From owner-methds-reagnts@net.bio.net Sat May 01 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!munnari.oz.au!ariel.ucs.unimelb.EDU.AU!ucsvc.ucs.unimelb.edu.au!u5216834 From: u5216834@ucsvc.ucs.unimelb.edu.au Newsgroups: bionet.molbio.methds-reagnts Subject: Ligation of Agarase purified DNA Message-ID: <1993May3.113651.5097@ucsvc.ucs.unimelb.edu.au> Date: 3 May 93 01:36:50 GMT Organization: The University of Melbourne Lines: 8 I have tried ligating DNA purified using Boehringer Mannheim 's AGARASE, with no success. Has any one encountered similar problems. The procedure used for purification was as described by B.M. Thank you. Clint E-mail : C.Grant@biochemistry.unimelb.edu.au From owner-methds-reagnts@net.bio.net Sat May 01 23:00:00 1993 Path: biosci!uwm.edu!cs.utexas.edu!uunet!newsflash.concordia.ca!mizar.cc.umanitoba.ca!hamel From: hamel@ccu.umanitoba.ca (Andre Hamel) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR/dot blot - removing mineral oil Message-ID: Date: 3 May 93 06:23:20 GMT References: Sender: news@ccu.umanitoba.ca Organization: University of Manitoba, Winnipeg, Manitoba, Canada Lines: 20 Nntp-Posting-Host: antares.cc.umanitoba.ca >Sorry everyone I missed the point of this thread in the orginal post. >Chloroform is of little use with most microtiter plates. > >Jim >J. Graham Still, is anyone aware of ANY type of microtitre plate that may well be CHCl3 AND heat resistant? I would think there'd be such an animal available out there somewhere ... (cliche's like "we can put people on moon, etc .. :') ---------------------------------------------------------------------------- Andre Hamel email: hamel@ccu.umanitoba.ca Manitoba Veterinary Services lab tel.: (204) 945-7630 Infectious Diseases Mol.Bio.Lab 545 University Crescent home tel.: (204) 275-1204 Winnipeg, Manitoba FAX: (204) 945-8062 CANADA R3T 5S6 ------------------------------------------------------------------------------ From owner-methds-reagnts@net.bio.net Sat May 01 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!darwin.sura.net!welchgate.welch.jhu.edu!danj From: danj@welchgate.welch.jhu.edu (Dan Jacobson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR/plates/oil Message-ID: <1993May2.144805.7133@welchgate.welch.jhu.edu> Date: 2 May 93 14:48:05 GMT References: Organization: Johns Hopkins Univ. Welch Medical Library Lines: 21 In article hamel@ccu.umanitoba.ca (Andre Hamel) writes: >I got an email from Patricia (Foster?) of Boston, before I could finish >reading her letter our system temporarily "crashed", resulting in loss >of her letter when I was able to log back on. A Netfind search I came up >empty. > [ . . . ] A little gophering finds the following for Patricia Foster: Patricia L. Foster Boston University School of Medicine Boston, MA USA pfoster@bu.edu Best of luck, Dan Jacobosn danj@welchgate.welch.jhu.edu From owner-methds-reagnts@net.bio.net Sat May 01 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!noc.near.net!das-news.harvard.edu!husc-news.harvard.edu!hsdndev!dartvax!grafton.dartmouth.edu!knight From: knight@grafton.dartmouth.edu (John Boswell) Newsgroups: bionet.molbio.methds-reagnts Subject: Texts on Cell Culture? Message-ID: Date: 2 May 93 23:43:29 GMT Sender: news@dartvax.dartmouth.edu (The News Manager) Organization: Dartmouth College, Hanover, NH Lines: 19 Hi, Could someone direct me towards any good references on basic Cell/ Tissue Culture techniques? In a couple of months I'll be starting up a cell culture facility in a new lab so we can overexpress some protein. I've done a bit of cell culture already, but I'm looking for stuff like how to tell when cells are confluent, when they are (*gasp*) dead, etc. Please bear in mind that I'm a chemist by training, so dealing with bugs is kinda new to me. Oh, I should say I'm looking for *mammalian* cell culture theory/ techniques. I've done quite a bit of *bacterial* cell culture, but that's different.... Thanks in advance for any pointers.... John Boswell knight@grafton.dartmouth.edu -- **************************************************************************** Dr. John Boswell knight@grafton.dartmouth.edu Dept. of Medicine/GI MCN C2104 Vanderbilt University, Nashville, TN 37232 615-322-6367 or 615-343-4747 From owner-methds-reagnts@net.bio.net Sat May 01 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!uunet!zaphod.mps.ohio-state.edu!howland.reston.ans.net!ux1.cso.uiuc.edu!usenet.ucs.indiana.edu!bronze.ucs.indiana.edu!jgraham From: jgraham@bronze.ucs.indiana.edu (the End) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR/dot blot - removing mineral oil Message-ID: Date: 2 May 93 23:40:10 GMT References: <1rk9bq$di7@usenet.INS.CWRU.Edu> <1rm7flINNkbd@lynx.unm.edu> Sender: news@usenet.ucs.indiana.edu (USENET News System) Organization: Indiana University Lines: 7 Nntp-Posting-Host: bronze.ucs.indiana.edu Sorry everyone I missed the point of this thread in the orginal post. Chloroform is of little use with most microtiter plates. Jim J. Graham . From owner-methds-reagnts@net.bio.net Sat May 01 23:00:00 1993 Path: biosci!uwm.edu!cs.utexas.edu!uunet!newsflash.concordia.ca!mizar.cc.umanitoba.ca!jrichmn From: jrichmn@ccu.umanitoba.ca (Joy Richman) Newsgroups: bionet.molbio.methds-reagnts Subject: 2nd posting, affinity chromat. for bFGF Message-ID: Date: 2 May 93 21:23:16 GMT Sender: news@ccu.umanitoba.ca Organization: University of Manitoba, Winnipeg, Canada Lines: 33 Nntp-Posting-Host: vogt.cc.umanitoba.ca This is the second time I am posting this question since I am still looking for answers to it. We are attempting to characterize expression of bFGF in the face using Western blots. We are homogenizing our early embryonic chick heads in 0.15 M Ammonium sulfate with protease inhibitors using a hand held homogenizer, spinning out the insoluble fraction in a microfuge, and then binding to heparin sepharose beads under high salt conditions (.6 M NaCl). We are using a batch method to bind to the beads rather than a column. The final elution is merely to boil the beads in sample buffer (SDS,glycerol, beta mercaptoethanol and dyes). Thus we should elute all forms of bFGF including any higher molecular weight forms. We have had intermittent success with this method. By success I mean that we can see a 18 kD band in some of our extracts but not in others. There is alot of variation between preparations. Some preparations have no low molecular weight bands but only 3-4 very large molecular weight bands (40-60 kD or greater!) Unfortunately we only have enough from each preparation to load in one lane of one gel. I am dissapointed in the lack of reproducibility and wonder if anyone has had experience with affinity chromatography and can offer suggestions? Oh our polyclonal antibodies are raised to the first 24 aminoacids of 146 aa form of bFGF conjugated to Keyhole Limpet Hemocyanin. The antibody cross reacts with chick and bovine bFGF and works well in our detection system (125I). We can pick up as little as 1 ng of bFGF. Joy Richman -- The most important event in life is not birth or death but gastrulation. Lewis Wolpert jrichmn@ccu.umanitoba.ca From owner-methds-reagnts@net.bio.net Sun May 02 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!ugle.unit.no!trane.uninett.no!nntp.uio.no!usenet From: TIMOTHYL@BIOMED.UIO.NO (Timothy Lavelle) Newsgroups: bionet.molbio.methds-reagnts Subject: polyethylenimine (polymin P)? Message-ID: <1s46u5$2hn@hermod.uio.no> Date: 3 May 93 22:39:01 GMT Organization: NORWEGIAN EMBNET NODE Lines: 8 NNTP-Posting-Host: biomed.uio.no X-News-Reader: VMS NEWS 1.24 Hello, I am interested in finding information on the reagent polymin P. It appears that it is used to precipitate various compounds. I would appreciate any references or descriptions concerning the action of this reagent. Thanks for your help. If you prefer to reply by e-mail my address is: Timothyl@biomed.uio.no From owner-methds-reagnts@net.bio.net Sun May 02 23:00:00 1993 Path: biosci!CBR.DWE.CSIRO.AU!MHOLLAND From: MHOLLAND@CBR.DWE.CSIRO.AU (Michael Holland) Newsgroups: bionet.molbio.methds-reagnts Subject: RE: quantitative westerns Message-ID: <930503172834.20e30@cbr.dwe.csiro.au> Date: 3 May 93 22:28:34 GMT Sender: kristoff@net.bio.net Reply-To: MHOLLAND@cbr.dwe.csiro.au (Michael Holland) Distribution: bionet Lines: 8 The easiest thing may be to use a second antibody linked to peroxidse (or anything else) and cut out your spot and dissolve in DMSO then count. Alternatively if the isotope is a good beta emitter simply transfer and put it down with some Saran wrap to stop the nitrocellulose sticking to your film. Both work fine. Good luck, Mike "Kangaroo" Holland From owner-methds-reagnts@net.bio.net Sun May 02 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!darwin.sura.net!udel!sbcs.sunysb.edu!max.physics.sunysb.edu!mhollowa From: mhollowa@ic.sunysb.edu (Michael Holloway) Newsgroups: bionet.molbio.methds-reagnts Subject: Sequencing primer for CAT? Message-ID: <1s3dth$8ln@max.physics.sunysb.edu> Date: 3 May 93 15:32:01 GMT Organization: State University of New York at Stony Brook Lines: 10 NNTP-Posting-Host: engws5.ic.sunysb.edu Does anyone know of a commercially available primer for sequencing upstream of CAT in a reporter construct? I KNOW I seen one somewhere and I can't find it in any catalog now (Grrrr). Lacking that, does anyone have any experience with a sequence at the 5' end of CAT that can be successfully used for priming? Thanks in advance. Mike From owner-methds-reagnts@net.bio.net Sun May 02 23:00:00 1993 Path: biosci!agate!mendel.berkeley.edu!mytelka From: mytelka@mendel.berkeley.edu ( Daniel Mytelka ) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: polyethylenimine (polymin P)? Message-ID: <1s4h15$eg@agate.berkeley.edu> Date: 4 May 93 01:31:17 GMT References: <1s46u5$2hn@hermod.uio.no> Organization: /etc/organization Lines: 16 NNTP-Posting-Host: mendel.berkeley.edu In article <1s46u5$2hn@hermod.uio.no> TIMOTHYL@BIOMED.UIO.NO (Timothy Lavelle) writes: > Hello, > I am interested in finding information on the reagent polymin P. It appears >that it is used to precipitate various compounds. I would appreciate any >references or descriptions concerning the action of this reagent. Thanks for >your help. > If you prefer to reply by e-mail my address is: > Timothyl@biomed.uio.no We use Polymin P to precipitate DNA and the associated E. coli RNA polymerase during our RNA Polymerase purification. See Burgess and Jendrisak (Biochemistry 14:4634), and references therein. Daniel Mytelka (mytelka@mendel.berkeley.edu) From owner-methds-reagnts@net.bio.net Sun May 02 23:00:00 1993 Path: biosci!Pharm.SOM.sunysb.edu!BILL From: BILL@Pharm.SOM.sunysb.edu (Bill) Newsgroups: bionet.molbio.methds-reagnts Subject: PCR microtiter plates Message-ID: Date: 3 May 93 13:03:56 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: Pharmacology, SUNY Stony Brook Lines: 7 Andre; I found a catalogue and Nunc does makr 120 ul wells. You may also want to get the tape they sell for sealing the reactions. 120 ul GeNunc #2-32549; tape: 232700. Bill Richards Bill@pharm.som.sunysb.edu From owner-methds-reagnts@net.bio.net Sun May 02 23:00:00 1993 Path: biosci!uwm.edu!cs.utexas.edu!uunet!mcsun!news.funet.fi!ousrvr.oulu.fi!pcc36145.oulu.fi!Kvist From: Kvist@lbiok-1.oulu.fi (L229) Newsgroups: bionet.molbio.methds-reagnts Subject: Cosmid Mapping with PFGE Summary: Help needeed Keywords: PFGE, Cosmid, Gene Mapping Message-ID: Date: 3 May 93 13:19:26 GMT Sender: news@ousrvr.oulu.fi Organization: Oulun yliopisto Lines: 10 Hi I have tried to map gene from cosmid clones (pWE15-vector) with PFGE but I haven't succeded to find proper conditions for PFGE. Does anyone heve experiences in this area? My inserts are about 40 kb long and I have to separate DNA's from 10 to 40 kb. Ari-Pekka Kvist Kvist@lbiok-1.oulu.fi or apk@phoenix.oulu.fi From owner-methds-reagnts@net.bio.net Mon May 03 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!munnari.oz.au!ariel.ucs.unimelb.EDU.AU!ucsvc.ucs.unimelb.edu.au!u2605163 From: u2605163@ucsvc.ucs.unimelb.edu.au (Jill Maddox) Newsgroups: bionet.molbio.methds-reagnts Subject: Dye slower than xylene cy or br blue? Message-ID: <1993May4.125916.5105@ucsvc.ucs.unimelb.edu.au> Date: 4 May 93 02:59:15 GMT Organization: The University of Melbourne Lines: 7 Does anyone know of a dye that migrates more slowly than bromophenol blue and xylene cyanol which can be seen during electrophoresis without resorting to UV light? Thanks Jill From owner-methds-reagnts@net.bio.net Mon May 03 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: vioque@cica.es (Agustin Vioque) Newsgroups: bionet.molbio.methds-reagnts Subject: lac promoter Message-ID: <1993May4.085251.25461@gserv1.dl.ac.uk> Date: 4 May 93 09:53:20 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 6 Content-Identifier: 133 Original-To: methods@uk.ac.daresbury Conversion: Prohibited Does anyone know if the lac promoter/operator sequence in pUC19 and derivatives is the lacUV5 mutant or the wild type? The lacUV5 is not subject to catabolite repression and thus can be fully induced in the presence of glucose. I want to know if I have to change the carbon source for full induction of the promoter. Thanks.Agustin. vioque@cica.es From owner-methds-reagnts@net.bio.net Mon May 03 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: vioque@cica.es (Agustin Vioque) Newsgroups: bionet.molbio.methds-reagnts Subject: CAT protein stability Message-ID: <1993May4.085620.25688@gserv1.dl.ac.uk> Date: 4 May 93 09:56:40 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 5 Content-Identifier: 134 Original-To: methods@uk.ac.daresbury Conversion: Prohibited I am using CAT as a reporter in the study of a bacterial promoter. Are there any data available about the stability of the CAT protein and its half life in the cell? Thanks.Agustin. vioque@cica.es From owner-methds-reagnts@net.bio.net Mon May 03 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!darwin.sura.net!ukma!ukcc.uky.edu!BIOSEE From: BIOSEE@ukcc.uky.edu (Stephanie Edelmann) Newsgroups: bionet.molbio.methds-reagnts Subject: Sort muscle/non-muscle in FACS: Markers? Message-ID: <16BC47DC0.BIOSEE@ukcc.uky.edu> Date: 4 May 93 12:56:32 GMT Sender: news@ms.uky.edu (USENET News System) Organization: The University of Kentucky Lines: 13 Nntp-Posting-Host: ukcc.uky.edu X-Newsreader: NNR/VM S_1.3.2 Here's another one for you guys, I am producing primary muscle cell cultures (dog) from tissue. Of course, there's always "contamination" of the muscle satellite cells with fibroblasts etc. So, I'd like to use a FACS (Fluorescent activated cell sorter) to pick out the muscle/non-muscle. I know that that's possible with humans: There, you use an antibody against m.. (I forget what:-(). Is there anything around that can do the same thing for dog cells? Thanks Stephanie From owner-methds-reagnts@net.bio.net Mon May 03 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!darwin.sura.net!udel!sbcs.sunysb.edu!max.physics.sunysb.edu!mhollowa From: mhollowa@ic.sunysb.edu (Michael Holloway) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: CAT protein stability Message-ID: <1s6105$2aj@max.physics.sunysb.edu> Date: 4 May 93 15:09:57 GMT References: <1993May4.085620.25688@gserv1.dl.ac.uk> Distribution: bionet Organization: State University of New York at Stony Brook Lines: 17 NNTP-Posting-Host: engws5.ic.sunysb.edu In article <1993May4.085620.25688@gserv1.dl.ac.uk> vioque@cica.es (Agustin Vioque) writes: >I am using CAT as a reporter in the study of a bacterial promoter. Are there >any data available about the stability of the CAT protein and its half life >in the cell? I can't be sure, but I seem to recall that the original Gorman papers had some data on that. The "common wisdom" is that the CAT protein has a long half life and seems to be relatively insensitive to in vivo protease activity. The mRNA is the converse. It's not supposed to be the reporter of choice for RNase protection assays. Luciferase is the converse of the CAT situation. The protein has a short half life. Wait a moment, this paper may be of use. It describes optimizing electroporation of mammalian cells but the shows how resistant CAT is relative to luciferase. DNA 7:557-562 (1982). Mike From owner-methds-reagnts@net.bio.net Mon May 03 23:00:00 1993 Path: biosci!ATGENOME.BIO.UPENN.EDU!cbell From: cbell@ATGENOME.BIO.UPENN.EDU (Callum Bell - Ecker lab) Newsgroups: bionet.molbio.methds-reagnts Subject: pcr in microtiter plates Message-ID: <9305041654.AA28952@atgenome.bio.upenn.edu> Date: 4 May 93 16:54:46 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 23 Can anyone recommend a good protocol for doing PCR in microtiter plates in a BIOS biocycler PCR oven? I am currently doing my reactions in 0.5 ml microfuge tubes in a Cetus 480, 20 ul per sample, using home-made PCR gems. The results are great but capping and uncapping the tubes is getting tedious. I'd also like to process a lot more samples. If possible I would like to keep the sample volume down, and that's where the problem is - 20 ul tends to just wet the bottom of the wells in the microtiter plates I have tried. The answer would be microtiter plates with much narrower wells. Does anyone know where these can be obtained? Any help will be gratefully received. Callum ---------------------------------------------------------------------- Callum Bell | cbell@atgenome.bio.upenn.edu Department of Biology | phone (215) 898-0946 University of Pennsylvania | FAX (215) 898-8780 PA 19104-6018 | ---------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Mon May 03 23:00:00 1993 Path: biosci!uwm.edu!cs.utexas.edu!usc!howland.reston.ans.net!torn!csd.unb.ca!news.ucs.mun.ca!kean.ucs.mun.ca!scarr From: scarr@kean.ucs.mun.ca Newsgroups: bionet.molbio.methds-reagnts Subject: READ Message-ID: <1993May4.182553.1@kean.ucs.mun.ca> Date: 4 May 93 21:55:53 GMT Sender: usenet@news.ucs.mun.ca (NNTP server account) Organization: Memorial University. St.John's Nfld, Canada Lines: 9 There's a commerical product called OilAway that causes the phases to invert so that the aqueous is on top - also stains the latter light blue according to the pH. From the smell I suspect it must contain Chloroform. I have reason to believe it's not compatible with some membrane purification systems (specifically Millipore UFCs). But it's cheap and worth a shot. Sorry, don't have the address handy. Steve Carr From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!bio.embnet.se!sunic!mcsun!Germany.EU.net!news.netmbx.de!mailgzrz.TU-Berlin.DE! math.fu-berlin.de!ira.uka.de!howland.reston.ans.net!ux1.cso.uiuc.edu!usenet.ucs.indiana.edu!bronze.ucs.indiana.edu!jgraham From: jgraham@bronze.ucs.indiana.edu (the End) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: READ Message-ID: Date: 5 May 93 16:28:18 GMT References: <1993May4.182553.1@kean.ucs.mun.ca> Sender: news@usenet.ucs.indiana.edu (USENET News System) Organization: Indiana University Lines: 11 Nntp-Posting-Host: bronze.ucs.indiana.edu Ahh, I have invented a new technique and am marketing it as a "kit" so that all the upcoming researchers can feel comfortable using it. It is called "Rid-Oil" and causes the phases to invert upon adding it to a completed PCR reaction. Just kidding :) We call that stuff "chloroform" around here. Jim J. Graham From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!pipex!uknet!pavo.csi.cam.ac.uk!cy-mac.welc.cam.ac.uk!cyk10 From: cyk10@cus.cam.ac.uk (Chong-Yee Khoo) Newsgroups: bionet.molbio.methds-reagnts Subject: Digoxigenin and In situ Hybridisations Message-ID: <1993May5.170635.1591@infodev.cam.ac.uk> Date: 5 May 93 17:06:35 GMT Sender: news@infodev.cam.ac.uk (USENET news) Organization: Wellcome/CRC Institute, Cambridge CB2 1QR, England Lines: 18 X-Xxmessage-Id: X-Xxdate: Wed, 5 May 93 18:06:37 GMT Nntp-Posting-Host: cy-mac.welc.cam.ac.uk X-Useragent: Nuntius v1.1.1d17 I am trying to do in situ hybridisation on Xenopus tissue sections and am having very random results. Some probes will work occasionally, one always works and others do not work at all. All probes will work in wholemount but this does not mean they will work on sections. Does anyone have a protocol? Please help my supervisor is going mad, because we've been trying for months now with no consistency. ======================================================================= ======= Chong-Yee Khoo E-mail: cyk10@cus.cam.ac.uk Wellcome/CRC Institute Talk: cyk10@cy-mac.welc.cam.ac.uk Tennis Court Road Phone: +44 223 334110 Cambridge CB2 1QR Fax: +44 223 334089 From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!SERVER.UGA.EDU!mcgraw%gandal.dnet From: mcgraw%gandal.dnet@SERVER.UGA.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: Protein staining with KCl and electroelution Message-ID: <9305051350.AA27328@server.uga.edu> Date: 5 May 93 13:50:43 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 25 Hi Netfolks, Anybody out there have experience staining protein gels with KCl and then electroeluting the band you want? This method was mentioned in a paper, but no details or reference were given. Maybe this is common knowledge for some people, but obviously not for me. They did say they used 25 mM KCl to "stain." I imagine you'd get white bands, but...What's the sensitivity? Any tricks to it? Also, assuming I can see something and cut it out, what's the best way to electroelute the protein? How much protein can one hope to recover from a gel slice? Assuming it's a fairly big (20cm x 20 cm x 1 mm) gel, how much should one load? I want to use the protein for making antibodies. I'm running the gel right now. Any comments, especially about the staining, would be greatly appreciated. Thanks, Al McGraw mcgraw@bscf.uga.edu From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!mcsun!Germany.EU.net!news.netmbx.de!mailgzrz.TU-Berlin.DE!math.fu-berlin.de!ira.uka.de!howland.reston.ans.net!usenet.ins.cwru.edu!magnus.acs.ohio-state.edu!cis.ohio-state.edu!news.sei.cmu.edu!bb3.andrew.cmu.edu!andrew.cmu.edu!mm6y+ From: mm6y+@andrew.cmu.edu (Morris F. Manolson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: KCl staining for protein Message-ID: Date: 5 May 93 15:26:02 GMT Organization: Sponsored account, Biological Sciences, Carnegie Mellon, Pittsburgh, PA Lines: 28 NNTP-Posting-Host: po4.andrew.cmu.edu > >Anybody out there have experience staining protein gels with KCl and then >electroeluting the band you want? KCl is used to precipitate SDS in the gel matrix leaving regions of low SDS (ie: high protein concentration) visible as clear bands. Following SDS-PAGE, the gels (1.5 mm thick) are incubated in 2M KCl until it becomes opaque (1-2 minutes). To visualize the clear bands, the gel is placed over a black background and is illuminated from the side. The bands must be quickly cut out as they are only visible for about 1 minute. WORK FAST! To stain the gel afterwards with coomassie blue the KCl must first be washed out of the gel by a 30 minutes incubation in 10% acetic acid. I crushed up the cut out gel slices by repeated passage through an 18 gauge needle and dialysed out the SDS and KCL. Best of luck! if you need more help just call. Morrie ****************************************************************************** Morris F. Manolson Tel: 416-813-6662 (office) Division of Cell Biology 416-813-5594 (lab) Hospital for Sick Children 416-813-5028 (FAX) 88 Elm St., McMaster building email: Morrie@resunix.ri.sickkids.on.ca Toronto, Ontario, Canada M5G 1X8 From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!enterpoop.mit.edu!gatech!howland.reston.ans.net!darwin.sura.net!fconvx.ncifcrf.gov!mayhew From: mayhew@ncifcrf.gov (George Mayhew) Newsgroups: sci.bio.technology,bionet.molbio.methds-reagnts Subject: U.S.E. Mutagenesis (Pharmacia)? Summary: Need help doing mutagenesis. Keywords: mutagenesis, plasmid Message-ID: Date: 5 May 93 15:51:31 GMT Organization: Frederick Cancer Research and Development Center Lines: 15 Xref: biosci sci.bio.technology:297 bionet.molbio.methds-reagnts:5355 Hi Netters, I'm in the process of attempting several point mutations in my gene of interest. I'm having some difficulty. I was thinking about trying the new Pharmacia U.S.E mutagenesis kit, but would like to discuss it with anyone who has used it. Please Email me direct. Thanks, George Mayhew -- -------------------------------------------------------------------------- | George Mayhew | MAYHEW@NCIFCRF.GOV | MAYHEW@FCONVX.NCIFCRF.GOV | | Toxinology Div. | ------------------------------------------------ | USAMRIID, Ft. Detrick | Disclaimer: All views expressed within are my | From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!enterpoop.mit.edu!gatech!howland.reston.ans.net!wupost!medicine.wustl.edu!bcserv!eric From: eric@bcserv.WUSTL.EDU (Hugo (Eric)) Newsgroups: bionet.molbio.methds-reagnts Subject: Multisubunit protein disassembly Summary: Anyone have any ideas on dissociating subunits Keywords: GroEL, denaturation Message-ID: <1s8ip2INN9go@medicine.wustl.edu> Date: 5 May 93 14:25:38 GMT Organization: Washington University School of Medicine Lines: 14 NNTP-Posting-Host: bcserv.wustl.edu Dear Bionetters, I am looking for tips on how to disassemble a multisubiunit protein (GroEL, 14mer). Standard denaturants (urea or guanidine) will break up the complex but unfortunately the conc. needed to cause disassembly leads to denaturation of the monomer as judged by CD spectropolarimetry. Does anyone out there have any gentle protocols they have used to disassociate or depolymerize various biopolymers. Please email any replies I will be happy to forward any information. Thanks for any ideas! Eric -- Eric R. Hugo, Postdoctoral Research Associate |eric@bcserv.wustl.edu Dept. of Biochemistry and Molecular Biophysics| Washington University School of Medicine | Box 8231, St. Louis, MO 63110_________________| (314) 362-3342 From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!usenet.ins.cwru.edu!cleveland.Freenet.Edu!bl275 From: bl275@cleveland.Freenet.Edu (Dan Diaz) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Make-your-own dNTPs? Message-ID: <1s87hb$h10@usenet.INS.CWRU.Edu> Date: 5 May 93 11:13:46 GMT References: Reply-To: bl275@cleveland.Freenet.Edu (Dan Diaz) Organization: Case Western Reserve University, Cleveland, OH (USA) Lines: 23 NNTP-Posting-Host: hela.ins.cwru.edu In a previous article, horton@molbio.cbs.umn.edu (Robert Horton) says: >Dear Kit-Hating BioTechnophiles; > >It occurred to me that, since dNTPs are the second most expensive ingredient >in a PCR next to the polymerase (or third, if you buy AmpliWax!), that it might >be worth considering purifying them myself. It seems like overkill to use >the incredibly pure dNTPs that most of us buy separately, then mix back >together again. Its not like they are rare or hard to find in nature, and since >we use 'em all together, you wouldn't need to purify them from each other, >which would be the hard part, but dNTPs vs. NTPs might not be so bad (?) > There MUST be published protocols, but I haven't been able to find >any; I assume this kind of work must have been done largely before the computer >databases for lit searches... > Any ideas? Whether you hate 'kits' or not is not the point. Time is money. Get yourself 0.1M solutions of HPLC purified dNTPs from either USB or Pharmacia. I have used both in PCRs, and colleagues use them in polymerase assays. Dont re-invent the wheel. -- Dan Diaz bl275@cleveland.freenet.edu 'Querer es poder' From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!ARTHUR.CITI2.FR!URA1335 From: URA1335@ARTHUR.CITI2.FR Newsgroups: bionet.molbio.methds-reagnts Subject: (none) Message-ID: <930505111818.20406f5b@ARTHUR.CITI2.FR> Date: 5 May 93 09:18:18 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 1 this is just a test From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!warwick!dcs.warwick.ac.uk!mrccrc!dmartin From: dmartin@crc.ac.uk (David Martin x3175) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: quantitative westerns Message-ID: <1993May5.075702.17995@crc.ac.uk> Date: 5 May 93 07:57:02 GMT References: <42F19B6AA4DF006FE4@HKUMD1.HKU.HK> Sender: news@crc.ac.uk Distribution: bionet Organization: MRC Human Genome Resource Centre Lines: 13 Nntp-Posting-Host: tin The ecl signal is semi quantitative.. but you have to run a standard curve on each western.. (and make sure you spread the ECL reagent evenly.) but then again most protein assays are semi quantitative, just some are more semi than quantitative. We have put a gradient of antigen on a blot and then densitometrically scanned it. the density corresponds to the antigen in a linear sort of fashion. Suck it and see is all I can suggest. Try a gradient of your antigen and see what sort of results you get. hope this helps ...d From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!trane.uninett.no!nntp.uio.no!tomk From: tomk@ulrik.uio.no (Tom Kristensen) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Texts on Cell Culture? Message-ID: <930405.083552.tomk@pcbiokj01> Date: 5 May 93 07:35:52 GMT Organization: University of Oslo Lines: 24 NNTP-Posting-Host: pcbiokj01.uio.no >Hi, > Could someone direct me towards any good references on basic Cell/ >Tissue Culture techniques? In a couple of months I'll be starting up a >cell culture facility in a new lab so we can overexpress some protein. I've >done a bit of cell culture already, but I'm looking for stuff like how >to tell when cells are confluent, when they are (*gasp*) dead, etc. Please >bear in mind that I'm a chemist by training, so dealing with bugs is >kinda new to me. Oh, I should say I'm looking for *mammalian* cell culture >theory/ techniques. I've done quite a bit of *bacterial* cell culture, >but that's different.... > Thanks in advance for any pointers.... > >John Boswell >knight@grafton.dartmouth.edu John, One comprehensive and not too expensive possibility is R.P.L. Adams: Cell Culture for Biochemists, in the series Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier 1990 (2nd. edition). Best wishes Tom Kristensen Dept. of Biochemistry, University of Oslo tom.kristensen@biokjemi.uio.no From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!darwin.sura.net!haven.umd.edu!purdue!mentor.cc.purdue.edu!bragg.bio.purdue.edu!PMIGUEL From: pmiguel@bragg.bio.purdue.edu Newsgroups: bionet.molbio.methds-reagnts Subject: nano-pipetting Message-ID: Date: 4 May 93 23:17:34 GMT Sender: news@mentor.cc.purdue.edu (USENET News) Reply-To: pmiguel@bragg.bio.purdue.edu Organization: Purdue University, Dept. of Biological Sciences Lines: 18 A researcher in our lab is attempting to do single chromosome random PCR. Most protocols for this require primers to be ligated on the digested DNA of the chromosome after a proteinase K digestion and subsequent phenol/chloroform extraction. All of these manipulations of liquids need to be done in a volume of less than 100 nanoliters. The pipetting device we are using seems incapable of doing this. Can anyone suggest how we can make or purchase such a device? Please either post answers or mail them to PMIGUEL@BILBO.BIO.PURDUE.EDU the address in the header is incorrect. Thanks in advance, Phillip From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!wupost!waikato.ac.nz!comp.vuw.ac.nz!canterbury.ac.nz!chmeds.ac.nz!mkennedy From: mkennedy@chmeds.ac.nz (Martin Kennedy) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Texts on Cell Culture? Message-ID: <1993May5.105453.237@chmeds.ac.nz> Date: 4 May 93 22:54:53 GMT References: Lines: 42 In article , knight@grafton.dartmouth.edu (John Boswell) writes: > Hi, > Could someone direct me towards any good references on basic Cell/ > Tissue Culture techniques? In a couple of months I'll be starting up a > cell culture facility in a new lab so we can overexpress some protein. I've stuff deleted.... > Thanks in advance for any pointers.... > > John Boswell > knight@grafton.dartmouth.edu John, Try: Paul, J. (1975) Cell and Tissue Culture (Churchill-Livingstone, Edinburgh and London). Methods in Enzymology 58 (1979) Cell culture. (And I think also one of the more recent volumes has useful information) Freshney,R: Animal Cell Culture, IRL Press "Practical Approach" series - sorry I don't know which one, but I know there has been a new edition in the last 3 years or so. Freshney, R (1987) Culture of animal cells: a manual of basic techniques (Alan R Liss NY). Pollard, J.W. and Walker, J.M. (eds) Animal Cell Culture. Methods in Molecular Biology v5. Humana Press, 1989. Cheers, Martin NNNN NN Martin A Kennedy (E-mail = mkennedy@chmeds.ac.nz) ZZZZZZZ NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ NN NN NN Christchurch School of Medicine ZZZ NN NNNN Christchurch, New Zealand ZZZZZZZ From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!micro.uct.ac.za!ED From: ED@micro.uct.ac.za ("Ed Rybicki") Newsgroups: bionet.molbio.methds-reagnts Subject: waismail Message-ID: Date: 5 May 93 13:55:21 GMT Sender: daemon@net.bio.net Reply-To: ed@micro.uct.ac.za Distribution: bionet Lines: 18 What can I say....it works! Within 5 min of getting the message, I had done my first search - and within 5 min I had my first reply! Kudos, bouquets and bubbly fermented stuff to the writers/programmers/those generally responsible. ____________________________________________________________________ | Ed Rybicki, PhD | "Lord, won't you buy me | | (ed@micro.uct.ac.za) | | | Dept Microbiology | A Mer-ce-des Benz..." | | University of Cape Town | | | Private Bag, Rondebosch | | | 7700, South Africa | - Janis Joplin | | fax: 27-21-650 4023 | | -------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!zaphod.mps.ohio-state.edu!moe.ksu.ksu.edu!kuhub.cc.ukans.edu!husc-news.harvard.edu!hsdndev!nmr-z!Frodo.MGH.Harvard.EDU!FINNEY From: finney@Frodo.MGH.Harvard.EDU (Michael Finney) Newsgroups: bionet.molbio.methds-reagnts Subject: re: microtiter plate PCR? Message-ID: <1993May5.023101.25288@nmr-z.mgh.harvard.edu> Date: 5 May 93 02:31:01 GMT Sender: usenet@nmr-z.mgh.harvard.edu (User for USENET news postings) Reply-To: finney@Frodo.MGH.Harvard.EDU Organization: Mass General Hospital Lines: 44 Nntp-Posting-Host: frodo.mgh.harvard.edu Warning--I am associated with MJ Research (but I won't try to sell you anything). I have been overseeing the development of our oil-free technology. With all the recent discussion of thermal cycling of microtiter plates without oil, I thought it would be a good idea to give you all the benefits of our trials and errors. There are two things necessary to do cycling without oil: a way of keeping the top hot, and a way of sealing-in the water vapor. Without an airtight seal, every time the tube is heated from ~60 deg to ~95 deg, a good deal of wet air escapes; when the tube is cooled, dry air is sucked in to replace it. The net effect is to lose about 0.5 microliter of water per cycle (for an 0.5 ml tube). For tubes, sealing is no problem, as the caps of most brands of tubes can easily hold the 1 atm of pressure generated in heating from room temp to 90+ deg. Sealing plates is a different matter. Tape is an obvious thing to try, but we haven't found any tapes that will stand up to the rigors of cycling. We're working on this, and may have a solution in a few months. In the mean time, the only 96-well format we know of that REALLY works (as opposed to sort-of works, see below) without oil is the 0.2 ml tubes (in our machine or Perkin-Elmer's). In some cases, it may be enough to have a system that sort-of works. If your plates don't have a good vapor seal, you may still be able to get the loss per cycle down to the point where it is tolerable. Use a plate with small-volume wells, start with large liquid volumes, use a low denaturation temperature, and don't do excessive numbers of cycles. Also, if the lid retains a lot of the wet air, so the wet air is sucked back in to the wells when the plate cools, then there will be less loss. But you will probably find more loss from edge wells, and therefore get uneven results. Finally, if anyone tries heating the tops of tubes with an iron, I have a couple of suggestions. First, the iron should be set to 100 to 105 deg. It has to be hot enough so that no condensation will form anywhere in the tube. Polypropylene tubes can take the heat with no problem. You may have difficulty measuring the surface temp, though--I leave that to you. Second, build a simple insulated chamber around the tubes. There's no way it will work if breezes can get to the tubes from the sides. Cheers, Mike Finney From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: IBELGAUFTS@vms.biochem.mpg.de Newsgroups: bionet.molbio.methds-reagnts Subject: human CD40 mAb source?? Message-ID: <1993May5.123926.21261@gserv1.dl.ac.uk> Date: 5 May 93 12:20:36 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 5 Original-To: methods@uk.ac.daresbury Original-Sender: IBELGAUFTS@de.mpg.biochem.vms dear netters does anyone know a source for monoclonal antibodies directed against human CD40 antigen? thanks Horst From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!mcsun!uunet!news.univie.ac.at!alijku11!aci.cvut.cs!mvax.uakom.cs!tuzvonet.so!tibor From: tibor@tuzvonet.so (Tibor WEIS,network manager) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: linear DNA Xformation Summary: Reference Message-ID: Date: 4 May 93 16:02:14 GMT References: <1993Apr19.094503.26490@gserv1.dl.ac.uk> Distribution: bionet,world Organization: Computer Center TU ZVOLEN Lines: 56 Nntp-Posting-Host: decpc.tuzvo.cs In article pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) writes: >Relay-Version: VMS News - V6.1B5 17/9/92 VAX/VMS V5.4-1; site mvax.uakom.cs >Path: mvax.uakom.cs!aci.cvut.cs!alijku11!news.univie.ac.at!scsing.switch.ch!ira.uka.de! > sol.ctr.columbia.edu!zaphod.mps.ohio-state.edu!darwin.sura.net!fconvx.ncifcrf.gov!fcsparc6!pnh >Newsgroups: bionet.molbio.methds-reagnts >Subject: Re: linear DNA Xformation >Message-ID: >From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) >Date: Mon, 19 Apr 1993 23:30:10 GMT >Sender: Paul N. Hengen >References: <1993Apr19.094503.26490@gserv1.dl.ac.uk> >Distribution: bionet >Organization: Frederick Cancer Research and Development Center >Summary: Reference >Nntp-Posting-Host: fcsparc6.ncifcrf.gov >Lines: 40 > >IBELGAUFTS@de.mpg.biochem.vms wrote: > >: Are there any reasons why transformation of bacteria with linear DNA >: should not work? Does it work? > >Mike Poidinger wrote: > >> It depends on why you want to do it. > >> My understanding is that Eukaryotic Xformations are done >> with linear DNA to improve the eficiency of integration into >> the chromosome. Since most bacterial Xformations involve >> autonomously replicating plasmids, linearizing the >> DNA would not serve much purpose. And if you actually wanted >> chromosomal integration, I would guess there are better ways >> than Xformation with linear DNA and then hoping :-) > >It could be you would like to force an integration or perhaps >cause a specific mutation within the bacterial chromosome. > >See the following article for a method to do such a thing: > >@article{Winans1985, >author = "S. C. Winans > and S. J. Elledge > and J. H. Krueger > and G. C. Walker", >title = "Site-directed insertion and deletion mutagenesis >with cloned fragments in Escherichia coli", >journal = "J. Bacteriol.", >volume = "161", >pages = "1219-1221", >year = "1985"} > > >Paul N. Hengen >National Cancer Institute >Frederick Cancer Research and Development Center >Frederick, Maryland 21702-1201 USA From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!biology.ucsc.edu!ludwig From: ludwig@biology.ucsc.edu (Bob Ludwig) Newsgroups: bionet.molbio.methds-reagnts Subject: zymolyase Message-ID: <9305051822.AA17467@biology.UCSC.EDU> Date: 5 May 93 18:22:11 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 16 Does anyone know of a vendor for zymolyase to protoplast yeast with a reasonable price? The best we can find is ICN, which wants $380 for 250 mg. Failing that, is their a zymolyase alternative wwwith which to protoplast yeast? Thanks for your input. Bob Ludwig Prof. molecular biology & biochemistry Sinsheimer Laboratories University of California Santa Cruz, CA 95064 USA From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!pipex!uunet!utcsri!utnut!torn!csd.unb.ca!news.ucs.mun.ca!engr!mhaddara From: mhaddara@engr Newsgroups: bionet.general,bionet.molbio.methds-reagnts Subject: ATCC on-line ( question) Message-ID: <1993May5.193223.1@engr> Date: 5 May 93 19:32:23 GMT Sender: usenet@news.ucs.mun.ca (NNTP server account) Organization: Memorial University of Newfoundland Lines: 15 Xref: biosci bionet.general:4766 bionet.molbio.methds-reagnts:5365 Hello everyone, I remember seeing a while ago, an on-line catalogue of ATCC. I have been unable to re-locate this catalogue in any of the gophers I've visited. I am trying to find a cell-line which is derived from 293s, but also contains lacI. The only one I've seen so far is A4/4 , but any other suggestions are most welcome ... Thanx in advance, Cheers, Wael Dept of Biology McMaster University From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!utcsri!newsflash.concordia.ca!mizar.cc.umanitoba.ca!hamel From: hamel@ccu.umanitoba.ca (Andre Hamel) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: microtiter plate PCR? Message-ID: Date: 6 May 93 01:08:22 GMT References: <01GXLKDMCM76000KRL@GW.AGR.CA> Sender: news@ccu.umanitoba.ca Distribution: bionet Organization: University of Manitoba, Winnipeg, Manitoba, Canada Lines: 23 Nntp-Posting-Host: antares.cc.umanitoba.ca In article <01GXLKDMCM76000KRL@GW.AGR.CA> EM609MBCPHL@NCCCOT7.AGR.CA writes: >(Sorry about that last partial message.) >I haven't seen anyone do PCR in microtiter plates. What machine(s) >can you use for this? a Perkin Elmer 9600? There's MANY machines ... MJR, Corbett, BioTherm to name a few. SOme are heat block, others cycling oven. >Pat Covello >Central Plant Health Lab >Agriculture Canada ---------------------------------------------------------------------- Andre Hamel email: hamel@ccu.umanitoba.ca Manitoba Veterinary Services lab tel.: (204) 945-7630 Infectious & Genetic Disease home tel.: (204) 275-1204 Mol.Biol.Lab FAX: (204) 945-8062 545 University Crescent Winnipeg, Manitoba CANADA R3T 5S6 ---------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!CBR.DWE.CSIRO.AU!MHOLLAND From: MHOLLAND@CBR.DWE.CSIRO.AU (Michael Holland) Newsgroups: bionet.molbio.methds-reagnts Subject: RE: Protein staining with KCl and electroelution Message-ID: <930506082651.21688@cbr.dwe.csiro.au> Date: 6 May 93 13:26:51 GMT Sender: kristoff@net.bio.net Reply-To: MHOLLAND@cbr.dwe.csiro.au (Michael Holland) Distribution: bionet Lines: 10 We have used this method successfully for the exact purpose you intend. The gel itself stains white and the proteins stay clear. The original procedure is described in an Analytical Biochem article from 1989 which, of course, I now can't find. Souuld be easy to find if you look under negative staining. Promega sells for an excessive price a kit which does the same thing. We recovered 4ug og protein from a complex mixture with about 50-60% recovery using an Amicon eluting device. The other trick is do a Western and Amido black stain then cut your bands and dissolve the nitrocellulose in DMSO add your adjuvant and inject. This works relly well and your staining and elution is one step with better recovery. Let me know how you go! Good luck Michael Holland From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!usenet.ins.cwru.edu!po.CWRU.Edu!ccy From: ccy@po.CWRU.Edu (Cheung C. Yue) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: zymolyase Message-ID: <1s9f4o$mf2@usenet.INS.CWRU.Edu> Date: 5 May 93 22:29:44 GMT References: <9305051822.AA17467@biology.UCSC.EDU> Reply-To: ccy@po.CWRU.Edu (Cheung C. Yue) Organization: Case Western Reserve University, Cleveland, OH (USA) Lines: 23 NNTP-Posting-Host: slc5.ins.cwru.edu In a previous article, ludwig@biology.ucsc.edu (Bob Ludwig) says: > >Does anyone know of a vendor for zymolyase to protoplast yeast with a >reasonable price? The best we can find is ICN, which wants $380 for >250 mg. > >Failing that, is their a zymolyase alternative wwwith which to >protoplast yeast? > Some years back I needed to extract DNA from a bunch of fungi and stumbled upon Mureinase which is available from USB. Price is considerably better than what you quoted. One minor problem seems to be degradation of DNA in the presence of magnesium although we never carefully sorted that out. I did not use it to protoplast yeast, but I gather it is possible with this enzyme mixture. I also vaguely recall reading somewhere that one of the active ingredient has been cloned, but I may be wrong. C Cho Yue -- From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!enterpoop.mit.edu!gatech!howland.reston.ans.net!usc!wupost!medicine.wustl.edu!wums.wustl.edu!wetsel_r From: wetsel_r@wums.wustl.edu Newsgroups: bionet.molbio.methds-reagnts Subject: RNase protection assays... Message-ID: <5MAY93.11573872@wums.wustl.edu> Date: 5 May 93 17:57:38 GMT Organization: MIT PLASMA FUSION CENTER Lines: 28 NNTP-Posting-Host: msnews.wustl.edu Hi Netters: I noticed that in addition to Ambion, Clonetech is now offering an RNase protection kit called the Guardian. Like Ambion's kit, literature from both company's is quite vague at the step where inactivation of the RNases takes place. (Sounds like proprietary kit information again!) Having done a number of protection assays, I'd love to avoid using protease-K and SDS in the assay, once precipitated neither disolve in anything very well. Has anyone ever just precipitated the double stranded RNA after the RNase treatment step with ethanol or isopropanol? I thought of trying a PEG PPT but realized that it wouldn't work as PEG won't PPT anything below 120-130 base pairs, even though it *may* inactivate the RNases. Does anyone familiar with RNase Protection assays have any thoughts to offer to get around not using protease-K and SDS? Anyone using either the Ambion or Clonetech kits care to "spill the beans" at this particular step? Thanks in advance... David haviland@kids.wustl.edu =========================================================================== + David L. Haviland, Ph.D. Internet:"haviland@kids.wustl.edu" + + Washington Univ. School of Med. A.K.A : The Compiler + + Dept. of Peds./Pulm. Box 8116 ICBM-Net : Just hit St. Louis + + 400 S. Kingshighway &-6 <- User is Brain Dead... + + St. Louis, MO 63110 FAX: 314-454-2476 + + (314) 454-6076 + =========================================================================== From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!s.u-tokyo!kuis!wnoc-kyo!sh.wide!sun-barr!cs.utexas.edu!usc!howland.reston.ans.net!darwin.sura.net!news-feed-1.peachnet.edu!gatech!destroyer!news.itd.umich.edu!usenet From: btroen@uv1.im.med.umich.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Sequencing primer for CAT? Message-ID: <1s9c5f$1lp@terminator.rs.itd.umich.edu> Date: 5 May 93 21:38:55 GMT References: <1s3dth$8ln@max.physics.sunysb.edu> Distribution: bionet Organization: University of Michigan Lines: 29 NNTP-Posting-Host: btroen.im.med.umich.edu In article <1s3dth$8ln@max.physics.sunysb.edu> mhollowa@ic.sunysb.edu (Michael Holloway) writes: >Does anyone know of a commercially available primer for sequencing upstream of >CAT in a reporter construct? I KNOW I seen one somewhere and I can't find >it in any catalog now (Grrrr). > >Lacking that, does anyone have any experience with a sequence at the >5' end of CAT that can be successfully used for priming? > >Thanks in advance. > >Mike > Mike - I have great success performing primer extensions and sequencing of CAT with an oligo called PR-1 (Pellegrino Rossi is the fellow who found that this worked very well). PR-1 5' - T G C C A T T G G G A T A T A T C A A C G G T G - 3' BP 4937-4960 of pSV2.CAT, anti-sense and heads upstream. Bruce Troen From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!HELIX.MGH.HARVARD.EDU!PECHAN From: PECHAN@HELIX.MGH.HARVARD.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: Hybridoma Bank Message-ID: <01GXTNW142J68WWSHA@HELIX.MGH.HARVARD.EDU> Date: 5 May 93 22:06:08 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 8 Dear Netters: Does anybody know the address of "Developmental Studies Hybridoma Bank" which is located in the States. For every message I would be very thankful. Peter Pechan Neurosci.Ctr,MGH Charlestown, MA PECHAN@helix.mgh.harvard.edu From owner-methds-reagnts@net.bio.net Tue May 04 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!mcsun!uknet!bnr.co.uk!bnrgate!nott!torn!howland.reston.ans.net!zaphod.mps.ohio-state.edu!magnus.acs.ohio-state.edu!cis.ohio-state.edu!news.sei.cmu.edu!bb3.andrew.cmu.edu!andrew.cmu.edu!mm6y+ From: mm6y+@andrew.cmu.edu (Morris F. Manolson) Newsgroups: bionet.molbio.methds-reagnts Subject: re: zymolyase Message-ID: <0fu1kP_00Uh_45yV8L@andrew.cmu.edu> Date: 5 May 93 19:57:15 GMT Organization: Sponsored account, Biological Sciences, Carnegie Mellon, Pittsburgh, PA Lines: 23 NNTP-Posting-Host: po4.andrew.cmu.edu >Does anyone know of a vendor for zymolyase to protoplast yeast with a >reasonable price? The best we can find is ICN, which wants $380 for >250 mg. No, just bargain with your ICN sales rep as best you can >Failing that, is their a zymolyase alternative wwwith which to >protoplast yeast? For large scale bichemical preps (ie: membrane preps, etc) where the protoplast are washed rapidely before using, (and where the cost would be prohibitive for one experiment with zymolyase) I use sigma's Lyticase cat#L8012. For more sensitive experiments like immunoflouresence, I still use ICN's zymolyzase. Hope this helps, Morrie ****************************************************************************** Morris F. Manolson Tel: 416-813-6662 (office) Division of Cell Biology 416-813-5594 (lab) Hospital for Sick Children 416-813-5028 (FAX) 88 Elm St., McMaster building email: Morrie@resunix.ri.sickkids.on.ca Toronto, Ontario, Canada M5G 1X8 From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!pipex!uknet!pavo.csi.cam.ac.uk!tjrc1 From: tjrc1@cus.cam.ac.uk (T.J.R. Cutts) Newsgroups: bionet.molbio.methds-reagnts Subject: CHO/human synkaryon karyotyping Keywords: hybrids, karyotype Message-ID: <1993May6.092140.17796@infodev.cam.ac.uk> Date: 6 May 93 09:21:40 GMT References: <1993May6.091025.17243@infodev.cam.ac.uk> Sender: news@infodev.cam.ac.uk (USENET news) Organization: U of Cambridge, England Lines: 6 Nntp-Posting-Host: apus.cus.cam.ac.uk I have some CHO/human hybrid cell lines, each with a full complement of CHO chromosomes and around 11 human chromosomes. I need to find a simple technique whereby I can stain chromosome spreads to determine how homogenous the karyotype is throughout the population. Any suggestions? Tim. From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!WORLDLINK.COM!wk01677 From: wk01677@WORLDLINK.COM (Condie E. Carmack) Newsgroups: bionet.molbio.methds-reagnts Subject: Polka dot background Message-ID: <9305070002.AA08952@worldlink.worldlink.com> Date: 7 May 93 00:02:59 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 10 I have been screening phage lifts on NC filters with kinased oligos and have been getting a lot of "polka dot" background. Does any one know what causes this and how to avoid it. I never get this with RNA or DNA probes. Condie E. Carmack ==================================================== GenPharm International ==================================================== 297 North Bernardo Avenue \/ Mountain View, CA 94043 ================= ================================= (415) 988-2434 ==================================================== From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!pipex!uknet!pavo.csi.cam.ac.uk!tjrc1 From: tjrc1@cus.cam.ac.uk (T.J.R. Cutts) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Ethidium Bromide mutagenesis Keywords: Ethidium bromide, mutation, mutagenesis Message-ID: <1993May6.091025.17243@infodev.cam.ac.uk> Date: 6 May 93 09:10:25 GMT References: Sender: news@infodev.cam.ac.uk (USENET news) Organization: U of Cambridge, England Lines: 6 Nntp-Posting-Host: apus.cus.cam.ac.uk I would imagine that EtBr mutagenesis occurs by a similar mechanism to most intercalating agents, in that it probably interferes with transcription and replication complexes, resulting in formation of DNA strand breaks. I don't have any references though... Tim. From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!usenet.ins.cwru.edu!magnus.acs.ohio-state.edu!zaphod.mps.ohio-state.edu!sol.ctr.columbia.edu!ira.uka.de!gmd.de!dearn!barilvm!vms.huji.ac.il!agri!marder From: marder@agri.huji.ac.il Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Protein staining with KCl and electroelution Message-ID: <1993May6.095444.1@agri.huji.ac.il> Date: 6 May 93 08:40:22 GMT References: <9305051350.AA27328@server.uga.edu> Distribution: bionet,world Organization: The Hebrew University of Jerusalem - Faculty of Agriculture Lines: 49 Nntp-Posting-Host: agri.huji.ac.il In article <9305051350.AA27328@server.uga.edu>, mcgraw%gandal.dnet@SERVER.UGA.EDU writes: > > Hi Netfolks, > > Anybody out there have experience staining protein gels with KCl and then > electroeluting the band you want? > > This method was mentioned in a paper, but no details or reference were > given. Maybe this is common knowledge for some people, but obviously > not for me. They did say they used 25 mM KCl to "stain." I imagine > you'd get white bands, but...What's the sensitivity? Any tricks to it? The white bands are due to localised precipitation of SDS. The technique is not very sensitive, and to see the band, I find that the best way is to put the gel on a BLACK background and illuminated from an oblique angle. BTW, the band will only be visible for a short time! Actually, my own experience is with the reverse procedure using 4M Sodium acetate to stain. The protein bands are clear against a white background. > > Also, assuming I can see something and cut it out, what's the best way > to electroelute the protein? One way is to equilibrate in electrophoresis sample buffer (you can drop the SDS conc. and omit bromophenol and glycerol) and then elute in Tris-Glycine running buffer. Put the gel strip in a dialysis bag, lay it across a flat-bed electrophoresis setup and give it 30-60 minutes at up to 10 Volts per cm (between electrodes). When you are done, reverse the field for about 30 seconds (to unstick protein from the dialysis bag) and empty the contents of the bag into a test tube. You may want to precipitate the protein with 10 per cent TCA. > > How much protein can one hope to recover from a gel slice? Assuming it's > a fairly big (20cm x 20 cm x 1 mm) gel, how much should one load? You could probably get up to 1 mg. > > I want to use the protein for making antibodies. I'm running the gel > right now. Any comments, especially about the staining, would be > greatly appreciated. > > Thanks, > > Al McGraw > mcgraw@bscf.uga.edu -- ' Jonathan B. Marder Internet: MARDER@AGRI.HUJI.AC.IL | Department of Agricultural Botany Bitnet: MARDER@HUJIAGRI | /\/ The Hebrew University of Jerusalem Phone: (08 or +9728) 481918 |/ \ Faculty of Agriculture Fax: (08 or +9728) 467763 / P.O.Box 12, Rehovot 76100, ISRAEL From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!zaphod.mps.ohio-state.edu!saimiri.primate.wisc.edu!zazen!post.its.mcw.edu!not-for-mail From: dmeier@post.its.mcw.edu (Daniel Meier) Newsgroups: bionet.molbio.methds-reagnts Subject: EtBr_Southern_Summary Message-ID: <1s9n07INNrct@post.its.mcw.edu> Date: 6 May 93 00:43:51 GMT Organization: Medical College of Wisconsin (Milwaukee, WI) Lines: 82 NNTP-Posting-Host: post.its.mcw.edu X-Newsreader: TIN [version 1.1 PL7] Hello All, My original question referred to whether ethidium bromide stain on nylon will interfere with subsequent hybridization. Fortunately, the following cognoscenti agreed that the stain would not be a problem. My thanks to them. Dan Meier A summary of responses follows: From bsh@med.pitt.edu Mon Apr 26 11:06:59 1993 Hi Dan: I have had this happen but it will not in any way affect your hybridizations. It just gets washed out during your hyb and other procedures. Regards. Raj. From nash@biologysx.lan.nrc.ca Mon Apr 26 12:27:30 1993 G'day, I just ignore the EtBr, and it seems to ignore me (i.e. it doesn't appear to interfere with anything). I wouldn't change anything unless I have a signal problem. cheers, John John Nash | Email: Nash@biologysx.lan.nrc.ca. Institute for Biological Sciences, | National Research Council of Canada, Cell Physiology Group. | Ottawa, Ontario, Canada. *** Disclaimer: All opinions are mine, not NRC's! *** From <@PUCC.PRINCETON.EDU:BMHTLAI@TWNAS886.BITNET> Mon Apr 26 16:34:08 1993 Hi! Despite that I do not know the theoretical basis, we are using a positively charged Nylon membrane GeneScreen Plus and we are routinely running gel with EtBr, blotting and seeing EtBr on the membrane. So far, our hybridization have not some seriously problem. Hence, I think you need not worry about it. Good Luck! Sincerely, Hsing-Tsu Lai From mayhew@fconvx.ncifcrf.gov Mon Apr 26 23:34:42 1993 Dan, How much EtBr were you using? It sounds to me like you may have over- stained your gel. Try using less and staining a wee bit longer. I doubt that you will get it off the membrane. > Will it interfere with subsequent hybridization? No. But if you are using a colourimetric development, it will look horrid. Radioactive or chemiluminescent reactions should be unaffected. > Is the problem worse with nylon vs. nitrocellulose? Don't know. Oops..lost the rest. The only major difference I have noticed about Nytran vs NC is that Nytran has a much higher background, esp. with colourimetric and chemiluminescent development. BMBs kit seems very prone to high background. Amersham's ECL has a high background as well, esp when using their Hybond+ (Nytran-like) membranes. Hope this helps, George George Mayhew | MAYHEW@NCIFCRF.GOV | From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!darwin.sura.net!sgiblab!munnari.oz.au!uniwa!kun.microbiol.uwa.edu.au!morag From: morag@arbo.microbiol.uwa.edu.au (Morag Lancaster) Newsgroups: bionet.molbio.methds-reagnts Subject: Dounce homogenizer Message-ID: Date: 6 May 93 02:29:33 GMT Organization: Dept of Microbiology, UWA Lines: 3 NNTP-Posting-Host: kun.microbiol.uwa.edu.au Hi, does anyone know where a Dounce homogenizer can be purchased or if a similar piece of equipment can be used to perform the same task? From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!utcsri!newsflash.concordia.ca!sifon!monod!francis From: francis@monod.Biol.McGill.CA (Francis Ouellette) Newsgroups: bionet.general,bionet.molbio.methds-reagnts Subject: Re: ATCC on-line ( question) Message-ID: Date: 6 May 93 02:04:12 GMT References: <1993May5.193223.1@engr> Sender: news@sifon.cc.mcgill.ca Organization: McGill University Lines: 87 Xref: biosci bionet.general:4767 bionet.molbio.methds-reagnts:5373 mhaddara@engr writes: > I remember seeing a while ago, an on-line catalogue of ATCC. I have >been unable to re-locate this catalogue in any of the gophers I've visited. Dear Wael there is a lot of ATCC info on gopher ... you have to loot at the right place :-) gopher merlot.welch.jhu.edu (the mother of biology gophers! [fly.bio.indiana.edu is the father :-]) --> 17. Usenet News and FAQs/ --> 5. Search messages to Usenet Newsgroups/ --> 2. Search Bionet articles (IG) use "atcc" as a search word, and get 123 hits ... --> 1. danj@welch Re: Re: ATCC info ?. 2. WMELCHIOR@ Re: ATCC internet access. 3. LDN@CU.NIH Re: ATCC online. 4. howard.kat Re: ftp'able atcc lists. 5. fgarbrec@P Re: Re: ATCC info ?. 6. dan@cubmol Re: ATCC online?. 7. pnh@fcspar Re: Re: ATCC online?. 8. SHICKLEY@V Re: Re: ATCC online?. 9. ZWIEB%JASO Re: PCR of ATCC samples. 10. p3ay@vax5. Re: Are ATCC listings on line?. 11. toms@fcs26 Re: Re: ATCC online. 12. eddy@mbcf. Re: Looking for FTP'able ATCC catalog.. the beginning of the first one reads: (another person who did not know how to use Gopher :-) Path: biosci!agate!ames!elroy.jpl.nasa.gov!sdd.hp.com!cs.utexas.edu!uunet!haven.umd.edu!darwin.sura.net!welchgate.welch.jhu.edu!danj From: danj@welchgate.welch.jhu.edu (Dan Jacobson) Newsgroups: bionet.general Subject: Re: ATCC info ? Message-ID: <1993Jan15.194021.23160@welchgate.welch.jhu.edu> Date: 15 Jan 93 19:40:21 GMT References: <1993Jan15.001929.251@midway.uchicago.edu> Distribution: usa Organization: Johns Hopkins Univ. Welch Medical Library Lines: 416 In article <1993Jan15.001929.251@midway.uchicago.edu > jm68@midway.uchicago.edu writes: >Help! Does anyone have an e-mail address for the American Type >Culture Collection (ATCC)? Or, better yet, a way to access >their catalog via Internet or dial-up? A hunt via Gopher was >fruitless..... Ah, you just didn't search the right part of gopher :-). You can search all the articles that have ever been posted to bionet by wais or gopher. Remembering that this question has come up before a search for a search for ATCC yields the following article posted by Bill Melchior last July. Best of luck, Dan Jacobson danj@welchgate.welch.jhu.edu =============== my $0.02 ... f. -- | B.F. Francis Ouellette * francis@monod.biol.mcgill.ca * | | "Je cherche a` comprendre" Jacques Monod From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!utcsri!newsflash.concordia.ca!mizar.cc.umanitoba.ca!hamel From: hamel@ccu.umanitoba.ca (Andre Hamel) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: microtiter plate PCR? Message-ID: Date: 6 May 93 01:22:10 GMT References: <1993May5.023101.25288@nmr-z.mgh.harvard.edu> Sender: news@ccu.umanitoba.ca Organization: University of Manitoba, Winnipeg, Manitoba, Canada Lines: 28 Nntp-Posting-Host: antares.cc.umanitoba.ca THANKS SO MUCH for sharing benefit of your experience ... nice to see from commercially associated folks who are obviously committed to the research field in general! >Warning--I am associated with MJ Research (but I won't try to sell you >anything). I have been overseeing the development of our oil-free >technology. With all the recent discussion of thermal cycling of >microtiter plates without oil, I thought it would be a good idea to give >you all the benefits of our trials and errors. >Mike Finney Thanks Mike! regards ---------------------------------------------------------------------- Andre Hamel email: hamel@ccu.umanitoba.ca Manitoba Veterinary Services lab tel.: (204) 945-7630 Infectious & Genetic Disease home tel.: (204) 275-1204 Mol.Biol.Lab FAX: (204) 945-8062 545 University Crescent Winnipeg, Manitoba CANADA R3T 5S6 ---------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!utcsri!newsflash.concordia.ca!mizar.cc.umanitoba.ca!hamel From: hamel@ccu.umanitoba.ca (Andre Hamel) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: pcr in microtiter plates Message-ID: Date: 6 May 93 01:15:16 GMT References: <9305041654.AA28952@atgenome.bio.upenn.edu> Sender: news@ccu.umanitoba.ca Distribution: bionet Organization: University of Manitoba, Winnipeg, Manitoba, Canada Lines: 23 Nntp-Posting-Host: antares.cc.umanitoba.ca >Can anyone recommend a good protocol for doing PCR in microtiter >plates in a BIOS biocycler PCR oven? If possible I would like >to keep the sample volume down, and that's >where the problem is - 20 ul tends to just wet the bottom of >the wells in the microtiter plates I have tried. The answer >would be microtiter plates with much narrower wells. Does >anyone know where these can be obtained? Any help will be >gratefully received. Please post responses if/when you get any ... I, and surely others, are interested in finding this out too! :-) cheers! ---------------------------------------------------------------------- Andre Hamel email: hamel@ccu.umanitoba.ca Manitoba Veterinary Services lab tel.: (204) 945-7630 Infectious & Genetic Disease home tel.: (204) 275-1204 Mol.Biol.Lab FAX: (204) 945-8062 545 University Crescent Winnipeg, Manitoba CANADA R3T 5S6 ---------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!anu.edu.au!Klaus.Matthaei From: Klaus.Matthaei@anu.edu.au Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Polka dot background Message-ID: <9305070037.AA26153@cscgpo.anu.edu.au> Date: 7 May 93 00:39:58 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 28 >I have been screening phage lifts on NC filters with kinased oligos and have >been getting a lot of "polka dot" background. Does any one know what causes >this and how to avoid it. I never get this with RNA or DNA probes. > > Condie E. Carmack ==================================================== > GenPharm International ==================================================== >297 North Bernardo Avenue \/ > Mountain View, CA 94043 ================= ================================= > (415) 988-2434 =================================================== Hi My guess would be excessively high levels of unincorporated nucleotides. Do you check your level of incorporation?? I have a rapid method for radiolabelled probes using a PEI tlc plate if you would like. Cheers, Klaus -------------------------------------------------------------------------- Klaus Matthaei Gene Targeting The John Curtin School of Medical Research The Australian National University E-mail: Klaus.Matthaei@anu.edu.au "If you don't do: you rust" -------------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pavo.csi.cam.ac.uk!tjrc1 From: tjrc1@cus.cam.ac.uk (T.J.R. Cutts) Newsgroups: bionet.molbio.methds-reagnts Subject: Enzymatic assay for deoxyribonucleotides? Keywords: nucleotides Message-ID: <1993May6.093558.18404@infodev.cam.ac.uk> Date: 6 May 93 09:35:58 GMT References: <1993May6.091025.17243@infodev.cam.ac.uk> Sender: news@infodev.cam.ac.uk (USENET news) Organization: U of Cambridge, England Lines: 22 Nntp-Posting-Host: apus.cus.cam.ac.uk I am trying to assay the levels of deoxypurine nucleotides in mammalian cells, following the method outlined in (1, 2). The assay works by incoroprating labelled nucleotides into DNA (using Pol I), the limiting factor being your supplied sample of one nucleotide, so if you're measuring dATP, you use a poly[dA.dT] template with hot dTTP. The amount of dATP in your sample is thus proportional to the amount of labelled DNA produced, once precipitated, washed and counted. The assay also includes AMP to inhibit any phosphatases. At present I am trying to get some standard curves before attempting to measure cell extracts, with little success. I end up with large amounts of label on the filters, even if no dATP is supplied. I'm using 3MM filters, and washing three times with 5%TCA/1%Na4P2O7, and then twice with 95% ethanol, all washes ice cold. Has anyone tried this assay and got it to work? Thanks all... Tim. References: 1. Hunting D, Henderson JF (1982) Methods Cancer Res 20 p. 245 2. Wilkinson YA, McKenna PG (1989) Leukemia Research 13/7 pp. 615-620 From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!micro.uct.ac.za!ED From: ED@micro.uct.ac.za ("Ed Rybicki") Newsgroups: bionet.molbio.methds-reagnts Subject: Re: READ Message-ID: Date: 6 May 93 07:30:39 GMT Sender: daemon@net.bio.net Reply-To: ed@micro.uct.ac.za Distribution: bionet Lines: 30 > From: jgraham@bronze.ucs.indiana.edu (the End) > Subject: Re: READ > I have invented a new technique and am marketing it as a "kit" so that > all the upcoming researchers can feel comfortable using it. It is called > "Rid-Oil" and causes the phases to invert upon adding it to a completed > PCR reaction. > > Just kidding :) We call that stuff "chloroform" around here. ...so what you waiting for? Market same, with alternative "Sorb-Oil" (=Parafilm), at $US100-00 the kit, guaranteed for 100 PCR reaction tubes. Or let ProAmStraUSPharBoeh GmBH Ltd do it for you, at $US200..... Don't you people know ANYTHING??? Disclaimer: I don't work ____________________________________________________________________ | Ed Rybicki, PhD | "Lord, won't you buy me | | (ed@micro.uct.ac.za) | | | Dept Microbiology | A Mer-ce-des Benz..." | | University of Cape Town | | | Private Bag, Rondebosch | | | 7700, South Africa | - Janis Joplin | | fax: 27-21-650 4023 | | -------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!NCBI.NLM.NIH.GOV!kristoff From: kristoff@NCBI.NLM.NIH.GOV (Dave Kristofferson) Newsgroups: bionet.molbio.methds-reagnts Subject: test of methods@net.bio.net - please ignore Message-ID: Date: 6 May 93 23:04:46 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 1 Checking the mailing address. Please ignore. From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!parc!decwrl!decwrl!elroy.jpl.nasa.gov!usc!howland.reston.ans.net!gatech!news-feed-1.peachnet.edu!bogus.sura.net!udel!sbcs.sunysb.edu!max.physics.sunysb.edu!mhollowa From: mhollowa@ic.sunysb.edu (Michael Holloway) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Large scale plasmid preps Message-ID: <1sbir2$rcn@max.physics.sunysb.edu> Date: 6 May 93 17:45:06 GMT References: <1993Apr27.160558.29038@ncsu.edu> <1993Apr30.200335.20206@ohsu.edu> Organization: State University of New York at Stony Brook Lines: 29 NNTP-Posting-Host: engws5.ic.sunysb.edu In article <1993Apr30.200335.20206@ohsu.edu> nishir@ohsu.edu writes: >In article <1993Apr27.160558.29038@ncsu.edu> jnppo@unity.ncsu.edu (James N >Petitte) writes: >> >>Greetings, >> >>Does anyone have experience with Qiagen's large scale plasmid prep columns? >>What are some of the realistic yields that can be expected? Is it useful for >>transfections? How clean is it? Are there any other methods beside CsCl >>that are equivalent? Look at Biotechniques 14(4). Two apparently different German groups show 3 and 4 fold better efficiency with Qiagen preps in transfections and that the Qiagen prep has much nicer looking circular plasmids than CSCl under electron microscopy. Qiagen just happens to be a German company. (Hmmmm...). I've experience with the Qiagen Maxi columns. They work as indicated and give enough of a yield to do a number of transfections with but the high volume of effluent is difficult to work with. They are damn expensive. I briefly reviewed expenses of materials in comparing it to CsCl without figuring in the cost of the ultracentrifuge and its upkeep. The Qiagen is easily twice as expensive. I've decided that at those prices, and since we already have an ultracentrifuge taking up space in the lab, it doesn't make any sense to keep shelling out megabucks for the Qiagen. With the Qiagen product available though, it wouldn't make any sense to buy an ultracentri- fuge for the expressed purpose of doing plasmid preps. Mike From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!relay.the.net!SHAUN%JASON.DECNET From: SHAUN%JASON.DECNET@relay.the.net ("Shaun D. Black") Newsgroups: bionet.molbio.methds-reagnts Subject: RE:Use of KCl for PAGE staining Message-ID: <01GXUU6XH88Y000EJT@nic.the.net> Date: 6 May 93 19:24:57 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 13 I thought I might add one further item to Al McGraw's question on KCl as a reagent for SDS-PAGE staining to recover protein for use as an antigen. There was a recent report in BioTechniques (12:564-573 (1992)) entitled "Reverse Staining of Sodium Dodecy Sulfate Po;yacrylamide Gels by Imadazole -Zinc Salts: Sensitive Detection of Unmodified Proteins". I haven't used the method, but their data look quite good. The stain uses readily accessible reagents, has very low background, is rapid, is as sensitive as a silver stain, is reversible, and can be used with Western Blotting and Edman sequencing. Let us know if anyone finds this useful. Cheers. -Shaun =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= = Shaun D. Black, PhD | Internet: shaun%jason.decnet@relay.the.net = = Dept. of Biochemistry | University of Texas Health Center at Tyler = =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!parc!decwrl!decwrl!sdd.hp.com!saimiri.primate.wisc.edu!zaphod.mps.ohio-state.edu!menudo.uh.edu!Elroy.UH.EDU!BCHS1B From: bchs1b@Elroy.UH.EDU (Michael Benedik) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: lac promoter Message-ID: <1sbfe6$h6l@menudo.uh.edu> Date: 6 May 93 16:47:02 GMT References: <1993May4.085251.25461@gserv1.dl.ac.uk> Reply-To: bchs1b@Elroy.UH.EDU Distribution: bionet Organization: University of Houston Lines: 18 NNTP-Posting-Host: elroy.uh.edu In article <1993May4.085251.25461@gserv1.dl.ac.uk>, vioque@cica.es (Agustin Vioque) writes: >Does anyone know if the lac promoter/operator sequence in pUC19 and derivatives >is the lacUV5 mutant or the wild type? The lacUV5 is not subject to catabolite >repression and thus can be fully induced in the presence of glucose. I want to >know if I have to change the carbon source for full induction of the promoter. >Thanks.Agustin. >vioque@cica.es I had always assumed that it was lacUV5. But when I read your question I decided to check. The sequence in the vector database for the pUC plasmids does not have the UV5 promoter but has the wild type promoter. So if you believe the sequence then it is wild type. ---------------------------------------------------------------------- Michael Benedik INTERNET: Benedik@uh.edu Dept. of Biochemical & Biophysical Sciences University of Houston BITNET: Benedik@uhou ----------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!pipex!zaphod.crihan.fr!univ-lyon1.fr!scsing.switch.ch!ira.uka.de!howland.reston.ans.net!wupost!medicine.wustl.edu!wums.wustl.edu!wetsel_r From: wetsel_r@wums.wustl.edu Newsgroups: bionet.molbio.methds-reagnts Subject: RE: human CD40 mAb source?? Message-ID: <6MAY93.11291652@wums.wustl.edu> Date: 6 May 93 11:29:16 GMT References: <1993May5.123926.21261@gserv1.dl.ac.uk> Organization: MIT PLASMA FUSION CENTER Lines: 17 NNTP-Posting-Host: msnews.wustl.edu In a previous article, IBELGAUFTS@vms.biochem.mpg.de wrote: >dear netters >does anyone know a source for monoclonal antibodies directed against human >CD40 antigen? >thanks >Horst --- YUP, I think I can help... a company called MONOSAN offers and anti-CD40 MAb in thier current catalog. page 48. Unfortunately it's an IgM Catalog number MON1046. Their antibodies appear to be marketed through CalTag laboratories, 436 Rozzi Place, So. San Francisco, CA 94080. 415-873-6106 phone and 415-873-2113 fax. Hope this helps... David haviland@kids.wustl.edu From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!pipex!uknet!edcastle!vls From: vls@festival (M Souter) Newsgroups: bionet.molbio.methds-reagnts Subject: Nitric Oxide Message-ID: <35335@castle.ed.ac.uk> Date: 6 May 93 16:04:45 GMT Sender: news@castle.ed.ac.uk Lines: 17 X-Newsreader: TIN [version 1.1 PL8] Speaking not as a scientist, but more as a clinician dipping his fingers into science - I'm interested in assays of nitric oxide from blood samples. This is in a situation which precludes the use of any synthase inhibitors or other similar measures. Obviously the halflife of the NO also renders direct estimation somewhat difficult. However I'm hoping some kind soul may have come across or be aware of any techniques (reliable) for assaying nitrate/nitrite end products or even citrulline/arginine ratios. I have even been told of a possible NO electrode, but cannot turn up any evidence of this. I'd be grateful if anyone could give me any info they have on this, even of the negative variety... Thanks. Mike Souter email vls@festival.ed.ac.uk Dept of Clin Neurosciences msouter@cix.compulink.co.uk Univ of Edinburgh From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!daresbury!bioftp.unibas.ch!urz.unibas.ch!bickle From: bickle@urz.unibas.ch Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Ethidium Bromide mutagenesis Message-ID: <1993May6.145845.42883@urz.unibas.ch> Date: 6 May 93 13:58:45 GMT References: <1993May6.091025.17243@infodev.cam.ac.uk> Organization: University of Basel, Switzerland Lines: 22 In article <1993May6.091025.17243@infodev.cam.ac.uk>, tjrc1@cus.cam.ac.uk (T.J.R. Cutts) writes: > I would imagine that EtBr mutagenesis occurs by a similar mechanism to most > intercalating agents, in that it probably interferes with transcription and > replication complexes, resulting in formation of DNA strand breaks. I don't > have any references though... > > Tim. Since this (very thin) thread started, I've done a little reading in the subject. It turns out that EtBr is not mutagenic in the Ames test unless it is S100 activated, when it scores just above background. It is good at curing plasmids in E. coli (like other intercalators) so it does enter the cell. It also eliminates mitochondrial DNA from yeast to give petite mutants and kinetoplast DNA from trypanosomal mitochondria. -- Tom Bickle Microbiology Dept Biozentrum, Basel University Klingelbergstrasse 70 CH-4056 Basel, Switzerland + 41 61 267 21 20 bickle@urz.unibas.ch From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!micro.uct.ac.za!ED From: ED@micro.uct.ac.za ("Ed Rybicki") Newsgroups: bionet.molbio.methds-reagnts Subject: Re: KCl staining for protein Message-ID: Date: 6 May 93 07:51:07 GMT Sender: daemon@net.bio.net Reply-To: ed@micro.uct.ac.za Distribution: bionet Lines: 61 > From: mm6y+@andrew.cmu.edu (Morris F. Manolson) > Subject: Re: KCl staining for protein > Date: 5 May 93 15:26:02 GMT > >Anybody out there have experience staining protein gels with KCl and then > >electroeluting the band you want? > > > > KCl is used to precipitate SDS in the gel matrix leaving regions of low > SDS (ie: high protein concentration) visible as clear bands. Following ... ...but it isn't very sensitive.... - much more sensitive is 0.3M CuCl2, which is a negative stain, is permanent (if you want it to be), and is easily destained (if you want to blot/elute/whatever). Simply soak gel straight after elec in 0.3M CuCl2 for as long as it takes to get good staining (+/-15min), then store in ddH2O for as long as you like: the proteins are insolubilised, so won't go anywhere. You can cut out and store bands (best visualised by indirect illumination against a black background) until you want to use them, or store gels until you want to blot them. To destain you simply wash a fewx5min in 250mM EDTA/500mM Tris pH 8.0: stain leaches out, and gel goes clear and prots are soluble once more.... And, it turns out, the gels stain better with Coomassie if copper stained first (with no destaining). It is the simplest, one of the cheapest, and most convenient (and quick) ways of staining SDS protein gels. Use and enjoy. Herewith ref: C. Lee, A. Levin and D. Branton: Copper staining: a five-minute protein stain for sodium dodecyl sulfate- polyacrylamide gels; Anal. Biochem. 166, 303-312; 1987 Gels may be stored in water for up to several months at room temperature with no problem or fading: proteins are immobilised as a Cu-SDS- polypeptide complex in the gel, which remains clear; the colour and background opacity are due to a Cu-SDS-Tris complex. Gels may be destained completely by repeated washing in 0.1-0.25 M Tris/0.25 M EDTA pH 8.0, and then electroblotted, or eluted from the gel for other purposes. NB: NATIVE PA GELS CAN ALSO BE STAINED : this is a less sensitive procedure, and bands are visible as faint blue opaque bands on a clear blue background. ELECTROBLOTS CANNOT BE STAINED WITH COPPER. ____________________________________________________________________ | Ed Rybicki, PhD | "Lord, won't you buy me | | (ed@micro.uct.ac.za) | | | Dept Microbiology | A Mer-ce-des Benz..." | | University of Cape Town | | | Private Bag, Rondebosch | | | 7700, South Africa | - Janis Joplin | | fax: 27-21-650 4023 | | -------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Wed May 05 23:00:00 1993 Path: biosci!parc!decwrl!decwrl!doc.ic.ac.uk!daresbury!daresbury!not-for-mail From: mbkxb@s-crim1.dl.ac.uk (K.C. Baker) Newsgroups: bionet.molbio.methds-reagnts Subject: Where can We get hold of CviJI. Message-ID: <1sarqlINNm90@s-crim1.dl.ac.uk> Date: 6 May 93 11:12:21 GMT Distribution: World Organization: Daresbury Lab., Warrington, U.K. Lines: 40 NNTP-Posting-Host: s-crim1.dl.ac.uk Good Morning Everybody I am posting this for a colleague who has no access, please will you reply direct to him at LAWA@IAPE.AFRC.AC.UK ************************************************************************** Subject: Where can We get hold of CviJI. A Colleague of mine wants to gt hold of an enzyme called Cvi JI. This cuts at rG'Cy. The reference he got it from is Nucl. Acids Res. Vol. 20, pp3753- 3762 (1992). The company that make it, CHIMERx appear to be one of the authors suggesting that it is in house research. Enzyme.dat reports no supplier for this enzyme. Can anyone suggest:- a) How we get in touch with CHIMERx or their representatives other than by snail-mail to the States. b) Where we can get hold of Cvi JI. c) Any other obscure alternatives? We want it to generate random genomic libraries better than by sonication, as described in the original reference. Thanks in advance, Andy Law ( LAWA @ IAPE.AFRC.AC.UK Big Nose in Edinburgh ) ***************************************************************************** Much obliged folks. Cheers, Ken. -- Dr Ken Baker JANET : UK.AC.DL.SEQNET::MBKXB Department of Protein Engineering INTERNET : MBKXB@SEQNET.DL.AC.UK AFRC Institute of Food Research TEL : (+44) 734 357139 Reading Berks RG6 2EF FAX : (+44) 734 267917 From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: skspoidn@reading.ac.uk Newsgroups: bionet.molbio.methds-reagnts Subject: Bgal fusions Message-ID: <1993May7.092140.9679@gserv1.dl.ac.uk> Date: 7 May 93 09:21:25 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 10 Original-To: methods@uk.ac.daresbury I recently constructed what I thought would be a fairly routine Bgal construct with my protein of interest in PUC18, with the aim of producing an immunoreactive fusion as a Wetsren blot control. I have sequenced thru the fusion site and everything is Thanks in advance, Mike Poidinger Dept of Microbiology University of Reading If only life were a kit...You could complain to the manufacturers and get a new one. From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!zaphod.mps.ohio-state.edu!moe.ksu.ksu.edu!kuhub.cc.ukans.edu!mbcf.stjude.org!bugg From: bugg@mbcf.stjude.org Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Site directed mutations Message-ID: <1993May6.182239.8455@mbcf.stjude.org> Date: 7 May 93 00:22:39 GMT References: <1993Apr20.121953.15040@alw.nih.gov> Distribution: bionet Organization: St. Jude Children's Research Hospital Lines: 45 In article <1993Apr20.121953.15040@alw.nih.gov>, jeffs@elsie.nci.nih.gov (Jeffrey Alan Silverman) writes: > Has anyone out there any experience with Enzymatic Inverse PCR? I am trying it > with little success. After making the primers - exact match for 20 bases containg a restriction site for a class !! enzyme- I get NO product in the pcr > reaction!! (despite trying the obvious manipulations in temperature, Mg, various > times of anneal and extensions etc).... So does anyone actually use this method? > how did you get it to work? > Related: if you don't use this method but rather favor another for site > directed mutagenesis... I'd be interested in hearing about that too... > Thanks ;-) > Jeff I use unique site elimination on double stranded plasmids very effectively. I have mutagenized plasmids for E.coli and mammalian expression by this method. The protocol is from Deng and Nickoloff. When I first started doing it someone in the lab had a Transformer kit from Clontech, which works just fine. However, when the reagents ran out, I didn't see any sense in spending that much again, so I just made them according to the Deng and Nickoloff paper and bought T4 DNA polymerase and T4 DNA ligase from Promega. The basic idea is that you simultaneously anneal your heat-denatured double- stranded plasmid with both a mutagenic primer and another primer (both PO4) which is designed to alter a unique restriction site in the vector (it must be in a non-essential area). Then, you use T4 DNA pol and T4 DNA ligase to extend around the circle and ligate to close up. You transform a mutS strain of E.coli with the in vitro reaction and grow up a "pool" with antibiotic selection overnight. The next day, you make a miniprep of the plasmid pool and digest to completion with the restriction enzyme whose site you are hoping to eliminate. A second transformation into a "good" E.coli strain like DH5 alpha is then plated out with antibiotic selection. This time you make up minipreps from individual clones and check for the loss of the site by restriction digest. Then, you confirm the mutation you want by sequencing. I have had anywhere from 1/6 to 6/6 of my minipreps lose the unique site. When sequenced, I have found that 100% of those that lost the unique site gained the mutation I wanted from the other primer. I think the variation in unique site elimination is due to specific annealing differences between the two primers that you design. This also is relevant to the question about transformation with linear DNA into E.coli. I always use electroporation for these USE experiments and I have not seen any evidence that linear DNA is effective for transformation. If it were, I would get huge numbers of transformants and they would all have the site I am trying to eliminate. I think the ones that still have the site have just escaped digestion in the linearization step. Barbara Bugg St. Jude Children's Research Hospital Memphis, TN USA From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!zaphod.mps.ohio-state.edu!moe.ksu.ksu.edu!kuhub.cc.ukans.edu!mbcf.stjude.org!bugg From: bugg@mbcf.stjude.org Newsgroups: bionet.molbio.methds-reagnts Subject: Re: recover DNA from gels using agarase Message-ID: <1993May6.180634.8454@mbcf.stjude.org> Date: 7 May 93 00:06:34 GMT References: <2109@rwja.umdnj.edu> Distribution: bionet Organization: St. Jude Children's Research Hospital Lines: 42 In article <2109@rwja.umdnj.edu>, bawagan@rwja.umdnj.edu (Hinayana Bawagan) writes: > > According to BM Biochemica March 93, recovery of DNA from low melting point agarose is about 80% efficient and is non-interfering with ligation reactions. Does anyone have any experience with this method? I am having problems with glass milk method. > Thank you. I was so late reading this posting that I thought, by now, someone might have mentioned my 2 cents' worth, but they haven't so here goes: The GELase agarase from Epicentre Technologies works very well for me in one particular instance. That is when I want to slice a particular band out of a gel, cut it with another restriction enzyme, and run it on another gel. This happens if you want to verify that a particular site is within a particular band. In this case, you run your first restriction digest on SeaPlaque or NuSieve in TAE buffer (I chill both the gel and the 1X buffer, so I can run it fast at room temp, just like for TBE). Then, you slice out the band of interest and dilute it as in the GELase protocol, make it 1X in restriction buffer and add the second enzyme and GELase enzyme simultaneously. After the GELase and restriction are complete (about 2 hours at 37C), you just add 2 volumes of cold ethanol and put on ice for 10 min. Microfuge at 12,500 Xg for 15 min, pour off supe, and vacuum dry the pellet. Now, you are ready to go on the second gel. I guess there are columns or some such gizmo that would also remove the agarose, but since you are planning a second digest anyway... AGAIN, I will just say that I do all of my ligations with the gel present by the method of Crouse, et. al and that SeaPlaque and NuSieve do not interfere with ligation. Another good and fast way to rid your DNA of agarose is to extract the melted slice with Tris-saturated phenol which has been pre-warmed to 65C. I just aliquot a small amount of the phenol into a microfuge tube under a fume hood, cap it and put it in the 65C water bath with the tube which contains the gel slice. After melting the slice for 10 min, the phenol is warm. Then, I just add an equal volume of phenol and vortex vigorously. After microfuging for 2 min to separate phases, you can see a whitish interface that has the agarose. I remove the upper aqueous phase to a new tube and then back-extract the organic phase with another equal volume of warm TE. After combining the 2 aqueous phases, I just precipitate with 2 volumes of cold ethanol on ice for 10 min and microfuge. I have not calculated the yield, but I can tell that it is very good. This method does not require multiple phenol extractions or even a single chloroform extraction. If the volume of the 2 aqueous phases is too large, all you have to do is butanol extract to reduce the volume before precipitating. That's all! Barbara Bugg St. Jude Children's Research Hospital Memphis, TN USA From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: skspoidn@reading.ac.uk Newsgroups: bionet.molbio.methds-reagnts Subject: Genomic library isolation Message-ID: <1993May7.093344.9981@gserv1.dl.ac.uk> Date: 7 May 93 09:28:40 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 17 Original-To: methods@uk.ac.daresbury I have screened an EMBL3A Rabbit genomic library, isolated 11 very obvious positives, extracted the DNA according to the manufacturers protocol with a bit of help from Promega's Magic Lambda preps (Puhlease! no kit flames here!), and got 11 identical preps all of which sem to have comletely kicked out their inserts. Why? Does lambda do this alot? Is there a way to avoid it? Yours in frustration, Mike Poidinger Dept of Microbiology University of Reading Have you noticed that Satan and Santa have the same letters in them...both wear Red and Black...You never see the 2 of them together in the same place...Think about it! From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!darwin.sura.net!sgiblab!munnari.oz.au!uniwa!uniwa!not-for-mail From: andrewh@uniwa.uwa.edu.au (Andrew Hobbs) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Enzymatic assay for deoxyribonucleotides? Message-ID: <1sci6h$dbf@uniwa.uwa.edu.au> Date: 7 May 93 02:40:17 GMT References: <1993May6.093558.18404@infodev.cam.ac.uk> Organization: The University of Western Australia Lines: 34 NNTP-Posting-Host: uniwa.uwa.edu.au X-Newsreader: Tin 1.1 PL5 T.J.R. Cutts (tjrc1@cus.cam.ac.uk) wrote: : I am trying to assay the levels of deoxypurine nucleotides in mammalian cells, : following the method outlined in (1, 2). The assay works by incoroprating : labelled nucleotides into DNA (using Pol I), the limiting factor being your : supplied sample of one nucleotide, so if you're measuring dATP, you use a : poly[dA.dT] template with hot dTTP. The amount of dATP in your sample is thus : proportional to the amount of labelled DNA produced, once precipitated, washed : and counted. The assay also includes AMP to inhibit any phosphatases. : : At present I am trying to get some standard curves before attempting to measure : cell extracts, with little success. I end up with large amounts of label on : the filters, even if no dATP is supplied. I'm using 3MM filters, and washing : three times with 5%TCA/1%Na4P2O7, and then twice with 95% ethanol, all washes : ice cold. : : Has anyone tried this assay and got it to work? Thanks all... : : Tim. : : References: : : 1. Hunting D, Henderson JF (1982) Methods Cancer Res 20 p. 245 : 2. Wilkinson YA, McKenna PG (1989) Leukemia Research 13/7 pp. 615-620 Hi, Sorry I haven't ever tried the technique but I would imagine you would get quite a reasonable incorporation as background anyway, since the poly(dA.dT) would self anneal and provide the primer. If the 'primer' ended with an 'A' then a 'T' would be added anyway without any extra dATP added. I would imagine it would be better to design a better template/primer combination. Either that or use the assay as it is but using labelled dATP in a competition type assay. From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!noc.near.net!saturn.caps.maine.edu!dartvax!grafton.dartmouth.edu!knight From: knight@grafton.dartmouth.edu (John Boswell) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Texts on Cell Culture? Message-ID: Date: 6 May 93 19:58:02 GMT References: <930405.083552.tomk@pcbiokj01> Sender: news@dartvax.dartmouth.edu (The News Manager) Organization: Dartmouth College, Hanover, NH Lines: 12 Hi, Thanks to everyone who replied to my request for texts on cell culture; I now have quite a few leads :):) have a good day! -John B. -- **************************************************************************** Dr. John Boswell knight@grafton.dartmouth.edu Dept. of Medicine/GI MCN C2104 Vanderbilt University, Nashville, TN 37232 615-322-6367 or 615-343-4747 From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!usc!sdd.hp.com!decwrl!decwrl!waikato.ac.nz!comp.vuw.ac.nz!canterbury.ac.nz!chmeds.ac.nz!mkennedy From: mkennedy@chmeds.ac.nz (Martin Kennedy) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Sequencing primer for CAT? Message-ID: <1993May7.112955.239@chmeds.ac.nz> Date: 6 May 93 23:29:55 GMT References: <1s3dth$8ln@max.physics.sunysb.edu> Lines: 29 In article <1s3dth$8ln@max.physics.sunysb.edu>, mhollowa@ic.sunysb.edu (Michael Holloway) writes: > > Lacking that, does anyone have any experience with a sequence at the > 5' end of CAT that can be successfully used for priming? > > Thanks in advance. > > Mike -- Mike, I have made and used the following primer successfully for sequencing from CAT in order to check constructs: 5'-AAT AAG CGG ATG AAT GGC AG-3' This falls in the 5' end of the CAT gene somewhere (I can't find my notes on it just now), and should give you the fusion site between your promoter and CAT. Cheers, Martin NNNN NN Martin A Kennedy (E-mail = mkennedy@chmeds.ac.nz) ZZZZZZZ NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ NN NN NN Christchurch School of Medicine ZZZ NN NNNN Christchurch, New Zealand ZZZZZZZ From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!darwin.sura.net!sgiblab!munnari.oz.au!uniwa!uniwa!not-for-mail From: andrewh@uniwa.uwa.edu.au (Andrew Hobbs) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Enzymatic assay for deoxyribonucleotides? Message-ID: <1scicu$dfr@uniwa.uwa.edu.au> Date: 7 May 93 02:43:42 GMT References: <1sci6h$dbf@uniwa.uwa.edu.au> Organization: The University of Western Australia Lines: 44 NNTP-Posting-Host: uniwa.uwa.edu.au X-Newsreader: Tin 1.1 PL5 Sorry about that. I got carried away and didn't indicate that it was from me. Andrew Hobbs (andrewh@uniwa.uwa.edu.au) wrote: : T.J.R. Cutts (tjrc1@cus.cam.ac.uk) wrote: : : I am trying to assay the levels of deoxypurine nucleotides in mammalian cells, : : following the method outlined in (1, 2). The assay works by incoroprating : : labelled nucleotides into DNA (using Pol I), the limiting factor being your : : supplied sample of one nucleotide, so if you're measuring dATP, you use a : : poly[dA.dT] template with hot dTTP. The amount of dATP in your sample is thus : : proportional to the amount of labelled DNA produced, once precipitated, washed : : and counted. The assay also includes AMP to inhibit any phosphatases. : : : : At present I am trying to get some standard curves before attempting to measure : : cell extracts, with little success. I end up with large amounts of label on : : the filters, even if no dATP is supplied. I'm using 3MM filters, and washing : : three times with 5%TCA/1%Na4P2O7, and then twice with 95% ethanol, all washes : : ice cold. : : : : Has anyone tried this assay and got it to work? Thanks all... : : : : Tim. : : : : References: : : : : 1. Hunting D, Henderson JF (1982) Methods Cancer Res 20 p. 245 : : 2. Wilkinson YA, McKenna PG (1989) Leukemia Research 13/7 pp. 615-620 : : Hi, : Sorry I haven't ever tried the technique but I would imagine you : would get quite a reasonable incorporation as background anyway, since the : poly(dA.dT) would self anneal and provide the primer. If the 'primer' : ended with an 'A' then a 'T' would be added anyway without any extra : dATP added. : : I would imagine it would be better to design a better template/primer : combination. Either that or use the assay as it is but using labelled : dATP in a competition type assay. : Andrew Hobbs Dept of Biochemistry University of Western Australia andrewh@uniwa.uwa.edu.au From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: skspoidn@reading.ac.uk Newsgroups: bionet.molbio.methds-reagnts Subject: Bgal Constructs Message-ID: <1993May7.101146.10890@gserv1.dl.ac.uk> Date: 7 May 93 10:11:34 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 16 Original-To: methods@uk.ac.daresbury Now here is the full version... I recently constructed what I thought would be a fairly routine Bgal construct with my protein of interest in PUC18, with the aim of producing an immunoreactive fusion as a Wetsren blot control. I have sequenced thru the fusion site and everything is in frame, but no protein is forthcoming. I have heard that some Bgal fusion just dont produce protein. Does anyone know why. or what to do to avoid it? Thanks in advance, Mike Poidinger Dept of Microbiology University of Reading If only life were a kit...You could complain to the manufacturers and get a new one. From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!usc!sdd.hp.com!nigel.msen.com!yale.edu!yale!hsdndev!newsfeed.rice.edu!bcm!ak990140 From: ak990140@SPECIALK.IAIMS.BCM.TMC.EDU (Arthur Kania) Newsgroups: bionet.molbio.methds-reagnts Subject: Help with Schneider S2 cells Message-ID: <1se8ef$2bv@gazette.bcm.tmc.edu> Date: 7 May 93 18:06:07 GMT Organization: Baylor College of Medicine, Houston, Tx Lines: 16 NNTP-Posting-Host: specialk.iaims.bcm.tmc.edu Originator: ak990140@SPECIALK.IAIMS.BCM.TMC.EDU I am working with Schneider S2 cells trying to express in them a cell adhesion molecule. The aim of the culture is to derive a stable cell line which will express my gene when induced. I am able to obtain transfectants which express the protein really well, but after several months of culture, the expression becomes very low and i have to retransfect new cells. The cells are kept under selection, so it's not just overgrowth with wild type cells. Does anyone have a similar problem with this cell line or is it just me ? Also, has anyone noticed that the S2 cells do not grow as a homogenous culture and a lot of differentiating cells (eg. fibroblast like) become visible soon after passage. thanks for your help a. kania From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!gatech!ukma!seqanal.mi.uky.edu!dtai From: dtai@seqanal.mi.uky.edu (Daniel Tai) Newsgroups: bionet.molbio.methds-reagnts Subject: Sequence for plasmid pCW? Message-ID: Date: 7 May 93 19:20:01 GMT Sender: news@ms.uky.edu (USENET News System) Organization: University Of Kentucky, Dept. of Math Sciences Lines: 9 Nntp-Posting-Host: seqanal.mi.uky.edu X-Newsreader: TIN [version 1.1 PL9] Does anyone have a copy of the sequence for the plasmid pCW? If you do, I'd appreciate it if you could send me a copy via. email! Thanks, Xia email: dtai@seqanal.mi.uky.edu From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!bldghsc.lan1.umanitoba.ca!GIETZ From: GIETZ@bldghsc.lan1.umanitoba.ca Newsgroups: bionet.molbio.methds-reagnts Subject: RE:Adding linkers to a vector Message-ID: <2BEAD623@adminbldg.lan1.umanitoba.ca> Date: 7 May 93 21:34:00 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 90 Hello! I would do a few things differently. 1) Cut your vector 2) then CIAP your vector. You may want to test ligate the vector to see if your CIAP is working properly. Do a kinase addition of the phosphate back to do positive control. Occasionally I have had problems with it going off after being about a year old. Here I have a question. Are your linkers phosphorylated? Not all linkers come phosphorylated. You have to treat with T4 PNK and ATP before ligation to do this. If this is your problem then it's solved. IF not then I would take your linkers and and the klenow in a single reaction and ligate. The key here is to make sure your Klenow is working (dies off easily even if you warm it upto 0 degrees from -20 a couple of time) So keep your Klenow in the freezer, never put it on ice. Set up the ligation as follows 200 ng of CIAP vector (phenol to remove CIAP) 1X nick translation buffer made with MgCl2 not MgSO4 100 micromolar dNTPs 20 ng of Kinased Linker to 20 microliters with H2O 1 unit of Klenow Incubate at RT for 20 mins then add 1/2 microliter of 50mM ATP NOT dATP 1/2 microliter of 250mM DTT mix 5 UNITS of Ligase (Boehringer Mannheim or equiv) Incubate overnight in a cooling water bath at 12-14 degrees (not in a heating water bath in the cold room causes condensation problems and stops ligase too early) The Key to blunt end ligations are the concentration of ends and the high levels of ligase (5 to 8 units) If you use 200 ngs of vector and an excess of linker then it should go. You can then take some of the ligation and transform. This should select for the plasmids that are stable in coli (not many linkers). YOu can then cut the miniprep DNA (PEG pptte to remove linkers) (Add 30 ul of 2.5 M NaCl 20% PEG 8000 to 50 ul of digestion Incubate in an ice water bath for 1 hr and then spin in a cool microfuge for 5 min at max, wash the pellet with 70% etoh and dry) and ligate at a low concentration to get rid multiple linkers. If you are committed to cutting the ligation prior to TRAFO then I would PEG pptte the DNA apres cutting (gets rid of the linkers) and then bring to probably 100 ul and ligate in 1 x ligase buffer and 1 unit of ligase. (this favours circularization) of single molecules. If you have specific questions please don't hesitate to ask. ______________________________________ R.Daniel Gietz Ph.D. Assistant Professor Department of Human Genetics University of Manitoba 770 Bannatyne Ave, Rm 250 Winnipeg, Manitoba, Canada R3E 0W3 Tel.: (204)788-6458 Fax.: (204)786-8712 E-mail GIETZ@BLDGHSC.LAN1.UMANITOBA.CA ________________________________________ ------------------------------------------------------------------------------ Here's the purpose: I have a vector in w/c the RE site used to linearize it for transcription is found in my insert. Had trouble doing partial digestion. So I want to remove that site and put in a linker in w/c the RE site is not found in my vector nor insert. Here's the procedure: 1. Linearize the vector at Spe I site. 2. Fill in w/ Klenow to produce blunt ends. 3. Dephosphorylate the vector w/ CIAP. 4. Ligation of deP vector and Pac I linkers. 5. Digestion w/ Pac I RE to remove the concatemers and leave sticky Pac I ends. 6. Circularization of the modified vector. 7. Transformation. At each step, phenol/CHCl3 extraction and Etol precipitation was done. Can't do it. Please help. Any advice will be useful and appreciated. From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!zaphod.mps.ohio-state.edu!menudo.uh.edu!Elroy.UH.EDU!BCHS1B From: bchs1b@Elroy.UH.EDU (Michael Benedik) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Adding linkers to a vector Message-ID: <1sea0e$b44@menudo.uh.edu> Date: 7 May 93 18:32:46 GMT References: <2147@rwja.umdnj.edu> Reply-To: bchs1b@Elroy.UH.EDU Distribution: bionet Organization: University of Houston Lines: 37 NNTP-Posting-Host: elroy.uh.edu In article <2147@rwja.umdnj.edu>, bawagan@rwja.umdnj.edu (Hinayana Bawagan) writes: >Here's the purpose: I have a vector in w/c the RE site used to linearize >it for transcription is found in my insert. Had trouble doing partial >digestion. So I want to remove that site and put in a linker in w/c >the RE site is not found in my vector nor insert. > >Here's the procedure: > >1. Linearize the vector at Spe I site. >2. Fill in w/ Klenow to produce blunt ends. >3. Dephosphorylate the vector w/ CIAP. >4. Ligation of deP vector and Pac I linkers. >5. Digestion w/ Pac I RE to remove the concatemers and > leave sticky Pac I ends. >6. Circularization of the modified vector. >7. Transformation. > >At each step, phenol/CHCl3 extraction and Etol precipitation was done. > >Can't do it. >Please help. Any advice will be useful and appreciated. Did you phosphorylate your linkers??? If you didn't then both your vector and your linkers don't have a terminal phosphate and you won't get any ligation products? If it still doesn't work, then kinase your linkers with some gamma-P32 and then ligate to your vector. You should be able to run a gel and see some counts ligated to the vector band after autoradiography. Using labelled linkers you can troubleshoot your different steps in the procedure. ---------------------------------------------------------------------- Michael Benedik INTERNET: Benedik@uh.edu Dept. of Biochemical & Biophysical Sciences University of Houston BITNET: Benedik@uhou ----------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!usenet.ins.cwru.edu!po.CWRU.Edu!txt15 From: txt15@po.CWRU.Edu (Tao Tao) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Bgal Constructs Message-ID: <1se5gf$blt@usenet.INS.CWRU.Edu> Date: 7 May 93 17:15:58 GMT Organization: Case Western Reserve University, Cleveland, Ohio (USA) Lines: 11 NNTP-Posting-Host: slc12.ins.cwru.edu The gene in pUC is only the alpha fragment with very little immumogenecity. It is hard to detect this using the ordinary polyclonal ab. You may be abel to get away with a mono ab that is specific to the alpha fragment. If you can find a source for such mab, I would like to know it too. Tao -- p From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!zaphod.mps.ohio-state.edu!uwm.edu!rutgers!rwja!bawagan From: bawagan@rwja.umdnj.edu (Hinayana Bawagan) Newsgroups: bionet.molbio.methds-reagnts Subject: Adding linkers to a vector Message-ID: <2147@rwja.umdnj.edu> Date: 7 May 93 16:33:11 GMT Distribution: bionet Organization: Robert Wood Johnson Medical School, Piscataway NJ Lines: 20 Here's the purpose: I have a vector in w/c the RE site used to linearize it for transcription is found in my insert. Had trouble doing partial digestion. So I want to remove that site and put in a linker in w/c the RE site is not found in my vector nor insert. Here's the procedure: 1. Linearize the vector at Spe I site. 2. Fill in w/ Klenow to produce blunt ends. 3. Dephosphorylate the vector w/ CIAP. 4. Ligation of deP vector and Pac I linkers. 5. Digestion w/ Pac I RE to remove the concatemers and leave sticky Pac I ends. 6. Circularization of the modified vector. 7. Transformation. At each step, phenol/CHCl3 extraction and Etol precipitation was done. Can't do it. Please help. Any advice will be useful and appreciated. From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!sas.upenn.edu!droos From: droos@sas.upenn.edu (David Roos) Newsgroups: bionet.molbio.methds-reagnts Subject: CAT sequencing primers Message-ID: <9305071647.AA07898@mail.sas.upenn.edu> Date: 7 May 93 16:47:36 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 7 **WARNING** -- Primer 5'-AATAAGCGGATGAATGGCAG-3' mentioned in a recent posting is located ~50 nt downstream of the stop codon, and therefore suitable for use only with downstream inserts (and only if your CAT cassette includes this much of the 3' non-coding region). For an antisense primer suitable for sequencing 5' inserts,try 5'-TGCCATTGGGATATATCAACGGTG-3', as mentioned a couple of days ago. -- D.S.Roos From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: skspoidn@reading.ac.uk Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Bgal Constructs Message-ID: <1993May7.134017.15575@gserv1.dl.ac.uk> Date: 7 May 93 13:37:20 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 30 Original-To: methods@uk.ac.daresbury re a personal reply to my original post from Alan Gerstein <76467.702@COM.CompuServe> ------ Explanations abound [about Bgal fusion problems]; solutions are more difficult to obtain. Possibilities include: -quality and quantity of IPTG insufficient -protein degraded -protein forms insoluble complexes -protein escaped detection; ie. if you looked at total cell extracts on a a PAGE gel, cellular proteins could be masking your Bgal fusion. -moody host cell strain And then it gets ugly. ---------- The situation is complicated/simplified by the fact that a Bgal fusion with a different version of the protein works fine. The difference between the 2 is that the one that works is fused at a site further down stream in the protein of interest, whilst the one that does not is fused further upstream (about 22aas apart). I would have simply made the latter like the former, but this particular version of the protein has a silent mutation removing the site at which the 1st fusion was made. It is definitely NOT a probelem with masking, IPTG or degradation or host cell strain Insoluble complexes are a possibility I guess, but I am bucking for the ugly :( Mike From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!MED.PITT.EDU!bsh From: bsh@MED.PITT.EDU (Basavaraju Shankarappa) Newsgroups: bionet.molbio.methds-reagnts Subject: RE:Adding linkers to a vector Message-ID: <9305072114.AA10493@phobos.med.pitt.edu> Date: 7 May 93 21:14:41 GMT References: <2BEAD623@adminbldg.lan1.umanitoba.ca> Sender: daemon@net.bio.net Distribution: bionet Lines: 30 If the insert is of decently smaller size, why not synthesize two primers from the begining of the transcription start site to the point where you want it terminated, PCR the insert and clean it up and use it for transcription. May be it is not as simple as this, anyway my thoughts. Raj Shankarappa bsh@med.pitt.edu > ------------------------------------------------------------------------------ > Here's the purpose: I have a vector in w/c the RE site used to linearize > it for transcription is found in my insert. Had trouble doing partial > digestion. So I want to remove that site and put in a linker in w/c > the RE site is not found in my vector nor insert. > > Here's the procedure: > > 1. Linearize the vector at Spe I site. > 2. Fill in w/ Klenow to produce blunt ends. > 3. Dephosphorylate the vector w/ CIAP. > 4. Ligation of deP vector and Pac I linkers. > 5. Digestion w/ Pac I RE to remove the concatemers and > leave sticky Pac I ends. > 6. Circularization of the modified vector. > 7. Transformation. > > At each step, phenol/CHCl3 extraction and Etol precipitation was done. > > Can't do it. > Please help. Any advice will be useful and appreciated. > From owner-methds-reagnts@net.bio.net Thu May 06 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!noc.near.net!uunet!utcsri!newsflash.concordia.ca!mizar.cc.umanitoba.ca!murphy.biochem.umanitoba.ca!user From: dotzlaw@ccu.umanitoba.ca (Helmut Dotzlaw) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Programs for image analysis of gels? Message-ID: Date: 7 May 93 13:11:43 GMT References: Sender: news@ccu.umanitoba.ca Followup-To: bionet.molbio.methds-reagnts Distribution: bionet Organization: University of Manitoba Lines: 23 Nntp-Posting-Host: murphy.biochem.umanitoba.ca In article , mligr@flash.LakeheadU.Ca (Martin Ligr) wrote: > > Hallo! > We are concidering purchase of IBM-PC software for analysis of images > of one dimensional protein gels. I would greatly appreciate if you > told me about your experince with this kind of software and gave > me some recomendations. Are there some shareware programs able to > perform this task? > > I recently posted a request to a few groups concerning the lack of shareware image analyses programs for pc. The result was "nothing." There are, of course commercial products, I think BioRad and Pharmacia et. al. - but pricey. For mac there is NIH Image - freeware. People at our u are buying a mac and using Image - it is actually less expensive to buy the computer than to buy the commercial pc software - go figure. Helmut Dotzlaw "You can observe a lot by just watchin.." Dept. Biochem. Mol.Bio. Yogi Berra University of Manitoba Winnipeg, Canada From owner-methds-reagnts@net.bio.net Fri May 07 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!uunet!europa.eng.gtefsd.com!howland.reston.ans.net!agate!ames!saimiri.primate.wisc.edu!usenet.coe.montana.edu!netnews.nwnet.net!news.u.washington.edu!remote0.rad.washington.edu!syverson From: syverson@u.washington.edu (dms) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Dounce homogenizer Message-ID: <1sfbohINN4ub@news.u.washington.edu> Date: 8 May 93 04:08:49 GMT References: Organization: University of Washington Lines: 14 NNTP-Posting-Host: remote0.rad.washington.edu X-UserAgent: Nuntius v1.1.1d11 X-XXMessage-ID: X-XXDate: Fri, 7 May 93 21:09:25 GMT Subject: Dounce homogenizer From: Morag Lancaster, morag@arbo.microbiol.uwa.edu.au Date: 8 May 1993 02:44:10 GMT In article Morag Lancaster, morag@arbo.microbiol.uwa.edu.au writes: >Hi, does anyone know where a Dounce homogenizer can be purchased or ig a >similar instrument can be used for the same task? I remember seeing it in the Fisher catalog. Give it a try. Wilas Nirunsuksiri UW, Seattle From owner-methds-reagnts@net.bio.net Fri May 07 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!uunet!zaphod.mps.ohio-state.edu!howland.reston.ans.net!agate!ames!saimiri.primate.wisc.edu!usenet.coe.montana.edu!netnews.nwnet.net!news.u.washington.edu!remote0.rad.washington.edu!syverson From: syverson@u.washington.edu (dms) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: nuclear run on Message-ID: <1sfbfgINN4ub@news.u.washington.edu> Date: 8 May 93 04:04:00 GMT References: <9304221618.AA16923@post.its.mcw.edu> Organization: University of Washington Lines: 24 NNTP-Posting-Host: remote0.rad.washington.edu X-UserAgent: Nuntius v1.1.1d11 X-XXMessage-ID: X-XXDate: Fri, 7 May 93 21:04:35 GMT Subject: nuclear run on From: Kathleen Hopp, khopp@POST.ITS.MCW.EDU Date: 22 Apr 93 16:18:44 GMT In article <9304221618.AA16923@post.its.mcw.edu> Kathleen Hopp, khopp@POST.ITS.MCW.EDU writes: >Fellow searchers: > >A collegue is struggling with the Red book protocol for nuclear run on. >Prostate fibroblast cells are clumping in the harvesting step and cause >subsequent problems. >Any advice, experience or better protocols out there? > >Thank you, >Kathie Hopp I had a similar problem with confluent skin cells and solved the problem by simply cutting the clump by sterile scissors before homogenizing the cells in a Dounce. It worked for me. Good luck. Wilas Nirunsuksiri UW, Seattle From owner-methds-reagnts@net.bio.net Fri May 07 23:00:00 1993 Path: biosci!HAL.HAHNEMANN.EDU!farleyp From: farleyp@HAL.HAHNEMANN.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: Disulfide Isomerase Message-ID: <0096C345.38573F00.23756@hal.hahnemann.edu> Date: 8 May 93 20:20:57 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 10 Hi Gang! Does anyone know where I can buy disulfide isomerase for use in in vitro translation reactions? I have heard that there may be a Japanese company called Tokata (?) that makes it, but I don't know a company that distributes it. I would appreciate any information you have. Thanks in advance! Patrick Farley Hahnemann University Philadelphia, PA From owner-methds-reagnts@net.bio.net Fri May 07 23:00:00 1993 Path: biosci!steele!news From: shapirop@ohsu.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Dounce homogenizer Message-ID: <1993May8.193647.22324@ohsu.edu> Date: 8 May 93 19:36:47 GMT References: <1sfbohINN4ub@news.u.washington.edu> Sender: news@ohsu.edu Organization: vollum institute/ohsu Lines: 24 Nntp-Posting-Host: 137.53.73.151 In article <1sfbohINN4ub@news.u.washington.edu> dms writes: > >Subject: Dounce homogenizer >From: Morag Lancaster, morag@arbo.microbiol.uwa.edu.au >Date: 8 May 1993 02:44:10 GMT >In article Morag Lancaster, >morag@arbo.microbiol.uwa.edu.au writes: >>Hi, does anyone know where a Dounce homogenizer can be purchased or ig a >>similar instrument can be used for the same task? > > >I remember seeing it in the Fisher catalog. Give it a try. > >Wilas Nirunsuksiri >UW, Seattle > Kontes (1-800-255-1672), and Thomas Scientific (1-800-345-2102) also sell Dounce homogenizers. Depending on the precise requirements of the cell/tissue homogenate a number of procedures and apparatuses MIGHT be substitiuted, e.g. "Polytron" homogenizer, sonication, Potter-Elvhjam teflon/glass homogenizer, hypotonic lysis, Nitrogen cavitation, detergent lysis, etc, etc.. From owner-methds-reagnts@net.bio.net Fri May 07 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!munnari.oz.au!uniwa!kun.microbiol.uwa.edu.au!morag From: morag@arbo.microbiol.uwa.edu.au (Morag Lancaster) Newsgroups: bionet.molbio.methds-reagnts Subject: Dounce homogenizer Message-ID: Date: 8 May 93 02:44:10 GMT Organization: Dept of Microbiology, UWA Lines: 2 NNTP-Posting-Host: kun.microbiol.uwa.edu.au Hi, does anyone know where a Dounce homogenizer can be purchased or ig a similar instrument can be used for the same task? From owner-methds-reagnts@net.bio.net Sat May 08 23:00:00 1993 Path: biosci!uwm.edu!zaphod.mps.ohio-state.edu!news.acns.nwu.edu!casbah.acns.nwu.edu!drmax From: drmax@casbah.acns.nwu.edu (Marianna Max) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RNase protection assays... Message-ID: <1993May9.101615.16630@news.acns.nwu.edu> Date: 9 May 93 10:16:15 GMT References: <5MAY93.11573872@wums.wustl.edu> Sender: usenet@news.acns.nwu.edu (Usenet on news.acns) Organization: Northwestern University, Evanston IL Lines: 38 Nntp-Posting-Host: casbah.acns.nwu.edu In article <5MAY93.11573872@wums.wustl.edu> wetsel_r@wums.wustl.edu writes: >Hi Netters: > >I noticed that in addition to Ambion, Clonetech is now offering an RNase >protection kit called the Guardian. Like Ambion's kit, literature from >both company's is quite vague at the step where inactivation of the RNases >takes place. (Sounds like proprietary kit information again!) Having done Ambion's instruction book that comes with the assay contains a complete list of all formulations, nothing proprietary at all (I'm refering to the first version of the kit not their second version, which I haven't used). The inactivation of RNase step in the kit is Prot K and SDS followed by a phenol extraction and a percipitation. I've never had problems with the RNA redisolving using this method. >a number of protection assays, I'd love to avoid using protease-K and SDS >in the assay, once precipitated neither disolve in anything very well. Has >anyone ever just precipitated the double stranded RNA after the RNase >treatment step with ethanol or isopropanol? I thought of trying a PEG PPT >but realized that it wouldn't work as PEG won't PPT anything below 120-130 >base pairs, even though it *may* inactivate the RNases. Does anyone >familiar with RNase Protection assays have any thoughts to offer to get >around not using protease-K and SDS? Anyone using either the Ambion or >Clonetech kits care to "spill the beans" at this particular step? > >Thanks in advance... > >David >haviland@kids.wustl.edu >=========================================================================== >+ David L. Haviland, Ph.D. Internet:"haviland@kids.wustl.edu" + >+ Washington Univ. School of Med. A.K.A : The Compiler + >+ Dept. of Peds./Pulm. Box 8116 ICBM-Net : Just hit St. Louis + >+ 400 S. Kingshighway &-6 <- User is Brain Dead... + >+ St. Louis, MO 63110 FAX: 314-454-2476 + >+ (314) 454-6076 + >=========================================================================== > From owner-methds-reagnts@net.bio.net Sat May 08 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!ira.uka.de!news.belwue.de!softserv.zdv.uni-tuebingen.de!studserv!zxmkr08 From: zxmkr08@studserv.zdv.uni-tuebingen.de (Cornelius Krasel) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Disulfide Isomerase Message-ID: Date: 9 May 93 15:06:11 GMT References: <0096C345.38573F00.23756@hal.hahnemann.edu> Distribution: bionet Organization: InterNetNews at ZDV Uni-Tuebingen Lines: 17 NNTP-Posting-Host: studserv.zdv.uni-tuebingen.de In <0096C345.38573F00.23756@hal.hahnemann.edu> farleyp@HAL.HAHNEMANN.EDU writes: >Does anyone know where I can buy disulfide isomerase for use in in vitro >translation reactions? I have heard that there may be a Japanese company >called Tokata (?) that makes it, but I don't know a company that distributes >it. I would appreciate any information you have. Thanks in advance! You probably mean Takada. They sell a lot of strange enzymes. Sorry, can't help you with the distribution -- I guess it wouldn't help you much if you'd know where you can buy their products in Germany :-) --Cornelius. -- /* Cornelius Krasel, Department of Physiological Chemistry, U T