please tell where I may buy or request the pBI101 vector series. Clontech does not offer them anymore. Thanks in advance. Ralf -- R. Weigel General Virology Institute of Genetics University of Hohenheim Emil Wolff Str.14 70599 Stuttgart Germany Tel.: +049/711/4592395 Fax: ~4592937 From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!chicago-news-feed2.bbnplanet.com!news.bbnplanet.com!news.itis.com!news.doit.wisc.edu!svetlov From: svetlov@oncology.wisc.edu (Vladimir Svetlov) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: His-tagged protein, urea & antibody production Date: Mon, 02 Feb 1998 14:19:20 -0600 Organization: McArdle Lab for Cancer Research, UW-Madison Lines: 44 Message-ID: References: <34D5883B.CCF@nospam.ic.ac.uk> NNTP-Posting-Host: alpha.oncology.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 In article <34D5883B.CCF@nospam.ic.ac.uk>, k.desmet@nospam.ic.ac.uk wrote: > Glenda Lawrence wrote: > > > > Hi. > > > > Does anyone have ideas or experience re- the best > > way to remove urea before injecting recombinant > > protein into rabbits for antibody production? > > How much urea can be tolerated? > > The protein is a His-tagged protein (insoluble) purified > > and eluted in 8M urea, 20mM Tris, 150mM NaCl and 200mM imidazole. > > > > Thanks, > > > > Glenda Lawrence > > > I don't know if this is the "best way", but we have succesfully produced > antisera by dialysing the urea away. In most cases, the recombinant > His-tagged protein preciptated, but that does not matter. YOu can still > make a suspension with incomplete Freunds adjuvant Well, it does not have to precipitate. One of the simplest solutions is to do a step-wise dialysis: 6M->4M->2M->0.7M->0M urea. If you don't use higher than 0.5 mg/ml concentration most of the proteins are likely to stay solubilized. Adding sarcosyl and Arg helps to refold some difficult proteins. Another way to refold you sticky protein is to refold it on the column by appying a shallow gradient of urea from 6 to 0M, or in the batch by using washings through the same steps as in dialysis above. Immobilization on the resin would not prevent proteins from collapsing on themselves, thus malfolded proteins may still result (as with any of these methods), but at least it antagonizes aggregation. Regards, V. -- Vladimir Svetlov McArdle Lab for Cancer Research Dept. Oncology UW-Madison 1400 University Ave. Madison, WI 53706 From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!207.5.0.44!nntp.mainstreet.net!news.pbi.net!news.pacbell.net!not-for-mail From: Susan Kang Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Free Power Supply Date: Mon, 02 Feb 1998 11:59:13 -0800 Organization: HIFAX Lines: 27 Message-ID: <34D62591.13E1@hifax.com> References: <34D5C4AC.51A2@hifax.com> Reply-To: order@hifax.com NNTP-Posting-Host: ppp-206-170-217-86.nhwd02.pacbell.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01C-PBWG (Win95; U) Susan Kang wrote: > > Dear Colleagues; > > As a new company, we decided to distribute our new PULSE POWER freely > to promote the customer recognition of the product. If you have any > interest please visit our web site www.hifax.com and send us e-mail. We > will send PULSE POWER to every 100th e-mailer. Furthermore, We will give > you a wonderful chance of spending vacation at Hawai. > > Susan Kang > Sales Manager > HIFAX.COM Dear Colleagues; Thank you for the innundation of your e-mails. However we found that some mailers did not visit our web site and just typed reply command from your e-mailer. It definitely is not working. Please visit our web site and find out the e-mail address from the front page and send your mail to that address. Many Thanks Susan Kang HIFAX From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!millipore.com!Jack_Leonard From: Jack_Leonard@millipore.com Newsgroups: bionet.molbio.methds-reagnts Subject: Re: streptavidin columns? Date: 2 Feb 1998 06:56:18 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 40 Sender: daemon@net.bio.net Distribution: world Message-ID: <8525659F.0051CC4D.00@MilliGust.Millipore.com> NNTP-Posting-Host: net.bio.net Check out GenoSys (no affiliation). They have (or did have) the streptavidin tips (AffiniTips?) you're looking for. Jack T. Leonard, Ph.D., Tech. Mgr Molecular Biology Group Millipore Corp. dzolek@ksu.edu on 02/01/98 02:50:16 PM To: methods@net.bio.net cc: (bcc: Jack Leonard/Bev/NA/Millipore) Subject: streptavidin columns? Some time ago I saw an advertisment for some mini-columns that had a streptavidin-impregnated matrix layer; the idea was that they fit on the end of a pipettor and one could dispense the "filtered" solution without a spin step. I sure could use them now, but I can't seem to find them. Does anyone know who makes them? Does anyone know of a similar product? TIA Dr. D. Z. Skinner USDA-ARS and Kansas State University 3006 Throckmorton Hall Manhattan, KS 66506-5501 Telephone (785) 532-7247 Fax: (785) 532-6167 http://naaic.org From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!194.72.7.126!btnet-peer!btnet!newsfeed.cableol.net!server4.netnews.ja.net!news.cc.ic.ac.uk!not-for-mail From: Koen De Smet Newsgroups: bionet.molbio.methds-reagnts Subject: Re: His-tagged protein, urea & antibody production Date: Mon, 02 Feb 1998 08:47:55 +0000 Organization: Department of Medical Microbiology Lines: 28 Message-ID: <34D5883B.CCF@nospam.ic.ac.uk> References: Reply-To: k.desmet@nospam.ic.ac.uk NNTP-Posting-Host: cb-22.sm.med.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; PPC) Glenda Lawrence wrote: > > Hi. > > Does anyone have ideas or experience re- the best > way to remove urea before injecting recombinant > protein into rabbits for antibody production? > How much urea can be tolerated? > The protein is a His-tagged protein (insoluble) purified > and eluted in 8M urea, 20mM Tris, 150mM NaCl and 200mM imidazole. > > Thanks, > > Glenda Lawrence I don't know if this is the "best way", but we have succesfully produced antisera by dialysing the urea away. In most cases, the recombinant His-tagged protein preciptated, but that does not matter. YOu can still make a suspension with incomplete Freunds adjuvant Koen De Smet ============================================================== ==>> To reply by email, remove "nospam." from the address <<== Imperial College School of Medicine at St Mary's http://www.sm.ic.ac.uk/medmicro/home. ============================================================== From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!newsfeed.eerie.fr!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!hgmp.mrc.ac.uk!jgreen From: jgreen@hgmp.mrc.ac.uk (Dr. J. Green) Newsgroups: bionet.molbio.methds-reagnts Subject: Heteroduplex mobility assays Date: 2 Feb 1998 19:40:43 GMT Organization: MRC Human Genome Mapping Project Resource Centre Lines: 11 Message-ID: <6b57fr$oj0$1@niobium.hgmp.mrc.ac.uk> NNTP-Posting-Host: tin.hgmp.mrc.ac.uk Hi, We're looking to use HMA to analyse small PCR products, the smallest being 120bp.We've tried Metaphor agarose and the PHAST PAGE system but haven't got particularly good resolution(we can differentiate products with upto approximately 80% identity but would like to raise the resolution to 90% identity). We could use standard PAGE gels but would prefer to use agarose. Is that feasible on products this small? Can anyone suggest alternative media. Any help appreciated Thanks Jon Green From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!news-peer.sprintlink.net!news-backup-west.sprintlink.net!news-in-west.sprintlink.net!news.sprintlink.net!Sprint!196.4.160.15!feeder.is.co.za!hermes.is.co.za!not-for-mail From: "Otto" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: stripping RNA probes Date: 2 Feb 1998 14:38:39 GMT Organization: OVI Lines: 21 Message-ID: <01bd2fe9$07f4a180$322660c0@virgo.ovi.ac.za> References: <34D0FAAE.A7FB05ED@maties.sun.ac.za> NNTP-Posting-Host: gauntlet.agric.za X-Newsreader: Microsoft Internet News 4.70.1155 Hi Judi Look at: www.ambion.com/RNA/html/strip-ez.html Cheers Otto Judi <9320393@maties.sun.ac.za> wrote in article <34D0FAAE.A7FB05ED@maties.sun.ac.za>... I would like to start hybridizations using RNA probes labelled with DIG or P-32 but have heard from the previous person who tried it in our lab that they do not stipp off the membrane very easily. I have to have a method where I can stripp membranes nicely because I have to probe my membranes with quite a few probes. I really wouldn't like to run a million Northerns!! If anyone has any experience with this please help. Thanks Judi From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!howland.erols.net!news.maxwell.syr.edu!news.mindspring.net!gatech!news-relay.ncren.net!ussun2n!news@ussun2n.glaxo.com From: Gary Kucera Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA isolation from hair Date: Mon, 02 Feb 1998 08:56:17 -0500 Organization: Glaxo Wellcome, Inc. Lines: 61 Message-ID: <34D5D081.7F9B@glaxowellcome.com> References: <01bd2d9f$66ccf500$d966c180@kortej> Reply-To: gtk10583@glaxowellcome.com NNTP-Posting-Host: 152.51.151.69 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (WinNT; I) To: John Korte The following article is from Biotechniqes and should give you the protocol you need. Good luck, Gary --------------------------------------------------------------------- Article Title Chelex 100 As A Medium For Simple Extraction Of DNA For Article Abstract CHELEX 100 AS A MEDIUM FOR SIMPLE EXTRACTION OF DNA FOR PCR-BASED TYPING FROM FORENSIC MATERIAL. Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the polymerase chain reaction (PCR). The procedures are simple, rapid, involve no organic solvents, and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using Proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method The Chelex method has been used with amplification and typing at the HLA DQ a locus genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swab s, hair and post-coital samples. The results of a concordance study are presented, in which the DQa genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two differ ent extraction methods were identical for all samples tested. Article Authors Higuchi, Russell; Metzger, David A.; Walsh, Sean P. Citation Research Report, Biotechniques, Vol.10, No.4, p.506-513 Topic Keywords DNA; PCR; Polymerase Chain Reaction; John Korte wrote: > > Greetings, > I was wondering if someone could send me a protocol for isolating DNA from > hair follicles. I am searching for a protocol that doesn't require > anything special other than the average chemicals found in a lab. > thanks, John Korte -- ______________________________ Gary Kucera, Research Scientist Functional Genetics/Transgenics Group Glaxo Wellcome, Inc. gtk10583@glaxowellcome.com From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!SCIBORG.UWATERLOO.CA!m3allen From: m3allen@SCIBORG.UWATERLOO.CA (Michael Allen) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA isolation from hair Date: 2 Feb 1998 06:23:30 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 56 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802021422.JAA20124@sciborg.uwaterloo.ca> NNTP-Posting-Host: net.bio.net >Date: Sun, 01 Feb 1998 15:35:32 >To: "John Korte" >From: Michael Allen >Subject: Re: DNA isolation from hair >Cc: methods@net.bio.net > >At 04:52 PM 1/30/98 GMT, you wrote: >>Greetings, >>I was wondering if someone could send me a protocol for isolating DNA from >>hair follicles. I am searching for a protocol that doesn't require >>anything special other than the average chemicals found in a lab. >>thanks, John Korte >> > >Hi John >We used this in an undergrad course I was TA' ing, and it seemed to work fine > >Place 2-3 hairs with good follicles in 100 ul of extraction buffer. >Incubate at 55 C for 1hour, and then at 95 C for 10 minutes. >Use 7.5 ul per 25 ul PCR reaction (if that's what you're using it for) > >Extraction buffer: > >50 mM KCl 1.86g >10 mM Tris 0.61g >2.5 mM MgCl2 0.25g >0.1 mg/ml gelatin 0.05g >0.45% NP40 2.25 ml >0.45% Tween 20 2.25 ml > >pH to 8.3 at room temperature. > >Add ddH2O to 500 ml and autoclave (it will look white until it cools). > >Hope this helps > >Mike > Sorry, I forgot to mention that there is also 6ug of proteinase K in every reaction Mike Michael Allen Ph. D. student University of Waterloo Waterloo, Ontario, Canada "Revolution is just a t-shirt away." -Billy Bragg From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!feeder.qis.net!btnet-peer!btnet!server2.netnews.ja.net!server6.netnews.ja.net!singer.cent.gla.ac.uk!usenet From: "F. Vaillant" Newsgroups: bionet.molbio.methds-reagnts Subject: FISH problem Date: Mon, 02 Feb 1998 10:56:43 +0000 Organization: University of Glasgow Lines: 16 Message-ID: <34D5A66B.A8C@pop-server.cent.gla.ac.uk> NNTP-Posting-Host: ewb1v.garscube.gla.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (WinNT; I) Hi, I have started doing FISH experiment. I have a problem with the final morphology of the chromosomes. They appear phffy and enlarged. They lose any distinctive features sometimes fusing in one mass. I believe that the denaturation step is the problem. I have reduced the temperature but without any significant changes. Is there any thing that I should do to avoid that? Any technical help of referal to the appropriate newsgroup is welcome. Thanks F. Vaillant From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!howland.erols.net!news.maxwell.syr.edu!Supernews60!supernews.com!Supernews73!supernews.com!nntp.snfc21.pbi.net!news.pbi.net!news.pacbell.net!not-for-mail From: Susan Kang Newsgroups: bionet.molbio.methds-reagnts Subject: Free Power Supply Date: Mon, 02 Feb 1998 05:05:48 -0800 Organization: HIFAX Lines: 11 Message-ID: <34D5C4AC.51A2@hifax.com> Reply-To: order@hifax.com NNTP-Posting-Host: ppp-206-170-217-232.nhwd02.pacbell.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01C-PBWG (Win95; U) Dear Colleagues; As a new company, we decided to distribute our new PULSE POWER freely to promote the customer recognition of the product. If you have any interest please visit our web site www.hifax.com and send us e-mail. We will send PULSE POWER to every 100th e-mailer. Furthermore, We will give you a wonderful chance of spending vacation at Hawai. Susan Kang Sales Manager HIFAX.COM From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!sunqbc.risq.qc.ca!newsfeed.eerie.fr!jussieu.fr!univ-lille1.fr!ciril.fr!morchella.scbiol.u-nancy.fr!user From: jacob@scbiol.u-nancy.fr (Christophe Jacob) Newsgroups: bionet.molbio.methds-reagnts Subject: packaging protocol Date: Mon, 02 Feb 1998 13:57:55 +0100 Organization: University H Poincare of Nancy Lines: 5 Message-ID: NNTP-Posting-Host: morchella.scbiol.u-nancy.fr Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit Hello! I am using the stratagen packaging kit GIGApack. At the end of the protocol, it is recommended to add chloroform after adding SM medium. Does anybody know the aim and the mechanism of this step. Thanks. Christophe and Sylvain. From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!howland.erols.net!news.maxwell.syr.edu!news-was.dfn.de!news-fra1.dfn.de!news-koe1.dfn.de!news.rhrz.uni-bonn.de!news.uni-stuttgart.de!news.urz.uni-heidelberg.de!not-for-mail From: January Weiner Newsgroups: bionet.molbio.methds-reagnts Subject: Re: sequencing Date: 2 Feb 1998 10:46:47 GMT Organization: University of Heidelberg, Germany Lines: 21 Distribution: bionet Message-ID: <6b486n$fc9@sun0.urz.uni-heidelberg.de> References: <6ahu0a$h7u@mserv1.dl.ac.uk> Reply-To: nospam_jweiner1@ix.urz.uni-heidelberg.de NNTP-Posting-Host: ix.urz.uni-heidelberg.de X-Newsreader: TIN [UNIX 1.3 unoff BETA 970820; AIX 4.2] Xcanpos: shelf.01/199802161301!0026973850 [Neil]: > Occassionally I notice that when applying fluorescent sequencing > techniques to miniprep DNA, I may get a fade out of signal. Samples > prepared in the same manner using the same quantity of DNA in reaction > will have completely different signal strengths. We are reasonably sure > this is not a PCR problem, so does anyone have any suggestions? From my experience with ABI 373 and adequate kits (ThermoSeq) it depends mostly on the primer used for sequencing. It is crucial to choose an optimal primer, e.g. using the programm Oligo you have always to check the primer manually, for the program sometimes chooses weird and inadequate primer as optimal. If the Tm lies high above 50 deg, or it builds sec. structeres the signal might fade out. Sequencuary -- ----)-\//-///-----------------------------------January-Weiner-3------- Good morning doctors. I have taken the liberty of removing Windows 95 from my hard drive. From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!newsgate.duke.edu!news.vt.edu!solaris.cc.vt.edu!news.cc.ukans.edu!news.starnet.net!axb.slu.edu!news From: Darren Tyson Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Experience with Renilla Luciferase? Date: Mon, 02 Feb 1998 13:21:09 -0600 Organization: Saint Louis University (remove ".nospam" from address) Lines: 49 Message-ID: <34D61C9F.2E59F08E@slu.edu> References: Reply-To: tysondr.nospam@slu.edu NNTP-Posting-Host: 165.134.82.91 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; I) Fern E. Murdoch wrote: > Hi, > > I'm considering using Promega's Dual luciferase system with Renilla > luciferase as the transfection efficiency control. I've had problems in > my cells with high endogenous beta-gal, and what looks to be competition > with my experimental plasmid's promoter. The dual luciferase system looks > attractive, but I'd like comments/advice from real users before I switch. > > Many thanks, > > Fern > > -- > Fern E. Murdoch > Biochemistry > Uniformed Services University > Bethesda, MD > e-mail: fmurdoch@erols.com I've used it. It works very well in my hands. The pRL-TK vector is a really good vector for use as an internal control... Not too strong, but strong enough, and the thymidine kinase promoter does not appear to squelch other promoters. The main drawback is cost. It is MUCH more expensive than the firefly luciferase assay alone. If money is not a problem, I strongly recommend it. **Standard disclaimer about no affiliation to Promega here** Cheers, Darren -- Darren Tyson, Ph.D. Candidate Cell and Molecular Biology Program Saint Louis University, St. Louis, MO tysondr@nospam.slu.edu (remove nospam. before mailing) http://users.primary.net/~darren/ From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 From: Sam Michaelson Newsgroups: bionet.molbio.methds-reagnts Subject: Re: storage of bacterial plates Date: Mon, 02 Feb 1998 16:38:45 +1100 Organization: Defence Science & Technology Organisation Lines: 29 Message-ID: <34D55BE3.438955E9@dsto.defence.gov.au.ANTISPAM> References: <6am7hn$6ge$1@enyo.uwa.edu.au> NNTP-Posting-Host: mbr-44-pc173.dsto.defence.gov.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) Path: biosci!agate!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!139.130.235.93!news.telstra.net!sa.news.telstra.net!news.camtech.net.au!ns.saard.net!fang.dsto.defence.gov.au!foxhound.dsto.defence.gov.au!usenet Tulene McCabe wrote: > I am performing colony blots with E.coli and I have heard there is a > method of storing master plates by freezing. Unfortunately my > attempts to > actually find this method have been unsuccessful. If anyone knows of > such a method, or has advice about long-term storage of the colonies I > am > screening, I'd be so pleased! Hiya Tulene A post-grad in my dept. was doing something that also required the storage of colonies on plates. She sealed the plates up with lab-film, then stored them in the cold room at 4 oC. E. coli is, as I understand it, generally a pretty robust sort of bug, and her plates sat for more than six months at a time without suffering any ill effect. I remember the bug storage thing, because the cold room was in my lab and I was 'keeper of the cold room' - I was forever on her case because she had boxes and boxes of plates in there, and never labelled anything. You should've seen her face when, after many dire warnings, I moved them all to another cold room - and told her that I'd cleaned out everything that was not properly labelled with the owner's name..... >:-) Sam From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!hgmp.mrc.ac.uk!jgreen From: jgreen@hgmp.mrc.ac.uk (Dr. J. Green) Newsgroups: bionet.molbio.methds-reagnts Subject: Automated nucleic acid extraction Date: 2 Feb 1998 19:38:07 GMT Organization: MRC Human Genome Mapping Project Resource Centre Lines: 12 Message-ID: <6b57av$ois$1@niobium.hgmp.mrc.ac.uk> NNTP-Posting-Host: tin.hgmp.mrc.ac.uk Hi Can anyone advise me as the current options for automation of nucleic acid extraction from clinical samples? What's available? Does it work? Is it reliable? Any advice gratefully received. Thank you Jon Green From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.sprintlink.net!news-backup-west.sprintlink.net!news-in-west.sprintlink.net!news.sprintlink.net!Sprint!199.125.85.9!news.mv.net!newspump.wustl.edu!news.starnet.net!axb.slu.edu!news From: Darren Tyson Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Small protein Western Analysis Date: Mon, 02 Feb 1998 13:16:39 -0600 Organization: Saint Louis University (remove ".nospam" from address) Lines: 19 Message-ID: <34D61B97.EF8A839A@slu.edu> References: <6aag78$sh1$1@bignews.shef.ac.uk> <34CB4F5B.4F34@medmicro.aau.dk> Reply-To: tysondr.nospam@slu.edu NNTP-Posting-Host: 165.134.82.91 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win95; I) > A P Smith wrote: > > > > Hi All > > > > I'm trying to detect two small proteins of 2.5 and 4.5 kDa by western > > analysis but I'm having a little problem resolving such small proteins > > on 15% and 20% PAGE. Anyone have any suggestions? > Try Tris-Tricine gels. Check out the Bio-Rad catalog for more information. -- Darren Tyson, Ph.D. Candidate Cell and Molecular Biology Program Saint Louis University, St. Louis, MO tysondr@nospam.slu.edu (remove nospam. before mailing) http://users.primary.net/~darren/ From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!europa.clark.net!128.158.254.10!news.msfc.nasa.gov!info.usuhs.mil!NewsWatcher!user From: fmurdoch@usuhs.mil (Fern E. Murdoch) Newsgroups: bionet.molbio.methds-reagnts Subject: Experience with Renilla Luciferase? Date: Mon, 02 Feb 1998 10:15:52 -0500 Organization: Uniformed Services University Lines: 18 Message-ID: NNTP-Posting-Host: 131.158.3.178 Hi, I'm considering using Promega's Dual luciferase system with Renilla luciferase as the transfection efficiency control. I've had problems in my cells with high endogenous beta-gal, and what looks to be competition with my experimental plasmid's promoter. The dual luciferase system looks attractive, but I'd like comments/advice from real users before I switch. Many thanks, Fern -- Fern E. Murdoch Biochemistry Uniformed Services University Bethesda, MD e-mail: fmurdoch@erols.com From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.14!news-pen-14.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!newsfeed.eerie.fr!jussieu.fr!saphir.jouy.inra.fr!not-for-mail From: Michael O'Donohue Newsgroups: bionet.molbio.methds-reagnts Subject: Pichia pains Date: Mon, 02 Feb 1998 18:08:33 +0100 Organization: Institut National de Recherche Agronomique Lines: 9 Message-ID: <34D5FD91.78DA@lille.inra.fr> Reply-To: odonohue@lille.inra.fr NNTP-Posting-Host: 194.199.64.141 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold [fr] (Win95; I) Hi everyone! Does any have any experience with the larger Pichia vectors (eg pHILS1, pPIC9 etc.)? I am having a load of trouble trying to clone a fragment into the EcoR1 /BamH1 sites of pHILS1. Such a simple cloning job!!!! I have lots of vector, lots of fragment......but not even one miserable colony. Is transformation efficiency at the root of the problem? Any ideas? From owner-methds-reagnts@net.bio.net Sun Feb 01 22:00:00 1998 Path: biosci!IX.NETCOM.COM!BSGolf From: BSGolf@IX.NETCOM.COM ("Web Promotion Spider") Newsgroups: bionet.molbio.methds-reagnts Subject: Web Promo Power for WebMasters Date: 2 Feb 1998 09:31:58 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 55 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802021655.KAA15540@dfw-ix13.ix.netcom.com> NNTP-Posting-Host: net.bio.net -------------------------------------------------------------------------- This mailing is sent conforming to a strict ethical code. 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Visit http://www.bunkershot.com/spider.htm From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!howland.erols.net!newsfeed.direct.ca!Supernews60!supernews.com!Supernews73!supernews.com!news.tamu.edu!news.utdallas.edu!nrchh45.rich.nt.com!bcarh189.bnr.ca!nott!uottawa!aix1.uottawa.ca!s535290 From: Robot-Boy Newsgroups: bionet.molbio.methds-reagnts Subject: inoue competent cells help ! Date: Tue, 3 Feb 1998 00:37:13 -0500 Organization: University d'/of Ottawa Lines: 12 Message-ID: NNTP-Posting-Host: aix1.uottawa.ca Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII hey all, just some quick help. I'm going to try making competent cells the inoue way, and only had the recipe for making the cells, but not for the actual transformation. Do I just do the transformation the "regular" way ? ie.thaw on ice, add DNA, wait 20-30 minutes, heatshock, chill, add SOC and grow at 37 deg for 60 minutes, plate on selective media. thanks in advance, Ed From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news1.chicago.iagnet.net!qual.net!iagnet.net!btnet-peer!btnet!newsfeed.cableol.net!server4.netnews.ja.net!news.cc.ic.ac.uk!not-for-mail From: Koen De Smet Newsgroups: bionet.molbio.methds-reagnts Subject: Re: His-tagged protein, urea & antibody production Date: Tue, 03 Feb 1998 12:24:05 +0000 Organization: Department of Medical Microbiology Lines: 41 Message-ID: <34D70C65.3541@nospam.ic.ac.uk> References: <34D5883B.CCF@nospam.ic.ac.uk> Reply-To: k.desmet@nospam.ic.ac.uk NNTP-Posting-Host: cb-22.sm.med.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; PPC) Vladimir Svetlov wrote: > > In article <34D5883B.CCF@nospam.ic.ac.uk>, k.desmet@nospam.ic.ac.uk wrote: > > > Glenda Lawrence wrote: > > > > > > Hi. > > > > > > Does anyone have ideas or experience re- the best > > > way to remove urea before injecting recombinant > > > protein into rabbits for antibody production? > > > How much urea can be tolerated? > > > The protein is a His-tagged protein (insoluble) purified > > > and eluted in 8M urea, 20mM Tris, 150mM NaCl and 200mM imidazole. > > > > > > Thanks, > > > > > > Glenda Lawrence > > > > > > I don't know if this is the "best way", but we have succesfully produced > > antisera by dialysing the urea away. In most cases, the recombinant > > His-tagged protein preciptated, but that does not matter. YOu can still > > make a suspension with incomplete Freunds adjuvant > > Well, it does not have to precipitate. One of the simplest solutions is to > do a step-wise dialysis: 6M->4M->2M->0.7M->0M urea. If you don't use higher > than 0.5 mg/ml concentration most of the proteins are likely to stay > solubilized. I tried that, but I had a protein that even precipitated in the 5M urea. But that doesn't matter for antiserum production. Finding the right conditions to refold into soluble, enzymatically active protein can be a pain. What works for one protein, doesn't work for the next one you purify. Koen De Smet ============================================================== ==>> To reply by email, remove "nospam." from the address <<== Imperial College School of Medicine at St Mary's http://www.sm.ic.ac.uk/medmicro/home. ============================================================== From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!193.174.75.126!news-was.dfn.de!news-fra1.dfn.de!news-ge.switch.ch!news.bme.hu!not-for-mail From: "Csaba Jeney" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Electrotransfer of high MW proteins Date: 3 Feb 1998 12:35:26 GMT Organization: Technical University of Budapest Lines: 21 Message-ID: <01bd30a0$32185d00$8333e0c1@moonsilver.sote.hu> References: <34C3938F.7FCF@leonardo.ls.huji.ac.il> <34c875a5.1118883@news.xs4all.nl> NNTP-Posting-Host: moonsilver.sote.hu X-Newsreader: Microsoft Internet News 4.70.1155 Tom Vink wrote in article <34c875a5.1118883@news.xs4all.nl>... > On Mon, 19 Jan 1998 17:55:42 +0000, Mrta Grifman > wrote: > > >I am trying to obtain an efficient way to electrotransfer high MW > >proteins (100-200Kd) using a semi-dry blotter. I have tryied several > >procedures with no success. I would be grateful if someone could send me > >a good protocol > >Thanks a lot!! > We most times use the phastgel system of Pharmacia and use so called > diffusion blotting,. This seems to work really fine for large > proteins. Especially a protein called von willebrand factor with > multimers upto 20 million can be transferred. > We use the PhastSytem gels and tranfer them with semidry BioRad system. We have not encountered any problem to transfer large aprox. 200 kD proteins. Maybe the thickness of the gel is the point. From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!newsfeed.internetmci.com!199.117.161.1!csn!nntp-xfer-1.csn.net!news.acsu.buffalo.edu!srv1.drenet.dnd.ca!crc-news.doc.ca!nott!uottawa!aix1.uottawa.ca!s535290 From: Robot-Boy Newsgroups: bionet.molbio.methds-reagnts Subject: Inoue help - again... Date: Tue, 3 Feb 1998 09:29:32 -0500 Organization: University d'/of Ottawa Lines: 18 Message-ID: NNTP-Posting-Host: aix1.uottawa.ca Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII sorry to post again...had forgotten something else... just some quick help. I'm going to try making competent cells the inoue way, and only had the recipe for making the cells, but not for the actual transformation. Do I just do the transformation the "regular" way ? ie.thaw on ice, add DNA, wait 20-30 minutes, heatshock, chill, add SOC and grow at 37 deg for 60 minutes, plate on selective media. I also wanted to ask what to use to bring the pH of the transformation buffer up to 6.7. I tried KOH and that didn't go well at all...I got some sort of precipitate (I guess some Manganese salt, since it has a purplish color). Should I use diluted KOH as opposed to 1 N KOH ? thanks in advance, Ed From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!131.103.1.116!news2.chicago.iagnet.net!qual.net!iagnet.net!144.92.88.12!newsspool.doit.wisc.edu!wiscnews.wiscnet.net!news.doit.wisc.edu!svetlov From: svetlov@oncology.wisc.edu (Vladimir Svetlov) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: His-tagged protein, urea & antibody production Date: Tue, 03 Feb 1998 12:12:15 -0600 Organization: McArdle Lab for Cancer Research, UW-Madison Lines: 22 Message-ID: References: <34D5883B.CCF@nospam.ic.ac.uk> <34D70C65.3541@nospam.ic.ac.uk> NNTP-Posting-Host: alpha.oncology.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 In article <34D70C65.3541@nospam.ic.ac.uk>, k.desmet@nospam.ic.ac.uk wrote: > > Well, it does not have to precipitate. One of the simplest solutions is to > > do a step-wise dialysis: 6M->4M->2M->0.7M->0M urea. If you don't use higher > > than 0.5 mg/ml concentration most of the proteins are likely to stay > > solubilized. > > I tried that, but I had a protein that even precipitated in the 5M urea. If that's the case, refolding on the resin is a good option. Cheers, V. -- Vladimir Svetlov McArdle Lab for Cancer Research Dept. Oncology UW-Madison 1400 University Ave. Madison, WI 53706 From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!westmed.wh.usyd.edu.au!glendal From: glendal@westmed.wh.usyd.edu.au (Glenda Lawrence) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: His-tagged protein, urea & antibody production Date: 3 Feb 1998 20:40:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 42 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Thankyou to everyone who provided information to my query about urea and antibody production. Below are a couple more responses that others may find helpful. 1. >I have routinely injected histagged proteins resuspended in 8 M Urea into >rabbits subcutaneously. The rabbits are fine and the antibody generation >is good. I would'nt worry about it. 2. >There is essentially no problem in injecting urea. It is a normal body >component, and in fact has been used therapeutically in humans as a >plasma volume expander (massive quantities infused iv). We have done >some work in rabbits with protein in 4M urea, and saw no effects, even >though we caefully looked (even to the point if shaving the inoc site, >and biopsying for histology). > >If urea still worries you, then try using the protein as a suspension >or precipitate (maybe with an oil-based adjuvant). Glenda >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Glenda Lawrence PhD Centre for Virus Research Westmead Institutes of Health Research Westmead Hospital Westmead NSW 2145 Australia Phone: (61 2) 9845 8086 (Office) 9885 6363 (Lab) Fax: (61 2) 9845 8300 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!smtpgwy.agric.nsw.gov.au!deborah_hailstones_at_emai From: deborah_hailstones_at_emai@smtpgwy.agric.nsw.gov.au Newsgroups: bionet.molbio.methds-reagnts Subject: Re: packaging protocol Date: 3 Feb 1998 16:28:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <9802048865.AA886546882@smtpgwy.agric.nsw.gov.au> NNTP-Posting-Host: net.bio.net Christophe and Sylvain wrote: Hello! I am using the stratagen packaging kit GIGApack. At the end of the protocol, it is recommended to add chloroform after adding SM medium. Does anybody know the aim and the mechanism of this step. Presumably this "gigapack" is a phage packaging kit..? the chloroform inhibits the growth of the (uninfected) bacteria (in the lawn or infection mix) so that they cannot overgrow your plaques/infected cells... i must admit tho that i didnt always perform the chloroform addition when i was using phage (mostly lamda and derviatives) and never had significant problems... Deb From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!news-peer.sprintlink.net!news-backup-east.sprintlink.net!news-in-east.sprintlink.net!news.sprintlink.net!Sprint!128.228.4.60!news.cuny.edu!nntp.upenn.edu!not-for-mail From: "Prakash K. Rao" Newsgroups: bionet.molbio.methds-reagnts Subject: jurkat transfections Date: Tue, 03 Feb 1998 18:22:33 +0000 Organization: University of Pennsylvania Lines: 5 Message-ID: <34D76069.702B@mail.med.upenn.edu> NNTP-Posting-Host: kadeschlab.humgen.upenn.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; U; PPC) is there a preferred method for Transfections into Jurkats when one considers CaPo4,cationic lipids, electroporation,etc.,etc. I am primarily interested in transient transfecvtions for reporter gene assays. Thanks. From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!ais.net!uunet!in2.uu.net!news.blarg.net!not-for-mail From: weazel@animal.blarg.net (Beth Wapelhorst) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Pichia pains Date: 3 Feb 1998 15:12:11 -0800 Organization: Blarg! Online Services - 206/441-9109 Lines: 29 Message-ID: <6b888b$o7j$1@animal.blarg.net> References: <34D5FD91.78DA@lille.inra.fr> NNTP-Posting-Host: animal.blarg.net X-Newsreader: NN version 6.5.1 (NOV) If it's your transformation, that's easy to check by transforming the intact vector as a control. If that's okay, then somethin's wacked with your ligation reaction. You didn't say what your insert frag was -PCR product? -cut from another vector? Are you phosphatasing your vector? Can you religate cut vector alone? It's them simple cloning jobs that get ya, because you figure it'll just pop in the first time, no problemo, so you get lazy and don't do your controls...then when it doesn't work, you have no idea why. I do this all the time, too...you'd think I'd learn ;) beth DNA Hacker's Resource: www.blarg.net/~weazel/DHR/dhr.htm Michael O'Donohue writes: >Hi everyone! >Does any have any experience with the larger Pichia vectors (eg pHILS1, >pPIC9 etc.)? I am having a load of trouble trying to clone a fragment >into the EcoR1 /BamH1 sites of pHILS1. Such a simple cloning job!!!! I >have lots of vector, lots of fragment......but not even one miserable >colony. Is transformation efficiency at the root of the problem? >Any ideas? -- sugar beth wapelhorst "I don't think I'm alone when I say I'd weazel@blarg.net like to see more and more planets fall www.blarg.net/~weazel under the ruthless domination of our solar system." - Jack Handey From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!usenet.INS.CWRU.Edu!news.apk.net!uunet!in4.uu.net!news-nyc.telia.net!masternews.telia.net!fci-se!fci!news.maxwell.syr.edu!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!207.5.0.44!nntp.mainstreet.net!news.pbi.net!news.pacbell.net!not-for-mail From: Susan Kang Newsgroups: bionet.molbio.methds-reagnts Subject: Re: inoue competent cells help ! Date: Tue, 03 Feb 1998 13:05:07 -0800 Organization: HIFAX Lines: 16 Message-ID: <34D78683.6B66@hifax.com> References: Reply-To: Order@hifax.com NNTP-Posting-Host: ppp-206-170-218-128.nhwd02.pacbell.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01C-PBWG (Win95; U) Robot-Boy wrote: > > hey all, > > just some quick help. I'm going to try making competent cells the inoue > way, and only had the recipe for making the cells, but not for the actual > transformation. Do I just do the transformation the "regular" way ? > ie.thaw on ice, add DNA, wait 20-30 minutes, heatshock, chill, add SOC > and grow at 37 deg for 60 minutes, plate on selective media. > > thanks in advance, > > Ed Why don't you visit www.hifax.com site? You will get a charming protocol for making competent cell From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!MASPO3.MAS.YALE.EDU!LolisE From: LolisE@MASPO3.MAS.YALE.EDU ("Lolis, Elias") Newsgroups: bionet.molbio.methds-reagnts Subject: mutagenesis strategy Date: 3 Feb 1998 11:50:52 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <34D77520@msmail3.mas.yale.edu> NNTP-Posting-Host: net.bio.net I would like to randomly mutate all 7 transmembrane regions of G protein-coupled receptors and then select for mutated receptors with functional properties. I was wondering whether anyone knows of any method that could target different regions of a cDNA for simultaneous random mutagenesis. One strategy I thought of requires a mutagenesis method that selectively targets either single-stranded or double-stranded DNA. For example, if there is a method that selectively targets single-stranded DNA, I could produce a single-stranded version of the cDNA, protect the non-transmembrane regions by synthesizing complementary DNA for these sequences, and applying the mutagenesis procedure to the remaining single-stranded DNA. If anyone knows of a method or article that answers this question please send me e-mail. Thanks. Elias Lolis From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!jsgpb1.path.cam.ac.uk!user From: jsg@mole.bio.cam.ac.uk (James) Newsgroups: bionet.molbio.methds-reagnts Subject: Liposomes Date: 3 Feb 1998 19:35:30 GMT Organization: Cambridge University Lines: 8 Message-ID: NNTP-Posting-Host: jsgpb1.path.cam.ac.uk Hello Do you know where I can buy liposome preparations to deliver DNA and protein in vivo? Most of the protocols I've come across in the literature involve vacuum dessication, and we don't have the apparatus. Thanks, James From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!newsgate.duke.edu!news.vt.edu!solaris.cc.vt.edu!newsrelay.netins.net!nntp.kreonet.re.kr!news.maxwell.syr.edu!newsfeed.eerie.fr!jussieu.fr!unilim.fr!cict.fr!not-for-mail From: Alexandra Manevski Newsgroups: bionet.molbio.methds-reagnts Subject: removing pigments Date: Tue, 03 Feb 1998 19:54:50 +0100 Organization: INRA Lines: 20 Message-ID: <34D767FA.22B0@toulouse.inra.fr> NNTP-Posting-Host: autan.toulouse.inra.fr Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.04 (X11; I; SunOS 5.5 sun4d) Hi, Could anyone tell me how to remove pigment from a protein extract of Arabidopsis thaliana cell suspension culture. Thanks Alex ____________________________________________________________________ Alexandra MANEVSKI Tel : 33 05 61 28 50 49 LBMRPM Fax : 33 05 61 28 50 61 UMR 215 CNRS-INRA Chemin de Borde-Rouge E-mail : manevski@toulouse.inra.fr BP 27 31326 Castanet-Tolosan France ____________________________________________________________________ From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!CHIMERX.COM!davidm From: davidm@CHIMERX.COM (David Mead) Newsgroups: bionet.molbio.methds-reagnts Subject: re: Commercial source for SV40 large T-ag? Date: 3 Feb 1998 09:13:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <32F4E5FA.24CE@chimerx.com> NNTP-Posting-Host: net.bio.net Ren=E9 Assenberg wrote: Hello, I'm currently setting up a project that will involve the usage of the SV40 large T-antigen but so far I haven't been able to locate a (preferably) commercial supplier. Could anyone help me with this? I'd appreciate it very much. Ren=E9 Assenberg University of Southampton, UK. Chimerx, Madison, Wisconsin (800-626-7833) supplies SV40 large T antigen. From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!192.41.171.5!newsserver.jvnc.net!ganglia.bms.com!NewsWatcher!user From: goldbers@bms.com (Steven Goldberg) Newsgroups: bionet.molbio.methds-reagnts Subject: Trying to find company/university to develop enzyme assays Date: Tue, 03 Feb 1998 13:08:47 -0500 Organization: Bristol-Myers Squibb Co. Lines: 12 Message-ID: NNTP-Posting-Host: 140.176.74.203 We do a lot of work with cloning and expression of bacterial enzymes (mainly in E. coli) and are mostly dependent upon HPLC or spectrophotometic assays to measure our activity. We would like to find a company or university willing to develop colorimetric and/or fluorimetric plate or whole cell assays as a means of rapidly screening libraries or overproducers of our enzyme. There is money available but I haven't had much luck identifying anyone who does this type of work. If you are interested or know of a place that could help me out, please let me know. Thanks. Steve Goldberg From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!usenet From: Nathan Siemers Newsgroups: bionet.molbio.methds-reagnts Subject: "Mortal" RNAse Needed Date: 03 Feb 1998 12:15:48 -0500 Organization: Bristol-Myers Squibb Lines: 23 Message-ID: NNTP-Posting-Host: rna.wi.mit.edu X-Newsreader: Gnus v5.3/Emacs 19.34 I am looking for an RNAse with the following properties: 1. Efficiently cleaves RNA in a RNA/DNA duplex environment. 2. Can be destroyed or potently inhibited in a simple manner after the digestion is complete. Thanks for any ideas, nathan -- Nathan Siemers, Ph.D. Research Investigator, Applied Genomics Bristol-Myers Squibb Pharmaceutical Research Institute Visiting Scientist Whitehead/MIT Center for Genome Research 617-252-1382 siemers@bms.com From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!nntp.news.xara.net!xara.net!server5.netnews.ja.net!leeds.ac.uk!news From: BGYCSW@leeds.ac.uk (C.S. Wilding) Subject: Hoefer TKO100 fluorometer Message-ID: <6b7k3q$8e0_001@leeds.ac.uk> NNTP-Posting-Host: bgy-pc042.leeds.ac.uk Organization: University of Leeds Date: Tue, 3 Feb 1998 17:28:29 +0000 (GMT) X-Newsreader: News Xpress Version 1.0 Beta #4 Lines: 12 We have a Hoefer TKO100 flurometer which is beginning to play up. A while ago the bulb started flashing on and off and conseqeuntly the machine never warmed up. After a while it appeared to behave itself but since the incident it now cannot be set to 1000 for a 1mg/ml standard- at full rotation of the scale the reading is only up to 500 or so. Prior to the bulb flashing incident it seemed fine so I presume the bulb is/has died (although I can see the light through the back). Pharmacia Biotech who have taken over Hoefer say they no longer hold any spares and cannot service the machine. Anybody any ideas of how to resurrect this machine, or where an aternative bulb may be bought? Craig. From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!howland.erols.net!news.maxwell.syr.edu!fu-berlin.de!uniol!news.uni-stuttgart.de!rz.uni-karlsruhe.de!news.urz.uni-heidelberg.de!usenet From: un691cs@genius.embnet.dkfz-heidelberg.de (Clemens Suter-Crazzolara) Newsgroups: bionet.molbio.methds-reagnts Subject: detection of protein Date: 3 Feb 1998 16:21:19 GMT Organization: University of Heidelberg, Germany Lines: 18 Message-ID: <6b7g5v$s68@sun0.urz.uni-heidelberg.de> NNTP-Posting-Host: 129.206.158.67 Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.8 (16bit) Xcanpos: shelf.01/199802172101!0010268466 Hi all, I want to detect a protein, which has a number of potential motifs potentially useful for detection ! These include sulfation, asn_glycosylation, myristylation and amidation. Is it easy to detect these modification sites and if yes, how do I do this ? Sulfation I have figured out: just add 32S (as H2SO3) to the culture and it (apparently) works. People here have some experience with that already. An additional problem is that I would like to be able to detect by pulse chase the protein amongst total cellular proteins, so f.i. what percentage of proteins is sulfated ? could you reply by e-mail directly as well ? Thanks, clemens suter-crazzolara, anatomy, heidelberg From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!blackbush.xlink.net!rz.uni-karlsruhe.de!news.urz.uni-heidelberg.de!usenet From: un691cs@genius.embnet.dkfz-heidelberg.de Newsgroups: bionet.molbio.methds-reagnts Subject: HIS tagged protein from baculo Date: 3 Feb 1998 16:43:23 GMT Organization: University of Heidelberg, Germany Lines: 24 Message-ID: <6b7hfb$s68@sun0.urz.uni-heidelberg.de> NNTP-Posting-Host: 129.206.158.67 Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.8 (16bit) Hi, we have cloned a HIS-tagged protein in baculovirus. It is a novel TGF-beta member, and dimeric. We get some expression; the protein is indeed dimeric and looks o.k. in a western. We can purify it with the his-tag. However, we need at least 100 micrograms for our initial assays, and this doesn't seem to work. question 1: what methods can we use to determine the amount of protein. It is very dilute. We have tried Bradford, but aren't in the linear range. Any pointer welcome here. I just want to know more or less exactly how much protein we have. question 2: how do I optimize my production of the protein ? what parameters to watch ? How much protein is normal, how much can YOU make. any tricks not found in the manuals ? Are certain proteins toxic to baculo ? Insect cells also produce TGF-betas. thanks, Clemens Suter-Crazzolara, Heidelberg From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!europa.clark.net!4.1.16.34!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!baron.netcom.net.uk!netcom.net.uk!server3.netnews.ja.net!is.bbsrc.ac.uk!news From: David Hills Newsgroups: bionet.molbio.methds-reagnts Subject: pYAC55 (Not I) availability Date: 3 Feb 1998 16:33:39 GMT Organization: Roslin Institute (Edinburgh) Lines: 13 Message-ID: <6b7gt3$g1j@is.bbsrc.ac.uk> NNTP-Posting-Host: pc0616.ri.bbsrc.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) Yo ! Anyone out there can send me some pYAC55....SIGMA no longer sell it in the UK. Will part exchange for aliquot of pYAC4...... Thanks Dave Dr D Hills Roslin institute Roslin Midlothian EH25 9PS U.K. From owner-methds-reagnts@net.bio.net Mon Feb 02 22:00:00 1998 Path: biosci!agate!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!fu-berlin.de!uniol!news.uni-stuttgart.de!news.urz.uni-heidelberg.de!usenet From: un691cs@genius.embnet.dkfz-heidelberg.de Newsgroups: bionet.molbio.methds-reagnts Subject: Re: ECACC Date: 3 Feb 1998 16:30:13 GMT Organization: University of Heidelberg, Germany Lines: 29 Message-ID: <6b7gml$s68@sun0.urz.uni-heidelberg.de> References: <69hsm7$17b@sun0.urz.uni-heidelberg.de> <01bd2277$2f2071f0$df236b92@pc2090> <34CE8C51.3163@rrk-berlin.de> <34CF2FAE.41C6@ggr.co.uk> <34CF3305.167E@ggr.co.uk> NNTP-Posting-Host: 129.206.158.67 Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.8 (16bit) Xcanpos: shelf.01/199802172101!0010423406 >> > > Michael (rosemann@gsf.de) >> > Sorry, but those sites (they are apparently one and the same) do not >> > work. Any time I have tried to search the catalog it only returns a "500 >> > Server Error". Mailing to the supposed server administrator has not >> > changed anything within the last three months. >> > I had asked ECACC whether they have a catalog on the net but they only >> > pointed me to a vesion that I could download to clutter up my hard disk >> > And no regular update. >> > So my opinion is: No, there is no ECACC catalog on the WWW. >> > Axel >> Err - I think the problem is your end - I've just used these links to >> access the ECACC catalogue without any problem at all ! >Oops ! I apologise,having delved further you're right it doesn't work ! >Sorry - Neil I was the original poster of this question, and I had also reached this level. Initially I thought I was stupid, but I am glad to learn it was the ECACC after all. Where can I complain to ECACC about this situation, now that their web page isn't working ? clemens From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!192.41.171.5!newsserver.jvnc.net!newsserver2.jvnc.net!news3.cac.psu.edu!usenet From: jdc6@psu.edu Newsgroups: bionet.molbio.methds-reagnts Subject: PFGE/Southers Date: 4 Feb 1998 17:23:25 GMT Organization: Penn State University Lines: 25 Sender: jdc6@0.0.0.0 Message-ID: <6ba86d$r92@r02n01.cac.psu.edu> NNTP-Posting-Host: thirtysixred.bmb.psu.edu X-Authinfo-User: jdc6@psu.edu X-Posted-From: InterNews 1.1@thirtysixred.bmb.psu.edu X-Authenticated: jdc6 on INN host 0.0.0.0 Dear Colleagues, I am currently trying to map, via PFGE, the region around a given gene. We have sequenced several thousand bp flanking this gene and it truns out that these sequences are highly repetitive. I have performed PFGE using a variety of restriction enzymes, followed by Southern blotting using this gene, as well as two linked genes, as a probe. In my Southerns, I am only getting hybridization to regions that apparently represent repetitive regions in the genome (bands between 48-145 kb), as well as to the compression zone. In standard gel electrophoresis, my probes work quite well. A very specific band with little or no background hybridization. I suspect (but I am not convinced) that the hybridization to the repetive regions is non-specific. Is it possible that, using a variety of enzymes, my probes consistently lights up repetitive regions, or is this just non-specific hybridization. If so, does anyone have any advice as to how I could improve the southern blotting. Thanks in advance, Joe Verica Penn State jdc6@psu.edu From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!howland.erols.net!news-peer.gip.net!news-penn.gip.net!news.gsl.net!gip.net!news.belnet.be!not-for-mail From: "Z.Huang" Newsgroups: bionet.molbio.methds-reagnts Subject: histidine aromatic? Date: Wed, 04 Feb 1998 11:26:33 -0800 Organization: Gut Hormone Lab, K.U.Leuven Lines: 7 Message-ID: <34D8C0E9.743D@med.kuleuven.ac.be> NNTP-Posting-Host: marvin.kulnet.kuleuven.ac.be Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: dalet.belnet.be 886587894 19828 (None) 134.58.127.3 X-Complaints-To: usenet@news.belnet.be X-Mailer: Mozilla 2.01 (Win16; I) Cache-Post-Path: marvin.kulnet.kuleuven.ac.be!unknown@gihvtr1.med.kuleuven.ac.be hello Can anybody tell me if histidine is an aromatic amino acid or not? thanks Peggy De Clercq From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!blackbush.xlink.net!rz.uni-karlsruhe.de!news.urz.uni-heidelberg.de!usenet From: un691cs@genius.embnet.dkfz-heidelberg.de (Clemens Suter-Crazzolara) Newsgroups: bionet.molbio.methds-reagnts Subject: stable transfections Date: 4 Feb 1998 08:01:00 GMT Organization: University of Heidelberg, Germany Lines: 12 Message-ID: <6b977s$9go@sun0.urz.uni-heidelberg.de> NNTP-Posting-Host: 129.206.158.67 Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.8 (16bit) Hello, I would like to make stable transfected (HEK or HNK) 293 cells. How much gentamicin will I have to use. How much gentamicin do you generally use for obtaining stable transfected cells ? Couls you reply directly as well by e-mail ? Thanks, Clemens, Anatomy heidelberg From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!newsgate.duke.edu!usenet From: "Michael J. Morales" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: "Mortal" RNAse Needed Date: Wed, 04 Feb 1998 11:58:46 -0500 Organization: Duke University, Durham, NC, USA Lines: 31 Message-ID: <34D89E46.736E8847@acpub.duke.edu> References: NNTP-Posting-Host: async249-113.async.duke.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) X-Corel-MessageType: EMail Nathan Siemers wrote: > > I am looking for an RNAse with the following properties: > > 1. Efficiently cleaves RNA in a RNA/DNA duplex environment. > > 2. Can be destroyed or potently inhibited in a simple manner after > the digestion is complete. > > Thanks for any ideas, Nathan, check out micrococcal nuclease. In think BM sells it. It is calcium dependendent, so it can be inhibited with EGTA. I'm not sure about duplex cleavage. Hope this helps, Mike Morales > > nathan > > -- > Nathan Siemers, Ph.D. > Research Investigator, Applied Genomics > Bristol-Myers Squibb Pharmaceutical Research Institute > Visiting Scientist > Whitehead/MIT Center for Genome Research > 617-252-1382 > siemers@bms.com From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!newsgate.duke.edu!usenet From: "Michael J. Morales" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Inoue help - again... Date: Wed, 04 Feb 1998 11:56:22 -0500 Organization: Duke University, Durham, NC, USA Lines: 36 Message-ID: <34D89DB6.9DCEE1F8@acpub.duke.edu> References: NNTP-Posting-Host: async249-113.async.duke.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) X-Corel-MessageType: EMail Robot-Boy wrote: > > sorry to post again...had forgotten something else... > > just some quick help. I'm going to try making competent cells the > inoue > way, and only had the recipe for making the cells, but not for the > actual > transformation. Do I just do the transformation the "regular" way ? > ie.thaw on ice, add DNA, wait 20-30 minutes, heatshock, chill, add SOC > and grow at 37 deg for 60 minutes, plate on selective media. > I have found that you can do almost anything you want. For routine subcloning (>10^8 colonies/ug), I add some of my ligation mix (PEG is no problem) to 50 ul cells in a 1.6 ml microfuge tube. 20 minutes on ice, 30 sec @ 42 degrees, 2 min on ice, then add 450 ul SOC & let recover at 37 degree for 30 minutes. This is with DH5 alpha. Hope this helps Mike Morales > I also wanted to ask what to use to bring the pH of the transformation > buffer up to 6.7. I tried KOH and that didn't go well at all...I got > some > sort of precipitate (I guess some Manganese salt, since it has a > purplish > color). Should I use diluted KOH as opposed to 1 N KOH ? > > thanks in advance, > > Ed > From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!204.59.152.222!news-peer.gip.net!news-dc.gip.net!news.gsl.net!gip.net!news.belnet.be!news.fundp.ac.be!biocell-viene.biocell.fundp.ac.be!user From: iernest@biocell.fundp.ac.be (Ernest Isabelle) Newsgroups: bionet.molbio.methds-reagnts Subject: tatgene/HIV-1 LTRreportergene Date: 4 Feb 1998 16:16:44 GMT Organization: FUNDP-Cellular biochemistry Lines: 16 Message-ID: NNTP-Posting-Host: biocell-viene.biocell.fundp.ac.be Hi, I should need some plasmid containing tat gene in order to express this protein in E. Coli and a reporter gene under the control of HIV-1 LTR in order to transfect Jurkat cells.Does anyone know where I could obtain them? Thank you Maggi Burton. mburton@biocell.fundp.ac.be Laboratory of cellular Biology, University of Namur (FUNDP) 61, rue de bruxelles, B-5000 Namur, Belgium From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!204.59.152.222!news-peer.gip.net!news-dc.gip.net!news.gsl.net!gip.net!news.belnet.be!news.fundp.ac.be!biocell-viene.biocell.fundp.ac.be!user From: iernest@biocell.fundp.ac.be (Ernest Isabelle) Newsgroups: bionet.molbio.methds-reagnts Subject: Tatgene/HIV-1LTR reporter Date: 4 Feb 1998 16:09:45 GMT Organization: FUNDP-Cellular biochemistry Lines: 14 Message-ID: NNTP-Posting-Host: biocell-viene.biocell.fundp.ac.be Hi I should need some plasmid containing tat gene in order to express this protein in E. Coli and a reporter gene under the control of HIV-1 LTR to tranfect Jurkat cells. Does anyone know where I could obtain this? Thank you by advance. Maggi Burton. Laboratory of Cellular Biology, University of Namur (FUNDP) 61, rue de bruxelles, B-5000 Namur, Belgium From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 From: "qin ling" Subject: PCR with degenerate primer Newsgroups: bionet.molbio.methds-reagnts Message-ID: <01bd3188$220e07a0$73a4e089@?.wau.nl> X-Newsreader: Microsoft Internet News 4.70.1155 NNTP-Posting-Host: flex115.dbh2.wau.nl Date: 4 Feb 98 16:09:09 GMT Lines: 10 Path: biosci!agate!newsgate.duke.edu!news.vt.edu!solaris.cc.vt.edu!fci-se!fci!news-out.internetmci.com!newsfeed.internetmci.com!192.87.106.104!surfnet.nl!wau.nl!flex115.dbh2.wau.nl Hi: I am trying to fish out the cDNA of which 20aa N-terminal seq is known. I have linkered cDNA as template. So in my PCR, one primer anneals to the linker, the other one is a set of nested degenerate primers designed based on aa seq. But I couldn't get any specific bands out of my PCR. Anybody has done similar experiments with success? Please give me some briliant suggestions. -- qin ling From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!AOL.COM!DOCTORHIM From: DOCTORHIM@AOL.COM Newsgroups: bionet.molbio.methds-reagnts Subject: Re: EDTA/Heparin & DNA stability Date: 4 Feb 1998 07:59:19 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <5c847ca.34d88ffe@aol.com> NNTP-Posting-Host: net.bio.net There is some literature on using heparinase to enhance PCR on blood samples. Check the entrez browser and search for PCR and heparinase. If I find the reference, I will send it. From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!uni-erlangen.de!newsserv.uni-bayreuth.de!not-for-mail From: Georg.Lipps@uni-bayreuth.de (Georg Lipps) Newsgroups: bionet.molbio.methds-reagnts Subject: removal of hexahistidine tag by peptidase Date: Wed, 04 Feb 1998 15:24:12 GMT Organization: Universität Bayreuth Lines: 10 Message-ID: <34d88733.1031107@newsserv.uni-bayreuth.de> NNTP-Posting-Host: btcbd4.che.uni-bayreuth.de X-Newsreader: Forte Free Agent 1.11/16.235 Hi everybody, does anyone has been successful in removing a C-terminal of N-terminal hexahistidine tag by limited exoproteolysis ? I have tried Aminopeptidase M and Carboxypeptidase A with my protein but have not been successful. Any hints are greatly appreciated ! Thanks in advance, Georg From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!server1.netnews.ja.net!ananke.salford.ac.uk!not-for-mail From: NoSpam@news.nospam.ac.uk (John) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: M -- please check my calculation Date: Wed, 04 Feb 98 20:27:33 GMT Organization: NoSpam Lines: 3 Message-ID: <6ba1db$mju$3@ananke.salford.ac.uk> References: <34c0d45f.133162109@nntp.ix.netcom.com> NNTP-Posting-Host: cockcroft-061.salford.ac.uk X-Newsreader: News Xpress Version 1.0 Beta #3 Looks Ok John From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!newsfeed.kornet.nm.kr!newsfeed.dacom.co.kr!newsfeed.direct.ca!torn!resunix.sickkids.on.ca!not-for-mail From: Randall C Willis Newsgroups: bionet.molbio.methds-reagnts Subject: Re: histidine aromatic? Date: Wed, 04 Feb 1998 09:12:43 -0500 Organization: The Hospital For Sick Children Lines: 34 Message-ID: <34D8775B.41C6@gandalf.psf.sickkids.on.ca> References: <34D8C0E9.743D@med.kuleuven.ac.be> NNTP-Posting-Host: gollum.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; IRIX 5.3 IP12) To: "Z.Huang" Z.Huang wrote: > Can anybody tell me if histidine is an aromatic amino acid or not? > > thanks > > Peggy De Clercq Peggy, Yes, but I believe that it depends upon the ionization state of the ring. Sorry that I can't be more helpful. Randy Randall C Willis, Researcher Biochemistry Research, Hospital for Sick Children 3522-555 University Ave Toronto, ON M5G 1X8 CANADA 416-813-5933 (ph) -5022 (fax) willis@gandalf.psf.sickkids.on.ca Randall C Willis, Publisher Aliquotes Press Aliquotes: A Journal Of Molecular and Biochemical Humour and Information 58 Balfour Ave. Toronto, ON M4C 1T6 CANADA 416-691-2921 (ph) 416-813-5022 (fax) roger@xybercom.net From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!LEONARDO.LS.HUJI.AC.IL!yoramg2 From: yoramg2@LEONARDO.LS.HUJI.AC.IL (Yoram Gerchman) Newsgroups: bionet.molbio.methds-reagnts Subject: Sequensing directly on the genom Date: 4 Feb 1998 06:39:59 -0800 Organization: Hebrew University Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <34D899AC.4D1C@leonardo.ls.huji.ac.il> Reply-To: yoramg2@leonardo.ls.huji.ac.il NNTP-Posting-Host: net.bio.net Greetings netters. Dose anyone know of a option of sequensing directly on genomic DNA (procariots), meaning witout prepering a librery first? From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!howland.erols.net!rill.news.pipex.net!pipex!join.news.pipex.net!pipex!ggr.co.uk!not-for-mail From: Fraser Dr N J Newsgroups: bionet.molbio.methds-reagnts Subject: Re: GFP Vector Date: Wed, 04 Feb 1998 13:37:28 +0000 Organization: Glaxo Wellcome Lines: 16 Message-ID: <34D86F18.41C6@ggr.co.uk> References: NNTP-Posting-Host: ukwbf80.ggr.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: ukwsv3.ggr.co.uk 886599272 10496 (None) 147.184.221.124 X-Complaints-To: usenet@ggr.co.uk X-Mailer: Mozilla 3.01 (X11; I; IRIX 5.3 IP22) To: a.doherty@bris.ac.uk Dr Andrew Doherty wrote: > > Hi every one out there > > Does anyone know of a mammalian expression vector which has GFP expressed > independently, like a selection marker? I would like to have a vector for > transient expression studies where I know which cells are tansfected. > > Ta in advance > > A.Doherty > > a.doherty@bris.ac.uk pTRACER vectors (Invitrogen) do this. Neil. From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 From: zjons@vetbio.unizh.ch (Zophonias O. Jonsson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: inoue competent cells help ! Message-ID: References: <34D78683.6B66@hifax.com> Organization: Universitat Zurich-Irchel NNTP-Posting-Host: 130.60.120.18 Date: 4 Feb 98 12:53:24 GMT Lines: 37 Path: biosci!agate!howland.erols.net!netnews.com!news.idt.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!feed1.news.luth.se!luth.se!news-ge.switch.ch!news-zh.switch.ch!rzunews1.unizh.ch!NewsWatcher!user In article <34D78683.6B66@hifax.com>, Order@hifax.com wrote: > Robot-Boy wrote: > > > > hey all, > > > > just some quick help. I'm going to try making competent cells the inoue > > way, and only had the recipe for making the cells, but not for the actual > > transformation. Do I just do the transformation the "regular" way ? > > ie.thaw on ice, add DNA, wait 20-30 minutes, heatshock, chill, add SOC > > and grow at 37 deg for 60 minutes, plate on selective media. > > > > thanks in advance, > > > > Ed > > Why don't you visit www.hifax.com site? You will get a charming protocol > for making competent cell A simpler answer would be ..... YES ! (If you want more deatil, 10 min on ice is enough and you can increase the number of transformants a little bit, by adding beta-mercapto-EtOH or DTT to 5-10 mM as you add the DNA) Zophonias _____________________________________________________________________ Zophonias O. Jonsson Institut fur Veterinarbiochemie Tel: (41-1)-635-54-75 Universitat Zurich-Irchel Fax: (41-1)-635-68-16 Winterthurerstrasse 190 CH-8057 Zurich Switzerland _____________________________________________________________________ From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!EU.net!Ireland.EU.net!web3.tcd.ie!gen093.gen.tcd.ie!user From: dmachugh@mail.tcd.ie (David MacHugh) Newsgroups: bionet.molbio.methds-reagnts Subject: EDTA/Heparin & DNA stability Date: Wed, 04 Feb 1998 12:09:27 +0000 Organization: Genetics Department, Trinity College, Dublin 2. Lines: 37 Message-ID: NNTP-Posting-Host: gen093.gen.tcd.ie Hi, We are currently looking at mtDNA variation in cheetahs sampled from various zoos around Europe and Africa. We have about 50 samples. All of these samples come from vacutainers which have been stored in a fridge since 1994 and in some cases since 1993. 18 of the 50 blood samples have been stored in vacutainers which have 15% K3EDTA as a preservative and the rest have lithium heparin. Because the samples were so old we did essentially an ancient DNA-type extraction. We used a whole-blood digestion buffer, A three-step phenol, phenol/CIA, CIA organic extraction followed by a purification using Boerhinger-Mannheim High-Pure columns normally used for cleaning up PCR products. Anyway we found that the 18 samples stored at 4C in EDTA gave DNA yields approx 100-fold more than those samples stored in lithium-heparin. This was reflected in the PCR amplifications also. The EDTA-stored samples gave beautiful mtDNA PCR products, while the lithium-heparin gave weak bands and in some cases failed to amplify at all. Has anyone else found EDTA to be a much better long-term storage medium for blood in this way? Also does anyone have any suggestions as to how the lithium-heparin samples may be retrieved more efficiently? Thanks in advance. David. David MacHugh Ph.D., Genetics Department, Trinity College, Dublin 2. Ireland. Phone: 353-1-608-2719 Fax: 353-1-679-8558 email: dmachugh@tcd.ie From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.nacamar.de!newscore.univie.ac.at!03-newsfeed.univie.ac.at!news.univie.ac.at!not-for-mail From: a8803349.nospam@unet.univie.ac.at (Martin Offterdinger) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: histidine aromatic? Date: Wed, 04 Feb 1998 12:29:28 GMT Organization: AKH Lines: 18 Message-ID: <34d85ef1.12993222@news.univie.ac.at> References: <34D8C0E9.743D@med.kuleuven.ac.be> NNTP-Posting-Host: grunt.imed1.akh-wien.ac.at X-Newsreader: Forte Free Agent 1.1/16.230 On Wed, 04 Feb 1998 11:26:33 -0800, "Z.Huang" wrote: >hello > >Can anybody tell me if histidine is an aromatic amino acid or not? > >thanks > >Peggy De Clercq YES, it is, because there are 6 delocalized electrons inside the ring Martin Offterdinger Internal Med.I,Dept. Oncology University of Vienna Austria E-Mail:a8803349.nospam@unet.univie.ac.at (remove nospam before mailing) From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!206.172.150.11!news1.bellglobal.com!news20.bellglobal.com!not-for-mail From: Joanne & Christopher Newsgroups: bionet.molbio.methds-reagnts Subject: Isolation advice Date: Wed, 04 Feb 1998 06:19:58 -0500 Organization: Bell Solutions Lines: 13 Message-ID: <34D84EDD.23D6124F@sympatico.ca> Reply-To: joanne.christopher@sympatico.ca NNTP-Posting-Host: 206.172.198.206 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: news21.bellglobal.com 886591284 15501 (None) 206.172.198.206 X-Complaints-To: usenet@news20.bellglobal.com X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) Hi, I'm planning on collecting some white blood cells so the DNA/RNA can be harvested later. The plan is to collect a blood in an EDTA tube, spin in the refrigerated centrifuge for 20 min, and then remove the buffycoat. This buffycoat would be frozen in a microcentrifuge tube, at -70C. I'm wondering if this will work OK, or if I should put a buffer on this prior to freezing? If I do require a buffer, which one, and could you please suggest a method? I am new to this type of work, and would appreciate any advice. Thanks, Joanne From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!logbridge.uoregon.edu!dispose.news.demon.net!demon!nntp.news.xara.net!xara.net!server5.netnews.ja.net!bris.ac.uk!usenet From: "Dr Andrew Doherty" Subject: GFP Vector X-Nntp-Posting-Host: anaajd.ana.bris.ac.uk Message-ID: X-Mimeole: Produced By Microsoft MimeOLE V4.71.1712.3 Sender: usenet@fsa.bris.ac.uk (Usenet) Organization: University of Bristol, England X-Newsreader: Microsoft Outlook Express 4.71.1712.3 Date: Wed, 4 Feb 1998 09:58:20 GMT Lines: 13 Hi every one out there Does anyone know of a mammalian expression vector which has GFP expressed independently, like a selection marker? I would like to have a vector for transient expression studies where I know which cells are tansfected. Ta in advance A.Doherty a.doherty@bris.ac.uk From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 From: aeb25@rrz.Uni-Koeln.DE (Kerstin Hoef-Emden) Newsgroups: bionet.molbio.methds-reagnts Subject: literature for rna secondary structure? Date: 4 Feb 1998 10:41:41 GMT Organization: Regional Computing Center, University of Cologne Lines: 32 Message-ID: <6b9gl5$d9h@news.rrz.Uni-Koeln.DE> NNTP-Posting-Host: axp1.rrz.uni-koeln.de X-Newsreader: TIN [version 1.2 PL2] Path: biosci!agate!logbridge.uoregon.edu!europa.clark.net!128.230.129.106!news.maxwell.syr.edu!fu-berlin.de!news.CS.Uni-Magdeburg.De!RRZ.Uni-Koeln.DE!aeb25 Hi everybody! I am looking for literature explaining the theoretical background of rna folding. We have got access to the GCG package here and my first attempts with mfold were successful. But I would like to get some idea what things should be considered to choose the correct settings and to avoid mistakes. Are there any books or publications about the method around which you recommend? Best wishes to all readers, Kerstin Hoef-Emden -- ################################################################## # # # Kerstin Hoef-Emden Universitaet zu Koeln # # Botanisches Institut # # Lehrstuhl I # # # # Kerstin.Hoef-Emden@uni-koeln.de Gyrhofstr. 15 # # Tel.: +49-221-470-4504 D-50931 Koeln # # Fax.: +49-221-470-5181 (Germany) # # # ################################################################## From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!daresbury!uninett.no!Cabal.CESspool!bofh.vszbr.cz!news.eecs.umich.edu!newsxfer3.itd.umich.edu!jobone!lynx.unm.edu!bubba.NMSU.Edu!NMSU.Edu!smori From: S. MORI Newsgroups: bionet.molbio.methds-reagnts Subject: Problems with Boehringers' DIG??? Date: 3 Feb 1998 16:51:02 GMT Organization: New Mexico State University Lines: 16 Message-ID: <6b7htm$pci@bubba.NMSU.Edu> NNTP-Posting-Host: hector.nmsu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 8bit X-Newsreader: TIN [UNIX 1.3 unoff BETA 970324; AIX 4.2] Has anyone had any recent problems with the new stocks of Boehringer's washing and blocking solutions??? I have been attempting southerns and northerns using their overpriced buffer/blocking solutions and I get GLOWING blots with high backgrounds and no signals.... -- DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA Shahram Mori Program in Molecular Biology Department of Chemistry and Biochemistry Box 3C New Mexico State University Las Cruces NM 88003 RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!POBOX.COM!profits1998 From: profits1998@POBOX.COM Newsgroups: bionet.molbio.methds-reagnts Subject: Earn $450 Commissions Part-time! Date: 4 Feb 1998 21:26:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 60 Sender: daemon@net.bio.net Distribution: world Message-ID: <199802050524.XAA15568@dfw-ix3.ix.netcom.com> NNTP-Posting-Host: net.bio.net Hello! I'm Ron Stewart, President of FPANY. I saw you listed on the internet and I believe that you can supplement your income by working with us. Please take a minute and read the following and contact me at your earliest convenience. EARN $450 COMMISSIONS... and more setting up businesses to accept Visa, MasterCard, American Express and Discover Credit cards with their own Merchant Account. We specialize in Internet Businesses, Home Based Businesses, etc. We offer the lowest discount rates in the industry and we have the best exclusive sales and marketing materials available plus web pages set up that you can use. You can also earn money with our Telephone Check Payment Systems, Inc. company which allows businesses to accept personal and business checks by phone, fax or internet. Phone-fax-email for FREE INFORMATION. IMPORTANT! For Free Information by Email, DO NOT HIT REPLY! Send your email to our special email address below! 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From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!AOL.COM!DOCTORHIM From: DOCTORHIM@AOL.COM Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Automatic Sequencing service wanted Date: 4 Feb 1998 19:19:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Try Midland Certified Reagents in Midland Texas. They have a web page and their prices are competitive. From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!intgwpad.nntp.telstra.net!news.telstra.net!harbinger.cc.monash.edu.au!bunyip.cc.uq.edu.au!not-for-mail From: K.Scott@botany.uq.edu.au Newsgroups: bionet.molbio.methds-reagnts Subject: Automatic Sequencing service wanted Date: Thu, 05 Feb 1998 11:36:29 +1100 Organization: University of Queensland Lines: 10 Message-ID: <34D9098D.A566DD46@botany.uq.edu.au> NNTP-Posting-Host: ripvan.ctpm.uq.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.03 [en] (Win95; I) Hi, I need to find information on companies and/or labs where we can have automatic sequencing reactions run out on ABI machines. Any information on providers and costs would be appreciated. Leon Scott From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!newsfeed.kornet.nm.kr!nntp.kreonet.re.kr!logbridge.uoregon.edu!sunqbc.risq.qc.ca!rcogate.rco.qc.ca!altitude!usenet From: Achim Recktenwald Newsgroups: bionet.molbio.methds-reagnts Subject: Re: EDTA/Heparin & DNA stability Date: Wed, 04 Feb 1998 18:43:58 -0500 Organization: Communication Accessible Montreal Lines: 27 Message-ID: <34D8FD3E.ADB5379C@cam.org> References: <5c847ca.34d88ffe@aol.com> NNTP-Posting-Host: dialup-691.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.04 [en] (Win16; I) DOCTORHIM@AOL.COM wrote: > There is some literature on using heparinase to enhance PCR on blood samples. > Check the entrez browser and search for PCR and heparinase. If I find the > reference, I will send it. I didn't see the original post, but second-guessing from the subject-title, if the question was what EDTA does to heparinase, I can tell you that it completely inhibits the activity of the enzyme. The enzyme is not killed; addition of calcium-ions will overcome the inhibition. One Ca-ion is part of its tertiary structure and directly involved in the enzyme activity. Achim ________________________________ Achim Recktenwald, Ph.D. 4469 avenue Marcil Montreal, Quebec, H4A 2Z9 Canada Phone: (514) 483-2807 Email: achim@cam.org From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!howland.erols.net!ais.net!ameritech.net!uunet!in4.uu.net!news.blarg.net!not-for-mail From: weazel@animal.blarg.net (Beth Wapelhorst) Newsgroups: bionet.molbio.methds-reagnts Subject: Have you seen these tip boxes?? Date: 4 Feb 1998 14:01:05 -0800 Organization: Blarg! Online Services - 206/441-9109 Lines: 14 Message-ID: <6baof1$443$1@animal.blarg.net> NNTP-Posting-Host: animal.blarg.net X-Newsreader: TIN [version 1.2 PL2] We've been looking for these 1-200ul tip boxes...they're grey and white marbled colored, and the bases are solid plastic - so the tips have no room to wobble. Does anyone know who sold these or if anyone makes tip boxes like that now? They're the best ones for multichannel work. thanks, beth The DNA Hacker's Resource www.blarg.net/~weazel/DHR/dhr.htm -- sugar beth wapelhorst "I don't think I'm alone when I say I'd weazel@blarg.net like to see more and more planets fall www.blarg.net/~weazel under the ruthless domination of our solar system." - Jack Handey From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!SCRIPPS.EDU!jkreps From: jkreps@SCRIPPS.EDU ("Joel A. Kreps") Newsgroups: bionet.molbio.methds-reagnts Subject: NEB "Improved" IMPACT T7 expression system Date: 4 Feb 1998 13:41:10 -0800 Organization: The Scripps Research Institute Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <34D8DF03.547B@scripps.edu> NNTP-Posting-Host: net.bio.net Hey- Has anyone been able to express and purify a eukaryotic protein with this system? Anyone had a terrible experience? And what is "improved" about it? Tom Darlington Grad Student TSRI c/o J Kreps From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!howland.erols.net!newsfeed.direct.ca!Supernews60!supernews.com!news.he.net!news.pagesat.net!news.itis.com!news.doit.wisc.edu!rumenbugs.dfrc.wisc.edu!user From: chriso@dfrc.wisc.edu (Chris Odt) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Problems with Boehringers' DIG??? Date: Wed, 04 Feb 1998 15:28:09 -0500 Organization: USDA/Dairy Forage Research Center Lines: 21 Message-ID: References: <6b7htm$pci@bubba.NMSU.Edu> NNTP-Posting-Host: rumenbugs.dfrc.wisc.edu In article <6b7htm$pci@bubba.NMSU.Edu>, S. MORI wrote: > Has anyone had any recent problems with the new stocks of Boehringer's > washing and blocking solutions??? I have been attempting southerns and > northerns using their overpriced buffer/blocking solutions and I get > GLOWING blots with high backgrounds and no signals.... > > -- > DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA$DNA > Shahram Mori > Program in Molecular Biology > Department of Chemistry and Biochemistry Box 3C > New Mexico State University > Las Cruces NM > 88003 > RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA$RNA Try using Amershans Rapid-hyb buffer instead. Even though it is formulated for use with isotopes, in my project, this hyb buffer worked better that Dig EZ hyb, with Lumiphos 530. Who knows why......... chris From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!sunqbc.risq.qc.ca!cyclone.mbnet.mb.ca!canopus.cc.umanitoba.ca!ummanley From: ummanley@news.cc.umanitoba.ca (Darren Mark Manley) Newsgroups: bionet.molbio.methds-reagnts Subject: protein assay in LDAO Date: 4 Feb 1998 19:57:09 GMT Organization: The University of Manitoba Lines: 17 Message-ID: <6bah6l$pid$1@canopus.cc.umanitoba.ca> NNTP-Posting-Host: toliman.cc.umanitoba.ca X-Newsreader: Tin 1.1 PL5 Does anyone know of a protein assay that can handle the presence of millimolar levels (5-10mM) of the detergent lauryldimethylamine oxide (LDAO), preferably a non-commercial, home-made protocol. Thanks ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ { } { Darren Manley Department of Chemistry } { phone (204) 474-6697 University of Manitoba } { fax (204) 474-7608 Winnipeg, MB., R3T-2N2 } { email ummanley@cc.umanitoba.ca Canada } { } { "Experience is something you don't get until } { just after you need it" } { } ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!sdu.edu.cn!lilab From: lilab@sdu.edu.cn (lab of MYXO) Newsgroups: bionet.molbio.methds-reagnts Subject: Simple Method to Determine the Action Mode of a New Drug NEEDED!!! Date: 4 Feb 1998 23:59:07 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <34D97157.6B40@sdu.edu.cn> Reply-To: lilab@sdu.edu.cn NNTP-Posting-Host: net.bio.net I am screening new bioactive products from myxobacteria. Now I have isolated one, which inhibites Mucor. The next I should do is to get an idea of its action mode, especially on DNA,RNA and protein synthesis. I have looked through many books and papers I can find, but I can not find a good way. Can anyone give me a good idea on the method to determine the action mode of a new drug? Please email me at your earliest convenience. Thanks for your time in advance! _______________________________________________ State Key Laboratory of Microbial Technology Department of Microbiology, Life Science College Shandong University Jinan 250100, P.R.China Tel:(86)(531)8564431(ext.411) Fax:(86)(531)8565234 Email: lilab@sdu.edu.cn _______________________________________________ From owner-methds-reagnts@net.bio.net Tue Feb 03 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news.gsl.net!gip.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!worldnet.att.net!news.u.washington.edu!not-for-mail From: rholmes@u.washington.edu (Rachel) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Hoefer TKO100 fluorometer Date: 4 Feb 1998 19:41:01 GMT Organization: University of Washington Lines: 15 Sender: -Not-Authenticated-[5683] Message-ID: <6bag8d$t5j$1@nntp3.u.washington.edu> References: <6b7k3q$8e0_001@leeds.ac.uk> NNTP-Posting-Host: 128.95.83.1 X-Trace: nntp3.u.washington.edu 886621261 29875 (None) 140.142.64.1 X-Complaints-To: help@cac.washington.edu X-Posted-From: InterNews 1.0.1@128.95.83.1 Xdisclaimer: No attempt was made to authenticate the sender's name. Craig, I talked to a tech at Pharmacia Biotech just the other day about similar problems our TKO100 is having. She was extremely helpful and told me that although they stopped selling them a few years back, they will still service them for another 4 years. I was also told that I could have ours serviced without a problem. I chose not to do it because of the cost. They do sell a replacement (DyNA Quant 200) for about $2500. Good luck, Rachel PS. I spoke to a tech named Nancy in case you wanted to speak to her directly From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!howland.erols.net!ais.net!uunet!in1.uu.net!ozemail!news.mel.aone.net.au!newsfeed-in.aone.net.au!news.mel.connect.com.au!news.uwa.edu.au!cyllene.uwa.edu.au!not-for-mail From: karenmk@cyllene.uwa.edu.au (Karen Kroeger) Newsgroups: bionet.molbio.methds-reagnts Subject: UV crosslinking Date: 6 Feb 1998 07:02:18 GMT Organization: The University of Western Australia Lines: 14 Message-ID: <6bechq$gdt$1@enyo.uwa.edu.au> NNTP-Posting-Host: cyllene.uwa.edu.au X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Hi, I am intersted in tyring UV- crosslinking as a method to try and determine the number and sizes (MW) of the proteins I see in complexes on gel shifts. If any one has successfully used this technique I would would be greatful for any advice. eg. what are the pitfalls, basic method. Also does anyone know of any other alternative methods which would allow me to do the same thing, without going through the task of protein purification. Any suggestions or information on other techniques,would help Thanks. Relpy to my email address directly would be preferred. Karen Kroeger Dept. Biochemistry University of Western Australia W.A. Australia Email: karenmk@cyllene.uwa.edu.au From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!Cabal.CESspool!bofh.vszbr.cz!serra.unipi.it!news-ge.switch.ch!news-zh.switch.ch!elna.ethz.ch!retrograde.ethz.ch!user From: ned@neuro.biol.ethz.ch (Ned Mantei) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: curious ligation question... Date: Fri, 06 Feb 1998 07:43:43 +0100 Organization: Dept. of Neurobiology, ETH-Zurich Lines: 19 Message-ID: References: NNTP-Posting-Host: retrograde.ethz.ch In article , Robot-Boy wrote: > > maybe this is a really dumb question, but let's say you are setting up a > ligation with vector that (in theory) shouldn't ligate to itself (ie.cut > with two different enzymes, needlessly dephosphorylated, *and* gel > purified).....etc. If the cells become saturated by the amount of DNA you put in, and most of this is insert not ligated to vector, the number of transformants could drop. You might want to read Revie, et al., Nucleic Acids Res, 16:10301-10321, "Kinetic analysis for optimization of DNA ligation reactions". -- Ned Mantei Dept. of Neurobiology, Swiss Federal Institute of Technology CH-8093 Zurich, Switzerland to reach me more quickly, replace my first name by my last name in my e-mail address. mantei@neuro.biol.ethz.ch Fax: +41-1-633-1046 From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!microbiology.unimelb.edu.au!p.homchampa From: p.homchampa@microbiology.unimelb.edu.au (Preecha Homchampa) Newsgroups: bionet.molbio.methds-reagnts Subject: Protein expression in pTrc99A, Please help !! Date: 5 Feb 1998 20:23:22 -0800 Organization: Uni of Melbourne Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <34DB2A8C.239@microbiology.unimelb.edu.au> NNTP-Posting-Host: net.bio.net Hi Sweet Netters, I need help with protein expression in pTrc99A. What I like to know is the followings: What is the best E.coli strain to use with this vector? What is the best induction conditions to use i.e IPTG concentration, what temperature, still or shaking culture and for how long etc.? I am going to express a protein subunit which will go to periplasm( it contains leader seq) and form multimer here. What is the best and practical method to get good yield of periplasmic proteins? Is there a way to enhance multimeric structure formation? Thank you very much for your help. Preecha From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!logbridge.uoregon.edu!news.maxwell.syr.edu!uninett.no!newscore.univie.ac.at!03-newsfeed.univie.ac.at!news.univie.ac.at!not-for-mail From: a8803349.nospam@unet.univie.ac.at (Martin Offterdinger) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: [q]digitonin Date: Fri, 06 Feb 1998 08:27:31 GMT Organization: AKH Lines: 24 Message-ID: <346c1059.3059432@news.univie.ac.at> References: NNTP-Posting-Host: grunt.imed1.akh-wien.ac.at X-Newsreader: Forte Free Agent 1.1/16.230 On 13 Nov 1997 05:19:17 GMT, ygen@nih.go.jp (Hisanori Yamamoto) wrote: > >Hi, >To permeabilize cells, i want to use digitonin. >Is digitonin-solution stable at R.T.? >How to prepare and stock digitonin-solution ? > >Hisanori >N.I.H.,japan There are several different qualities of dig available on the market, which dissolve differently. I used to dissolve dig from sigma at 1% in isotonic tris buffer with heating to 60°C filter afterwards and store the solution at 4°C for several weeks. Upon prolonged storage a precipitate forms which I took as a sign to discard the solution an prepare a new one.(Carefully, very toxic!!!) Martin Martin Offterdinger Internal Med.I,Dept. Oncology University of Vienna Austria E-Mail:a8803349.nospam@unet.univie.ac.at (remove nospam before mailing) From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!EARTHLINK.NET!webby1 From: webby1@EARTHLINK.NET (Tom Webster) Newsgroups: bionet.molbio.methds-reagnts Subject: Plate assays for B-lactamase Date: 5 Feb 1998 18:23:09 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <34DA727D.D51025E0@earthlink.net> NNTP-Posting-Host: net.bio.net I am looking for an assay system to detect B-lactamase in agar plates. I have read about a method involving PADAC. Has anyone ever used this reagent? Any information would be greatly appreciated. Thanks, Miriam Braunstein braunste@aecom.yu.edu From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!csn!nntp-xfer-1.csn.net!news.acsu.buffalo.edu!srv1.drenet.dnd.ca!crc-news.doc.ca!nott!uottawa!aix1.uottawa.ca!s535290 From: Robot-Boy Newsgroups: bionet.molbio.methds-reagnts Subject: curious ligation question... Date: Thu, 5 Feb 1998 19:35:58 -0500 Organization: University d'/of Ottawa Lines: 28 Message-ID: NNTP-Posting-Host: aix1.uottawa.ca Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII hi all, maybe this is a really dumb question, but let's say you are setting up a ligation with vector that (in theory) shouldn't ligate to itself (ie.cut with two different enzymes, needlessly dephosphorylated, *and* gel purified). You set up two ligations with a test insert, use fixed amounts of vector : one ligation has 5 times more insert than the other one. I haven't mentioned anything about vector/insert ratios because for my experiment, the amount of real insert is *very* limited. Anyway...these test ligations were done so that I could get an idea as to the number of transformants expected when the amount of insert is as limited as mine is. Anyhow, barring any mislabelling of my tubes, I obtained a slightly higher number of transformants from the ligation where the insert was was "limiting". And this was surprising to me. I would have figured that the number of transformants obtained would increase linearly with the amount of insert used, given the fact that in this case, the vector was kept constant and it was in excess with respect to the insert. Any thoughts ? Again, the only reason that I am using an excess of vector, is the fact that my insert is limiting and I would think that the chances of ligating a vector (that can't self-ligate) to a given insert would only increase as you increase the amount of available insert for the ligation. puzzled and bewildered.... TIA, Ed From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!kitts.u.arizona.edu!hes From: Harold E Smith Newsgroups: bionet.molbio.methds-reagnts Subject: Re: mutagenesis strategy Date: Thu, 5 Feb 1998 15:11:31 -0700 Organization: The University of Arizona Lines: 8 Message-ID: References: <34D77520@msmail3.mas.yale.edu> NNTP-Posting-Host: kitts-l.u.arizona.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII To: "Lolis, Elias" In-Reply-To: <34D77520@msmail3.mas.yale.edu> Elias- You could try mutagenic PCR of the region of interest. Engineer restriction sites flanking the TM region, and include the same sites in your PCR primers. Amplify under conditions of reduced fidelity (by either biased nucleotide ratios or addition of manganese to the reaction), cut, and ligate. Will find references for the PCR if you're interested. -Harold Smith From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!bloom-beacon.mit.edu!senator-bedfellow.mit.edu!usenet From: Nathan Siemers Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Plate assays for B-lactamase Date: 05 Feb 1998 22:42:18 -0500 Organization: Bristol-Myers Squibb Lines: 35 Message-ID: References: <34DA727D.D51025E0@earthlink.net> NNTP-Posting-Host: rna.wi.mit.edu X-Newsreader: Gnus v5.3/Emacs 19.34 Miriam, If you just want a yes/no answer for colonies/plaques, and don't need to do anything with the plates afterwards, you can simply coat the plates with a dilute solution of nitrocefin, and observe the red color generated where there is lactamase activity. I think I tried it with padac also, and got similar results. Works like a charm. nathan webby1@EARTHLINK.NET (Tom Webster) writes: > > I am looking for an assay system to detect B-lactamase in agar plates. > I have read about a method involving PADAC. Has anyone ever used this > reagent? > Any information would be greatly appreciated. > > Thanks, > > Miriam Braunstein > braunste@aecom.yu.edu > -- Nathan Siemers, Ph.D. Research Investigator, Applied Genomics Bristol-Myers Squibb Pharmaceutical Research Institute Visiting Scientist Whitehead/MIT Center for Genome Research 617-252-1382 siemers@bms.com From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!howland.erols.net!news.maxwell.syr.edu!dispose.news.demon.net!demon!rill.news.pipex.net!pipex!join.news.pipex.net!pipex!ggr.co.uk!not-for-mail From: Fraser Dr N J Newsgroups: bionet.molbio.methds-reagnts Subject: RT-PCR from incredibly small amounts of tissue Date: Thu, 05 Feb 1998 15:44:10 +0000 Organization: Glaxo Wellcome Message-ID: <34D9DE4A.167E@ggr.co.uk> NNTP-Posting-Host: ukwbf80.ggr.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: ukwsv3.ggr.co.uk 886693274 9090 (None) 147.184.221.124 X-Complaints-To: usenet@ggr.co.uk X-Mailer: Mozilla 3.01 (X11; I; IRIX 5.3 IP22) Lines: 3 Does anyone have a protocol/reference that would be suitable for RNA isolation/RT-PCR from a micro-dissected sample of fixed pancreas ? Thanks - Neil From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!cardiff.ac.uk!Oviedo-Orta From: Oviedo-Orta@cardiff.ac.uk (ERNESTO OVIDEO ORTA) Newsgroups: bionet.molbio.methds-reagnts Subject: Lymphocytes Date: 5 Feb 1998 07:31:48 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <2BD749C5F71@wmcu1.uwcm.ac.uk> Reply-To: oviedo-orta@cardiff.ac.uk NNTP-Posting-Host: net.bio.net Hi, I would like to know if someone can help me to find the following methods: * Non-FACS, non-magnetic beads, human B lymphocytes isolation and purification. * Fast double immunofluorescence for lymphocytes in suspension. Thank you in advance, Ernesto. From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!join.news.pipex.net!pipex!ggr.co.uk!not-for-mail From: Fraser Dr N J Newsgroups: bionet.molbio.methds-reagnts Subject: How much total RNA ? Date: Thu, 05 Feb 1998 15:28:21 +0000 Organization: Glaxo Wellcome Lines: 1 Message-ID: <34D9DA95.41C6@ggr.co.uk> NNTP-Posting-Host: ukwbf80.ggr.co.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: ukwsv3.ggr.co.uk 886692325 8658 (None) 147.184.221.124 X-Complaints-To: usenet@ggr.co.uk X-Mailer: Mozilla 3.01 (X11; I; IRIX 5.3 IP22) How much total RNA can I expect to get from a rat cortex and cerebellum? From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!daresbury!not-for-mail From: "Wolfgang Schechinger" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Simple Method to Determine the Action Mode of a New Drug NEE Date: 5 Feb 1998 15:11:03 -0000 Organization: Med Clinic iV, Dept. of Pathobiochemistry, Lines: 49 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <6bckq7$rgn@mserv1.dl.ac.uk> Reply-To: wgschech@med.uni-tuebingen.de Comments: Authenticated sender is X-Authentication-Warning: drakkar.med.uni-tuebingen.de: smap set sender to using -f X-mailer: Pegasus Mail for Windows (v2.52) MIME-Version: 1.0 In-reply-to: <982502125.~INN-PVDa00213.bionet-news@dl.ac.uk> Original-To: lilab@sdu.edu.cn (lab of MYXO), lilab@sdu.edu.cn, methods@dl.ac.uk Hi. Just some basic ideas: My suggestion for an approach is: Try to find out what cellular machines are necessary for your drug to work. Block mRNA synthesis, protein synthesis, kick out mitochondria, stop the cell cycle at different points and look what's happening. Shurely here in the net are people who know more about this. Good luck! Wolfgang > I am screening new bioactive products from myxobacteria. Now I have > isolated one, which inhibites Mucor. The next I should do is to get > an idea of its action mode, especially on DNA,RNA and protein > synthesis. I have looked through many books and papers I can find, > but I can not find a good way. Can anyone give me a good idea on the > method to determine the action mode of a new drug? Please email me > at your earliest convenience. Thanks for your time in advance! > _______________________________________________ State Key Laboratory > of Microbial Technology Department of Microbiology, Life Science > College Shandong University Jinan 250100, P.R.China > > Tel:(86)(531)8564431(ext.411) > Fax:(86)(531)8565234 > Email: lilab@sdu.edu.cn > _______________________________________________ > > ----- This message is RNAse free - please don't touch! ----- Wolfgang Schechinger University of Tuebingen email: wgschech@med.uni-tuebingen.de http://www.medizin.uni-tuebingen.de/~wgschech/research.htm ------- !Junk mail is *not* appreciated! SPAMs will be answered automatically by sending my 128MByte virtual memory file. This is *NO* joke. ------ usual disclaimers apply ------ From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!nih.gov!not-for-mail From: "Darren A. Natale" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: HIS tagged protein from baculo Date: Thu, 05 Feb 1998 09:41:27 -0400 Organization: NIH Lines: 37 Message-ID: <34D9C13B.523E@box-d.nih.gov.nospam> References: <6b7hfb$s68@sun0.urz.uni-heidelberg.de> NNTP-Posting-Host: ally.nei.nih.gov Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) un691cs@genius.embnet.dkfz-heidelberg.de wrote: > we have cloned a HIS-tagged protein in baculovirus. It > is a novel TGF-beta member, and dimeric. We get > some expression; the protein is indeed dimeric and looks o.k. > in a western. We can purify it with the his-tag. > However, we need at least 100 micrograms > for our initial assays, and this doesn't seem to work. > > question 1: what methods can we use to determine the amount of > protein. It is very dilute. We have tried Bradford, but aren't > in the linear range. Any pointer welcome here. I just want to > know more or less exactly how much protein we have. > > question 2: how do I optimize my production of the protein ? > what parameters to watch ? How much protein is normal, how much > can YOU make. any tricks not found in the manuals ? Are certain > proteins toxic to baculo ? Insect cells also produce TGF-betas. I presume you are using spinner flasks? I found out the hard way that you have to keep the volume lower than you may expect given the volume size of the flask. For example, when I tried expressing a protein in Sf9 using a 1 L culture in a 1 L flask, I got nothing! If I do the same protein, same cells, same flask, but drop the culture volume down to 400 mL, it works fine. Bear in mind that I was relying on the spinner to provide aeration. I think this problem can be avoided by bubbling air through the liquid, but I never tried it that way (I don't have the equipment). I have also seen reference to problems if the MOI is too high. There tends to be some degradation and/or low expression. D. Natale (remove ".nospam" from address to reply by mail) > > thanks, Clemens Suter-Crazzolara, Heidelberg From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!logbridge.uoregon.edu!news-peer.gip.net!news-lond.gip.net!news.gsl.net!gip.net!baron.netcom.net.uk!netcom.net.uk!server3.netnews.ja.net!ucl.ac.uk!bcc.ac.uk!lud36.ludwig.ucl.ac.uk!user From: "Stuart Rison" Subject: sequence for pBAG Sender: news@ucl.ac.uk (Usenet News System) Message-ID: Date: Thu, 5 Feb 1998 12:13:34 GMT Organization: Ludwig Institute for Cancer Research (UCL) X-Newsreader: Microsoft Internet Mail and News for Macintosh - 1.1 (34) Lines: 19 Hello, Does anyone have the sequence (I can cope with pretty much any format) for the vector pBAG (ref. Price, Turner and Cepko, (1987) PNAS 84:156-160). cheers, Stuart. +-------------------------+--------------------------------------+ | Stuart Rison | Ludwig Institute for Cancer Research | | Tel. (0171) 878 4127 | Courtauld Building | | Fax. (0171) 878 4040 | 91 Riding House Street | +-------------------------+ London, W1P 8BT | | stuart@ludwig.ucl.ac.uk | UNITED KINGDOM. | +-------------------------+--------------------------------------+ - and there's a NO_JUNK bit in my return address - From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!agate!newsfeed.kornet.nm.kr!nntp.kreonet.re.kr!news.maxwell.syr.edu!rill.news.pipex.net!pipex!server1.netnews.ja.net!news.nott.ac.uk!pdxkgs From: pdxkgs@pdn1.gene.nottingham.ac.uk (Karen Spink) Newsgroups: bionet.molbio.methds-reagnts Subject: Band-shift assay Date: Thu, 05 Feb 1998 12:52:26 +0000 Organization: University of Nottingham Lines: 24 Message-ID: NNTP-Posting-Host: ppdirg.nottingham.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 I am very confused! Recently, I have been trying to purify a DNA-binding protein from yeast and have constructed a specific DNA affinity column, attaching the binding site for my protein to CnBr-activated Sepharose. I bound some of my partially purified protein extract to this column and was successfull in retrieving a band-shift with one of the fraction. SDS-PAGE informed me that I now had a dozen or so bands in this fraction, so I re-applied this fraction to the column and eluted with a narrower range of salt concentrations, but my protein has now DISAPPEARED! It is neither in the flow through nor any of the fractions. I have read in a couple of publications that loss of activity in a band-shift can occur with a highly pure DNA binding protein and that activity can be regained by the addition of 1mg/ml casein to the binding reactions. I am hoping someone can enlighten me on this phenomena. 1) Does this really work ? 2) Why does this work, (BSA supposedly does not have this effect on activity) 3) Is it better to use casein or B-casein I do not really understand how the addition of casein, rather than BSA, can affect activity in this way and am hoping not to have to do the whole purification thing again Thanks very much for your time, Karen Spink From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!daresbury!not-for-mail From: "Wolfgang Schechinger" Newsgroups: bionet.molbio.methds-reagnts Subject: Q: differentiating and evaluating different GFPs with fl Date: 5 Feb 1998 10:48:57 -0000 Organization: Med Clinic iV, Dept. of Pathobiochemistry, Lines: 25 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <6bc5ep$c47@mserv1.dl.ac.uk> Reply-To: wgschech@med.uni-tuebingen.de Comments: Authenticated sender is X-Authentication-Warning: drakkar.med.uni-tuebingen.de: smap set sender to using -f X-mailer: Pegasus Mail for Windows (v2.52) MIME-Version: 1.0 Original-To: methods@dl.ac.uk Hi all! I have done some mutagenesis on the GFP-part of a GFP fusion protein. Is it possible to screen for positive mutants (higher quantum yield and / or color shift) by measuring (trypsinized?) cells in a fluorimeter? Any ideas, comments, hints and protocols are highly appreciated! Wolfgang ----- This message is RNAse free - please don't touch! ----- Wolfgang Schechinger University of Tuebingen email: wgschech@med.uni-tuebingen.de http://www.medizin.uni-tuebingen.de/~wgschech/research.htm ------- !Junk mail is *not* appreciated! SPAMs will be answered automatically by sending my 128MByte virtual memory file. This is *NO* joke. ------ usual disclaimers apply ------ From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!howland.erols.net!rill.news.pipex.net!pipex!server1.netnews.ja.net!newsfeed.ed.ac.uk!leeds.ac.uk!news From: BGYCSW@leeds.ac.uk (C.S. Wilding) Subject: Re: literature for rna secondary structure? Message-ID: <6bbtbb$8e0_001@leeds.ac.uk> NNTP-Posting-Host: bgy-pc041.leeds.ac.uk Organization: University of Leeds Date: Thu, 5 Feb 1998 08:30:39 +0000 (GMT) References: <6b9gl5$d9h@news.rrz.Uni-Koeln.DE> X-Newsreader: News Xpress Version 1.0 Beta #4 Lines: 33 Kerstin, for a start you could try Hickson, R. E., C. Simon, A. Copper, G. S. Spicer, J. Sullivan and D. Penny. 1996. Conserved sequence motifs, alignment, and secondary structure for the 3rd-domain of animal 12S ribosomal RNA. Mol. Biol. Evol. 13:150-169. This has some stuff about folding relating mtDNA rRNA to nuclear rRNA structures. Craig. In article <6b9gl5$d9h@news.rrz.Uni-Koeln.DE>, aeb25@rrz.Uni-Koeln.DE (Kerstin Hoef-Emden) wrote: > >Hi everybody! > > >I am looking for literature explaining the theoretical background of rna >folding. We have got access to the GCG package here and my first attempts >with mfold were successful. But I would like to get some idea >what things should be considered to choose the correct settings and to >avoid mistakes. > >Are there any books or publications about the method around which you >recommend? > > >Best wishes to all readers, > >Kerstin Hoef-Emden > > From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!daresbury!uninett.no!Cabal.CESspool!bofh.vszbr.cz!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!207.5.0.44!nntp.mainstreet.net!news.pbi.net!news.pacbell.net!not-for-mail From: Susan Kang Newsgroups: bionet.molbio.methds-reagnts Subject: Pulse power supply Date: Thu, 05 Feb 1998 13:51:08 -0800 Organization: HIFAX Lines: 57 Message-ID: <34DA344C.6CED@hifax.com> Reply-To: Order@hifax.com NNTP-Posting-Host: ppp-206-170-217-211.nhwd02.pacbell.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01C-PBWG (Win95; U) Dear colleagues; Once we announced the free gift of our PULSE POWER power supply to every 100th visitor of our web site www.hifax.com. However, since not many biology researchers does not know the concept of this kind of apparatus, I decided to explain the basic concept. 1. Decentralize Molecular biology Lab. I have experienced waiting several ten minutes or hours till somebody finished their gel running to use the limited number of power supply in the Lab. Of course you can do other things while you are waiting. However, we know that we loose some efficiency if we loose our momentum. My idea was having my own power supply, and finally dream came true after spending several hundred dollars (of course that was my boss's money). I am quite sure everybody want to has her/his own power supply. 2. Should be inexpensive, and small I don't have to say inexpensive. Now the Lab. is becoming crowded and the bench space became one of the most precious assets of the institute. If everybody wants to have own power supply, it should be as small as possible. 3. Not many peopled need variable voltage setting. We know that once the gel running box is selected usually the optimal voltage follows. That means gel box is the limiting factor and not the power supply. That is the reason why we recommended less than 20 cm of electrode distance. 4. Only one diode can do perfect job. If you want to convert AC to DC, you have to use diodes, several condensers and resistances to stabilize the fluctuation of the current. However, if you think once more about your electrophoresis, you will recognize that flat current is not necessary just for moving and separating molecules. Our goal is separating molecules and not having fancy apparatus. 5. Is it difficult to make your own apparatus? Never, I think almost everybody in the lab can make this apparatus without any difficulties. 6. Am I an inventor of this product? No, I just improved. 7. Who am I I am the founder of HIFAX and the guy who once posted the method how to make less than 1 dollar gel box. Best regards Chulho Kang, Ph.D. From owner-methds-reagnts@net.bio.net Wed Feb 04 22:00:00 1998 Path: biosci!daresbury!not-for-mail From: "Wolfgang Schechinger" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR with degenerate primer Date: 5 Feb 1998 20:56:08 -0000 Organization: Med Clinic iV, Dept. of Pathobiochemistry, Lines: 48 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <6bd918$m9k@mserv1.dl.ac.uk> Reply-To: wgschech@med.uni-tuebingen.de Comments: Authenticated sender is X-Authentication-Warning: drakkar.med.uni-tuebingen.de: smap set sender to