From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!bcm!cs.utexas.edu!howland.reston.ans.net!usenet.ins.cwru.edu!lerc.nasa.gov!purdue!news.bu.edu!med-pharm5.bu.edu!user From: pmclean@darwin.bu.edu (Pam McLean) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: E-mail address for EMBL submissions Date: 30 Jan 1995 23:52:59 GMT Organization: Boston University Dept. of Pharmacology Lines: 27 Message-ID: References: <1995Jan30.151219.114355@rs6000.cmp.ilstu.edu> NNTP-Posting-Host: med-pharm5.bu.edu X-Newsreader: Value-Added NewsWatcher 2.0b24.0+ In article <1995Jan30.151219.114355@rs6000.cmp.ilstu.edu>, ajotsuka@rs6000.cmp.ilstu.edu (Anthony Otsuka) wrote: > For some strange reason the Journal of Cell Biology requires > submission of sequences to the EMBL data bank. Please > inform me of the appropriate e-mail address and format > to provide DNA sequence data to EMBL. The EMBNet computer > "felix" is apparently not accessible to me. Thanks > for the help. ajotsuka@rs6000.cmp.ilstu.edu If you use the Authorin program provided by NCBI you can prepare your submission and submit to GenBank, EMBL and the DNA Data Bank of Japan. To receive a copy of Authorin e-mail: authorin@ncbi.nlm.nih.gov (it's free!) The addresses for submission are included in the handbook, but here they are anyway: GenBank: gb-sub@ncbi.nlm.nih.gov Tel: (301) 402-1301 EMBL: datasub@embl-heidelberg.de Tel: +49 6221 387 258 ---Pam -- "Welcome to all things Scottish. If it's not Scottish, it's CRAP!" -Mike Myers, Saturday Nite Live. -- e-mail: pmclean@darwin.bu.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!bcm!cs.utexas.edu!swrinde!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!lll-winken.llnl.gov!unixhub!news.Stanford.EDU!hsdndev!dartvax.dartmouth.edu!usenet From: James.J.Taub@dartmouth.edu (James J. Taub) Newsgroups: bionet.molbio.methds-reagnts Subject: C.elegans cDNA lib. in yeast vector Date: 30 Jan 1995 23:14:31 GMT Organization: Dartmouth College, Hanover, NH Lines: 11 Message-ID: <3gjrsn$91b@dartvax.dartmouth.edu> NNTP-Posting-Host: at-3-sn-165.dartmouth.edu X-Posted-From: InterNews 1.0.2@dartmouth.edu I would like to clone the C. elegans homologs of two yeast genes and I would prefer to to do it by complemeting the yeast mutants. Does anyone know if there is a C.elegans cDNA library in a yeast vector? Any info would be a great help. I would prefer an E-mail responce as I don't normally monitor this group. Thanx in advance James J Taub james.taub@dartmouth.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!uwm.edu!lll-winken.llnl.gov!enews.sgi.com!decwrl!tribune.usask.ca!quartz.ucs.ualberta.ca!unixg.ubc.ca!steevlab.generes.ca!user From: mrott@unixg.ubc.ca (Mike Rott) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: A GOOD KIT FOR PCR CLONING Date: 30 Jan 1995 21:56:44 GMT Organization: ubc Lines: 17 Distribution: world Message-ID: References: NNTP-Posting-Host: steevlab.generes.ca > In article , reedc@iac.net (Reed > R. Corderman) wrote: > > > In article , > > xz@KEPLER.UNH.EDU (Xin-Hua Zhou) wrote: > > > > > I am looking for a good kit for PCR cloning. Can you give me some ideas > > > , based on your EXPERENCE? I have only used the Invitrogen TA cloning kit. it works every time, what more can I say. The kit is kind of expensive but I have found that you can cut back all the reagents in half and get twice as many cloning reactions as advertised. mike From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!rutgers!netnews.upenn.edu!dsinc!newsfeed.pitt.edu!gatech!usenet.ins.cwru.edu!cleveland.Freenet.Edu!ch994 From: ch994@cleveland.Freenet.Edu (Parke K. Flick) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: KlenTaq/Pfu for LA-PCR? Date: 2 Feb 1995 19:29:55 GMT Organization: Case Western Reserve University, Cleveland, Ohio (USA) Lines: 8 Message-ID: <3grbrj$ao0@usenet.INS.CWRU.Edu> NNTP-Posting-Host: piglet.ins.cwru.edu You can do just as well with regular Taq polymerase and deep vent from NEB. P. Flick -- Parke K. Flick, Ph.D. ch994@cleveland.freenet.edu USB/Amersham Life Sciences, Inc. Cleveland, OH 44128 Ph: 216-464-9277; FAX: 216-360-0975 From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!news.cs.umb.edu!hsdndev!purdue!news.bu.edu!med-pharm16.bu.edu!user From: leach@bu.edu (Martin Leach) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA purification: Quiagen, glassmilk etc Date: 2 Feb 1995 18:25:42 GMT Organization: Boston University Dept. of Pharmacology Lines: 42 Message-ID: References: NNTP-Posting-Host: med-pharm16.bu.edu In article , marchaa@agric.nsw.gov.au (A.Marchant) wrote: > A couple of questions about DNA purification: > > 1) What's the best (ie, best ratio of > (yield,quality):(effort,expense,difficulty)) way of purifying DNA > from a complex dirty mixture? What is the difference between 'glassmilk' and > 'silica gel'? Is Quiagen really as good as their advertising says? Do > Quiagen sell their resin separately from their prepared tips, so that I > can make up my own format with it? > > 2) What is the chemistry of the binding/release of DNA from Quiagen resin > and from silica? > > 3) How clean does your homogenate have to be before you can use this > stuff? Is cation concentration the only important thing involved in the > specific binding and release? > I have not an email address of qiagen...however, their is a company called the Nest Group...that sells a rival resin column...nestgrp@world.std.com...they may be able to answer some of the chemistry questions.... M -- ..... Martin Leach Email:leach@bu.edu _|____ Dept. of Pharmacology Phone: (617) 638-5323 / o / Boston Univ. School of Med. Fax: (617) 638-4329 _/ |-/__==/ 80 E. Concord St. (L603) (BULLDOZER) \_ Boston MA 02118 "Not the old underpants on your USA head.....WIBBLE" -BLACKADDER p.s. try BioMOO (virtual biology on the internet - telnet bioinfo.weizmann.ac.il 8888) My home page: http://155.41.115.114/Leach.html From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!news.cs.umb.edu!hsdndev!purdue!news.bu.edu!med-pharm16.bu.edu!user From: leach@bu.edu (Martin Leach) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: S: Addresses of bio-companies Date: 2 Feb 1995 18:18:50 GMT Organization: Boston University Dept. of Pharmacology Lines: 43 Distribution: world Message-ID: References: <3gopdi$l0i@sunserver.lrz-muenchen.de> NNTP-Posting-Host: med-pharm16.bu.edu In article <3gopdi$l0i@sunserver.lrz-muenchen.de>, t7845ag@cd1.lrz-muenchen.de (Hermann Rochholz) wrote: > Hello! > > I am searching a list of addresses of companies > producing biological materials like primers, dyes, etc. > > I know, that such a searchable index exists on the > World-wide-web (including search-index), > but I can't remember the www-address. > And I didn't find the address at pedros. try telnetting to biotechnet telnet biotechnet.com username biotech pw technique there are a whole mess of companies with email addresses here...e.g. ambion, clonetech, owl, tropix to name but a few.... ciao Martin -- ..... Martin Leach Email:leach@bu.edu _|____ Dept. of Pharmacology Phone: (617) 638-5323 / o / Boston Univ. School of Med. Fax: (617) 638-4329 _/ |-/__==/ 80 E. Concord St. (L603) (BULLDOZER) \_ Boston MA 02118 "Not the old underpants on your USA head.....WIBBLE" -BLACKADDER p.s. try BioMOO (virtual biology on the internet - telnet bioinfo.weizmann.ac.il 8888) My home page: http://155.41.115.114/Leach.html From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!news.cs.umb.edu!hsdndev!purdue!news.bu.edu!med-pharm16.bu.edu!user From: leach@bu.edu (Martin Leach) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Who konws "PCR walking"? Date: 2 Feb 1995 18:15:32 GMT Organization: Boston University Dept. of Pharmacology Lines: 37 Distribution: world Message-ID: References: NNTP-Posting-Host: med-pharm16.bu.edu In article , beeman@crunch.usgmrl.ksu.edu (Dick Beeman) wrote: > In article , > wusp@GATE.SINICA.EDU.TW (San-pin Wu) wrote: > > > Hello: > > Does anyone know about "PCR walking"? I would like to clone the > > upstream region of spinach chloroplast psaA-psaB gene. > > I woould very appreciate if anyone could tell me where or how could > > I get the protocol and related information about this technique. > > Thank you very much and HAPPY CHINESE NEW YEAR! > > there are many variations of this.... try 'rapid amplification of genomic ends' in Biotechniques..(6 months ago?). Inverse PCR....in the Erlich PCR Technology book. Panhandle PCR (about 3 yrs ago in nucleic acids res...) Many more exist...mail me for more info...eg.refs -- ..... Martin Leach Email:leach@bu.edu _|____ Dept. of Pharmacology Phone: (617) 638-5323 / o / Boston Univ. School of Med. Fax: (617) 638-4329 _/ |-/__==/ 80 E. Concord St. (L603) (BULLDOZER) \_ Boston MA 02118 "Not the old underpants on your USA head.....WIBBLE" -BLACKADDER p.s. try BioMOO (virtual biology on the internet - telnet bioinfo.weizmann.ac.il 8888) My home page: http://155.41.115.114/Leach.html From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!THORIN.UTHSCSA.EDU!HARDIES From: HARDIES@THORIN.UTHSCSA.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: re: Ampicillin addition for plasmid Date: 2 Feb 1995 11:34:29 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 37 Sender: daemon@net.bio.net Distribution: world Message-ID: <950202133550.20ccda@thorin.uthscsa.edu> NNTP-Posting-Host: net.bio.net Deshpande Milind writes: > I am growing E. coli at 100 liter scale for plasmid preparation. Generally I add about 50 microgram ampicilline every hour. Does anybody know about the dose required to maintain plasmid, how fast amp. is depleted from the medium? At the end of 6 hours we are looking at 1.5 kg of E. coli, hence wonder about how much ampicilline should be added. I assume you mean 50 ug/ml. I don't think seggregation is fast enough to hurt you in the last 6 hr. of growth, so I'd concentrate on making sure you managed to grow the preculture without seggregants. I think 100 ug/ml at the beginning of the preculture is probably enough; but I suppose an additional 50 ug/ml in the 100L media for safety wouldn't hurt. I'd worry a lot more about T1 infestation than plasmid seggregation at this scale. 100 L fermentors aren't really sterile. Phage T1 grows aggressively on most coli strains making plaques the size of a quarter. T1 survives dessication so it can spread on air currents and has been known to contaminate walls and ventillation systems for years. So if you clear a 100L culture with T1, it ends up blowing all around the building and essentially shuts down all work with ordinary coli strains for a few years. This makes you very unpopular. I don't know how frequently this happens, but I have heard of a couple of cases. The tonA mutation renders strains resistant to T1. The only strain that has it that I know off the top of my head is C600. You can select tonA- strains by resistance to T5. As a corallary, anyone who has a culture clear mysteriously: don't open it; sterilize it immediately. Cheers, Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio Hardies@uthscsa.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!daresbury!not-for-mail From: MIcha Ron Newsgroups: bionet.molbio.methds-reagnts Subject: ABI 377 Date: 2 Feb 1995 19:44:55 -0000 Lines: 10 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3grcnn$qct@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk Just saw a brochure for ABI's new sequencer, 377. It seems that the main advantage is faster gel runs.Has anbody any experience with this new machine? Appreciate any feedback. Mark Band ARO Volcani Center Israel email: michar@indycc1.agri.huji.ac.il From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!rutgers!gatech!newsxfer.itd.umich.edu!zip.eecs.umich.edu!caen!reeve.research.aa.wl.com!dom006.dom.aa.wl.com!user From: pumiglk@aa.wl.com (Kevin Pumiglia) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: reference manager? Followup-To: bionet.molbio.methds-reagnts Date: 2 Feb 1995 12:25:57 GMT Organization: U of Michigan/Parke-Davis Lines: 23 Message-ID: References: <3goj7f$1c7c@bigblue.oit.unc.edu> NNTP-Posting-Host: dom006.dom.aa.wl.com In article <3goj7f$1c7c@bigblue.oit.unc.edu>, guest@halley.sph.unc.edu wrote: > I'm sorry if this is an inappropriate post, but I imagine that some of you must > have some experience with reference managers that are specifically formatted > for scientific journals. I'm looking for the best software package to manage > references, create bibliographies with different formats (depending on the > journal I'm submitting to), accept downloaded info from current content > searches, etc. We use IBMs in the lab, so the program has to be compatible with > DOS and Window. I've heard of EndNote, ProCite and Papyrus(?). Do any of you > use these? What do you think about them? > Thanks for your help. Camella > You can email me directly at cbailey@uncvx1.oit.unc.edu I use Papyrus! Inexpensive, reliable, and flexible.. if not the slickest interface in the world. The program runs in Dos, but seamlessly interfaces with windows through the clipboard. It is the most stable program I have ever run in a Dos window-others software companies should take note. The links to wordperfect (my preference are very good) as are the importfunctions from paperchase (Medline). Has filters for all of the common imports, e.g. Silver platter, current contents etc. and most major word processors. Very satisfied customer every time I write! No afiliation etc.. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!bcm!cs.utexas.edu!swrinde!howland.reston.ans.net!gatech!udel!news-4.nss.udel.edu!strauss.udel.edu!not-for-mail From: mcdonald@strauss.udel.edu (John H McDonald) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Dye-Deoxy Cycle Sequencing-ramp time? Date: 30 Jan 1995 18:02:56 -0500 Organization: University of Delaware Lines: 22 Message-ID: <3gjr70$hjo@strauss.udel.edu> References: NNTP-Posting-Host: strauss.udel.edu In article , James Gray wrote: > Does anyone know how important ramp time REALLY is in Taq >Dye-Deoxy cycle sequencing? I am using an MJ Research Minicycler using >the 0.2ml tubes and the default ramp times on the cycler. This machine >has a noticeably faster ramp time than the recommended Perkin-Elmer >machine (1C/sec). I find that my reads fall off at about 90 bases and >the background is high. > We do dye-terminator cycle sequencing for the ABI 373 using an Idaho Technologies thermal cycler, which has much faster ramp times than your poky ol' MJ, and it works fine. Make sure your extension time is at least three minutes, since the modified nucleotides used in dye-terminator sequencing get added quite slowly. Also, try to quantify your template; noisy sequence that starts strong, then drops off rapidly can be caused by too much template DNA. John H. McDonald Department of Biology University of Delaware From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!bcm!cs.utexas.edu!convex!news.duke.edu!godot.cc.duq.edu!newsfeed.pitt.edu!dsinc!netnews.upenn.edu!msunews!NewsWatcher!user From: 22313tcn@ibm.cl.msu.edu (Thomas Newman) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Dye-Deoxy Cycle Sequencing-ramp time? Followup-To: bionet.molbio.methds-reagnts Date: Mon, 30 Jan 1995 17:51:39 -0500 Organization: MSU-DOE Plant Research Lab Lines: 20 Message-ID: <22313tcn-300195175139@35.8.197.53> References: NNTP-Posting-Host: 35.8.197.53 In article , James Gray wrote: > Does anyone know how important ramp time REALLY is in Taq > Dye-Deoxy cycle sequencing? I am using an MJ Research Minicycler using > the 0.2ml tubes and the default ramp times on the cycler. This machine > has a noticeably faster ramp time than the recommended Perkin-Elmer > machine (1C/sec). I find that my reads fall off at about 90 bases and > the background is high. > We just ran some DNA samples (0.2ug/um promega wizard prepped) on the new MJR DNA Engine, with 3C/sec ramp times and the results were fine. If the initial peaks from the sequencer are very strong, you may be starting with too much DNA in the sample which results in early garbage. Tom Newman MSU-DOE Plant Research Lab Sequencing Facility From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!hubcap.clemson.edu!biosci!bcm!cs.utexas.edu!swrinde!howland.reston.ans.net!vixen.cso.uiuc.edu!uchinews!kimbark!jm68 From: jm68@kimbark.uchicago.edu (Jim Mensch) Subject: Re: gt11 blue/white plaques? Message-ID: <1995Jan30.224552.29636@midway.uchicago.edu> Summary: alpha complementation on Y1090? Sender: news@uchinews.uchicago.edu (News System) Reply-To: jm68@midway.uchicago.edu Organization: University of Chicago References: <1995Jan27.180623.18151@midway.uchicago.edu> Distribution: na Date: Mon, 30 Jan 1995 22:45:52 GMT Lines: 8 Warren, Thanks for your interest. The plating described was indeed done on Y1090, the expression specific host strain. Would blue plaques on this host necessarily indicate non-recombinant phage? We have not re-amplified this library, it is as we obtained it from ATCC. Thanks again. -- -- Jim Mensch From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!MOLBIO.UOREGON.EDU!KORTE From: KORTE@MOLBIO.UOREGON.EDU ("JOHN A. KORTE") Newsgroups: bionet.molbio.methds-reagnts Subject: Plant Genomic DNA (CTAB?) Date: 30 Jan 1995 14:55:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <950130153845.20c54848@molbio.uoregon.edu> NNTP-Posting-Host: net.bio.net Greetings, By any chance does anyone out there have protocols for isolating genomic DNA from plants that leave the DNA clean and very restrictable (is that a proper term?)? I believe that people have mentioned CTAB methods before, but, did not elaborate. If anyone has a protocol could you send it to me? Thanks, John From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!bcm!cs.utexas.edu!howland.reston.ans.net!ix.netcom.com!netcom.com!csus.edu!news.ucdavis.edu!usenet From: Chris Barry Newsgroups: bionet.molbio.methds-reagnts Subject: Re: fluorescent in situ PCR - bacteria Date: 30 Jan 1995 20:27:17 GMT Organization: University of California, Davis Lines: 16 Message-ID: <3gji35$rt1@mark.ucdavis.edu> References: NNTP-Posting-Host: modem174.ucdavis.edu alpc@liverpool.ac.uk (Mr. D.A. Deere) wrote: > > Has anyone out there come across any successful conclusive > work in which individual bacterial cells could be visualised from mixed > background populations using in situ PCR? I don't know is this answers your question or not. But I have done some FISH in eukaryotes and this is how it works for me. Label your probe with one color fluorophore (ie: Texas Red, Cy3, fluorocine, etc.) and use a filter on an epi-fluorescence microscope that allows you to visualize that color. The prokaryote you are looking for should stand out from the others. Chris Barry From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!daresbury!not-for-mail From: (Dave Johnston) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: fluorescence microscopy Date: 2 Feb 1995 08:21:07 -0000 Lines: 29 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3gq4lj$mmu@mserv1.dl.ac.uk> X-POPmail-Charset: British Original-To: cgrunau%postman.riken.go.jp@earn-relay.ac.uk, methods@dl.ac.uk On 1 Feb 95 04:58:16 GMT, Christoph Grunau writes: > >Does anyone has experience using DABCO (1,4-diazabicyclo [2.2.2] - octane >or p-Phenylenediamine in fluorescence microscopy. Which of both can you >recommend or in which special case is one better than the other one. > See J. Immunol Methods 55 (1982), 231-242 for comarative data. Basically PPD is most potent but DABCO is cheap, stable, readily available and, IMHO, brilliant! RECIPE Glycerol: 9 parts PBS: 1 part (Dulbecco's "A" PBS or 0.15M NaCl in 0.01M phosphate buffer) DABCO: 2.5g / 100mls pH final solution to 8.6 (optimum for FITC fluorescence) - this is a bit (very) difficult due to viscosity. In my experience with FITC labelled antibodies staining microtubules, I could still clearly see and photograph microtubules after 30 minutes continouus exposure to the excitation beam. DAJ David A. Johnston Research Fellow, Dept of Zoology, The Natural History Museum, Cromwell Road, South Kensington, London SW7 5DB. England (tel 071 9389297, fax 071 9388754, email daj@nhm.ac.uk) From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!zenith.Berkeley.EDU!mytelka From: mytelka@zenith.Berkeley.EDU ( Daniel Mytelka ) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: his tag protein storage Date: 2 Feb 1995 08:10:35 GMT Organization: University of California, Berkeley Lines: 47 Message-ID: <3gq41r$gam@agate.berkeley.edu> References: NNTP-Posting-Host: zenith.berkeley.edu In article , *.* wrote: >>Date: 24-JAN-1995 15:08 >>From: sl16s@cc.usu.edu (NAME RAVI PRAKASH .D) >>Description: his tag protein storage >> >>I am trying to purify His tag fusion protein in pET vectors. >>I would be great full if someone can tell me how I can store >>the protein for long term. If anyone has any expertise in this >>matter I would be happy toar from you. >>thank you vey much. >> > Hi, there. You forgot to mention, at which conditions >(native/denaturated) you purify your protein. > > Since sprig last year, I have my proteins samples stored at -85. >Routinely I`ve freeze and thaw one tube and for short term storage used a >-20 freezer. As long as 6 month late I compared the samples in SDS-PAGE, and >in ligand blot experiments. The gel after SDS-PAGE I stained by silver >nitrate. There was no difference between the samples in both experiments. >However, I`d recommend to aliquot the samples and store them at -85 (read >-70, in our department all deep freezers adjusted to -85), and for routine >experiments store sample at -20. >The samples mentioned above were in elution buffer from Ni-NTA resin (8 M >Urea; 0.1 M NaH2PO4; 10 mM Tris; 10 mM b-MEt; 0.1 mM PMSF, 1 mM Benz*HCl; pH >4.5). > In case you purify the protein at native conditions, I`d recommend >following extra addition to the basic buffer: 2-5 mM b-Met; 40-30% Glycerol; >1-0.2% NP-40; 5-10 mM MgCl2; the NaCl and KCl should be at physiological >concentrations (as in PBS). >----------- > Nikolai. > >Child says: "If All Fails Carefully Read the Manual" >----------------------------------------------------------- The more general answer is to just try and find what your protein likes. Find a protein that's as simialr to your protein as possible that has already been purified and find out what it has been stored in. Vary things and look at stability. I have one protein that's stable in a simple column elution buffer on ice for a year, and another that dies in a week at in 50% glycerol at -20. NP-40, for example, can cause problems with some multimeric proteins, but increases solubility for others. Make sure you include any necessary ions/cofactors. Dan Mytelka From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!news.cs.umb.edu!hsdndev!purdue!news.bu.edu!med-pharm16.bu.edu!user From: leach@bu.edu (Martin Leach) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: TA Cloning - old product Date: 2 Feb 1995 18:12:33 GMT Organization: Boston University Dept. of Pharmacology Lines: 31 Message-ID: References: <9500307915.AA791514934@tcplink.nrel.gov> NNTP-Posting-Host: med-pharm16.bu.edu In article , mes@zoo.toronto.edu (Mark Siddall) wrote: > I am attempting to clone my pcr product with the TA system. First > attempt failed. > Product was about a week old but had been at -20C the whole time. > Now it's about 2 weeks old though still at -20C. > Any body know what the likelihood of having lost the adenosine > residues might be? > I think it should be fine...i've used older products...if in doubt..incubate the purified product with dATP and Taq pol fro 1-2hrs at 72C in PCR buffer. Martin -- ..... Martin Leach Email:leach@bu.edu _|____ Dept. of Pharmacology Phone: (617) 638-5323 / o / Boston Univ. School of Med. Fax: (617) 638-4329 _/ |-/__==/ 80 E. Concord St. (L603) (BULLDOZER) \_ Boston MA 02118 "Not the old underpants on your USA head.....WIBBLE" -BLACKADDER p.s. try BioMOO (virtual biology on the internet - telnet bioinfo.weizmann.ac.il 8888) My home page: http://155.41.115.114/Leach.html From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!news.cs.umb.edu!hsdndev!purdue!news.bu.edu!med-pharm16.bu.edu!user From: leach@bu.edu (Martin Leach) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Need Protocol for Nucleus isolation from rat spleen lymphocytes. Date: 2 Feb 1995 18:10:42 GMT Organization: Boston University Dept. of Pharmacology Lines: 31 Message-ID: References: NNTP-Posting-Host: med-pharm16.bu.edu In article , hadimani@powai.cc.iitb.ernet.in (shreeshail kumar) wrote: > I need a protocol for isloating nuclei from rat spleen lymphocytes, beginning from the sacrificing of the rats. Also the protocols for the marker enzyme assays needed. I would be very grateful if I could get these at the earliest. > Thanx. > n > :p > :q I believe current protocols has something on preparation of nucleii../nuclear extracts... Martin -- ..... Martin Leach Email:leach@bu.edu _|____ Dept. of Pharmacology Phone: (617) 638-5323 / o / Boston Univ. School of Med. Fax: (617) 638-4329 _/ |-/__==/ 80 E. Concord St. (L603) (BULLDOZER) \_ Boston MA 02118 "Not the old underpants on your USA head.....WIBBLE" -BLACKADDER p.s. try BioMOO (virtual biology on the internet - telnet bioinfo.weizmann.ac.il 8888) My home page: http://155.41.115.114/Leach.html From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!daresbury!not-for-mail From: spehr Newsgroups: bionet.molbio.methds-reagnts Subject: Needed: T7-RNA-Polymerase-gen Date: 2 Feb 1995 18:56:43 -0000 Lines: 10 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3gr9tb$o3m@mserv1.dl.ac.uk> X-Minuet-Version: Minuet1.0_Beta_14 X-POPMail-Charset: English Original-To: methods@dl.ac.uk I am looking for an active T7-RNA-Polymerase-gen, located on a plasmid. I need it for expression-studies in E. coli. In the mean time I welcome any suggestions that you may have. Please respond by e-mail to volker.spehr@uni-duesseldorf.de Thanks in advance Volker From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!news.cs.umb.edu!hsdndev!purdue!haven.umd.edu!news.umbc.edu!usenet From: Thomas O'Neill Newsgroups: bionet.molbio.methds-reagnts Subject: LexA antibodies Date: Thu, 2 Feb 1995 12:04:19 -0500 Organization: University of Maryland, Baltimore County Lines: 4 Message-ID: NNTP-Posting-Host: f-umbc8.umbc.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: toneil1@umbc8.umbc.edu Anyoneknow where I can buy some (good) LexA antibodies for Western blot analysis? Thanks, T.O'N. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!BORCIM.WUSTL.EDU!saboteur From: saboteur@BORCIM.WUSTL.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: re: ramp time & cycle seqn Date: 2 Feb 1995 09:40:02 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <199502021739.AA19067@wugate.wustl.edu> NNTP-Posting-Host: net.bio.net James Gray wrote: > Does anyone know how important ramp time REALLY is in Taq >Dye-Deoxy cycle sequencing? I am using an MJ Research Minicycler using >the 0.2ml tubes and the default ramp times on the cycler. This machine >has a noticeably faster ramp time than the recommended Perkin-Elmer >machine (1C/sec). I find that my reads fall off at about 90 bases and >the background is high. I tried the ABI single-tube seqn reactions on a stratagene machine we had out on a test drive, one of those ones that has 4 blocks and a robotic arm that physically picks up the tubes from one block and drops them onto another. Presumably this "eliminates" ramp time. The reactions didn't work. Using the same melt/anneal/extension times & temps on a PE 9600 we also had out on loan, they did work, and beautifully. Note this machine still ramps, but is one of the fastest "rampers" around. My conclusion is that it probably is important to have *some* ramp time, but you don't need much. We got the PE 9600. Brett Lindenbach Lucille P. Markey Student in Human Pathobiology Program in Immunology Washington University - St Louis brett@borcim.wustl.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!caen!hearst.acc.Virginia.EDU!murdoch!usenet From: "R. John Lye" Subject: Re: Shearing DNA to ~2Kb X-Nntp-Posting-Host: bootp-17-100.bootp.virginia.edu Message-ID: Sender: usenet@murdoch.acc.Virginia.EDU Organization: University of Virginia References: <3gp2jj$1bk@pith.uoregon.edu> Date: Thu, 2 Feb 1995 16:55:25 GMT Lines: 11 > I would like to shear a sample of genomic DNA down to about 2 Kb and > don't know of any shearing method that will get it down that small. Can > anybody recommend a shearing method that will give me the desired > results? > Schriefer, et al. N.A.R. 18:7455-7456 (1990) describe a French press method that I've used with good results. cheers, John From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!agate!susana From: susana@nature.Berkeley.EDU (Susan Abrahamson) Newsgroups: bionet.molbio.methds-reagnts Subject: CHO DNA Date: 31 Jan 1995 00:23:30 GMT Organization: U.C. College of Natural Resources Lines: 8 Message-ID: <3gjvu2$r8u@agate.berkeley.edu> NNTP-Posting-Host: nature.berkeley.edu We have been having trouble detecting genomic CHO DNA in a dot-blot hybridization assay. When decreasing amounts of pure genomic DNA are applied to a positively charged Nylon membrane and detected by hybridization with the same DNA labeled with 32-P, we find a 10-fold lower sensitivity with CHO DNA (5 pg) as compared to mouse or human genomic DNA (0.5 pg). Has anyone else ever had this problem with CHO DNA? Suggestions? From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!scripps.edu!NewsWatcher!user From: drjon@clone.scripps.edu (Jon Chappel) Newsgroups: bionet.molbio.methds-reagnts Subject: beta lactamase fusions Date: Mon, 30 Jan 1995 16:15:59 -0800 Organization: The Scripps Research Institute Lines: 12 Message-ID: NNTP-Posting-Host: collossus.scripps.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit Dear All Has anyone out there had any success purifying beta-lactamases using phenyl boronate sepharose. I bought some some Phe/bo resin from Mobitec and followed their (Kolmar et al.) instructions and only managed to get a meager amount of protein to bind to the resin. No use at all !! Anyone got any tips? TIA, Jon From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!rutgers!gatech!swrinde!pipex!uunet!news.inhouse.compuserve.com!news.production.compuserve.com!news From: Dr. Ulrich Thoenes <100331.3506@CompuServe.COM> Newsgroups: bionet.molbio.methds-reagnts Subject: List of restriction enzymes Date: 31 Jan 1995 00:07:30 GMT Organization: via CompuServe Information Service Lines: 15 Message-ID: <3gjv02$r3v$1@mhadg.production.compuserve.com> Hello to you all Is there anybody out there who knows about a list of restriction enzymes with the following informations: The name The availabolity from several companies The number of units per ul The buffer conditions The price Thanks a lot for any answer !! My e-mail is: 100331.3506@compuserve.com Best Regards, Ulli Thoenes From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!YODA.NIDR.NIH.GOV!moore From: moore@YODA.NIDR.NIH.GOV (Marilyn Moore) Newsgroups: bionet.molbio.methds-reagnts Subject: RE: RNA sequencing Date: 2 Feb 1995 08:58:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <199502021658.IAA07150@net.bio.net> NNTP-Posting-Host: net.bio.net I have successfully sequenced RNA using the basic methodology of primer extension. I was able to read about 30 bases 5' of the clone that I had (which was the length predicted from primer extension). There is a description of this method by Hahn et al. in Methods of Enzymology, 180:120. It was not an easy method for sequencing, and until I had all of the conditions ideal for each nucleotide it took 4 trys...but it did work. Good luck, -- Marilyn L. Moore NIH/NIDR/CIPCB moore@yoda.nidr.nih.gov From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!msunews!netnews.upenn.edu!wal6000a.udc.upenn.edu!ljohnson From: ljohnson@udcemail.udc.upenn.edu (Lois C. Johnson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: HELP: PLASMIDS & Transfromation Date: 2 Feb 1995 16:12:04 GMT Organization: University of Pennsylvania Lines: 16 Distribution: bionet Message-ID: <3gr08k$ndb@netnews.upenn.edu> References: NNTP-Posting-Host: wal6000a.udc.upenn.edu X-Newsreader: TIN [version 1.2 PL2] Just a thought: Has anyone had problems with transforming plasmids with BRL ME DH5a cells? Do you have problems with varience; that is one time it works and another time it doesn't!? Does anyone have any hints? Could it be too much antibiotic? Could I have "spread them wrong"? It's something weird & I've taxed my brain trying to figure out the source. All ideas & helpful hint will be greatly appreciated. ThANKS IN ADVANCE! Lois Johnson Monell Chemical Senses Center Phila., PA 19104 215-573-3328 21-898-2084 (fax) ljohnson@udcemail.udc.upenn.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!agate!howland.reston.ans.net!Germany.EU.net!Belgium.EU.net!ub4b!idefix.CS.kuleuven.ac.be!infoserv.rug.ac.be!news From: dieter.deforce@rug.ac.be Newsgroups: bionet.molbio.methds-reagnts Subject: Heeeeeeeeeeelp nucleoprobs Date: 2 Feb 1995 15:50:09 GMT Organization: Pharmaceutical Biotechnology Lines: 16 Distribution: world Message-ID: <3gquvh$cig@infoserv.rug.ac.be> NNTP-Posting-Host: farmbio2.rug.ac.be X-Newsreader: Hello, A question: I'm digesting DNA to mononucleotides with enzymes So I need to buffer it After the reaction I have to clean up the nucleotides and get rid of the enzymes and the buffer ions MG of the nucleos 340 reaction volume 1 ml What do I use dialysis has a cut of of 1000 MW Gelfiltration??? Thanks From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!agate!newsxfer.itd.umich.edu!gatech!udel!news-4.nss.udel.edu!strauss.udel.edu!not-for-mail From: mcdonald@strauss.udel.edu (John H McDonald) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: ABI 48cm strech problem Date: 2 Feb 1995 11:25:55 -0500 Organization: University of Delaware Lines: 27 Message-ID: <3gr12j$b5n@strauss.udel.edu> References: <9502021431.AA00258@phage.cshl.org> NNTP-Posting-Host: strauss.udel.edu In article <9502021431.AA00258@phage.cshl.org>, Muhammad Lodhi wrote: >Lately we are having lots of trouble with our 48 cm strech. We have tried >running gels with Taq terminator and dye primer rxns on it and most of the >sequences, even though look good on gel, are pretty bad. Sequences are >noisy, base spacing is -12 and peaks tend to overlap. This is mostly but >not entirely true for rxns with forward primers. I am wondering if anyone >has any idea how to correct it or what are we doing wrong. Is there any >problem with the sequencer matrix of filter set A. Any help/advice will be >appreciated. If the Gel Image looks good, but the analyzed data are crappy and the base spacing is -12, your gel probably ran either too fast or too slow. The version of the Analysis program that I have throws up its hands in confusion at any peak spacing less than 9 or greater than 15 scans/peak and uses a default value of 12. Since peak spacing is an important parameter in the algorithm, this leads to junky looking analyzed sequence. Check the EPT windows to see if your voltage is different now than it used to be when things were working well, since higher voltage=faster running gels=fewer scans/peak. My guess is that either someone made up a new batch of buffer at a slightly different ionic strength, or someone forgot to filter out the Amberlite beads before adding buffer to the gel mix, or someone has been messing with the settings on the machine. John H. McDonald Department of Biology University of Delaware From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!torn!ccshst05.cs.uoguelph.ca!ccshst01.cs.uoguelph.ca!awright From: awright@uoguelph.ca (Andre-Denis G Wright) Newsgroups: bionet.molbio.methds-reagnts Subject: Removing CTAB from Nucleic Acids Date: 2 Feb 1995 15:50:23 GMT Organization: University of Guelph Lines: 8 Message-ID: <3gquvv$7h6@ccshst05.cs.uoguelph.ca> NNTP-Posting-Host: ccshst01.cs.uoguelph.ca X-Newsreader: TIN [version 1.2 PL2] Dear Netters! I extracted DNA using CTAB to remove polyusacchardides, but the CTAB coprecipitated with my nucleic acids. I kept the NaCl conc above the recommended 0.7M so this wouldn't happen, but it still did. Any suggestions? Andre From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts From: Duncan@genesys.demon.co.uk (Duncan Clark) Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!pipex!peernews.demon.co.uk!genesys.demon.co.uk!Duncan Subject: Re: TA Cloning - old product References: <9500307915.AA791514934@tcplink.nrel.gov> Organization: GeneSys Ltd. Reply-To: Duncan@genesys.demon.co.uk X-Newsreader: Newswin Alpha 0.7 Lines: 21 X-Posting-Host: genesys.demon.co.uk Date: Thu, 2 Feb 1995 16:03:35 +0000 Message-ID: <800474327wnr@genesys.demon.co.uk> Sender: usenet@demon.co.uk In article: mes@zoo.toronto.edu (Mark Siddall) writes: > > > > I am attempting to clone my pcr product with the TA system. First > attempt failed. > Product was about a week old but had been at -20C the whole time. > Now it's about 2 weeks old though still at -20C. > Any body know what the likelihood of having lost the adenosine > residues might be? > Easy solution. Run an extension at 72C for 15mins with just dATP. Single base should be added. Duncan ----------------------------------------------------------------------------- My mind's made up. Don't confuse me with the facts! From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!torn!newshost.uwo.ca!julian.uwo.ca!ovanham From: ovanham@julian.uwo.ca (o. van-ham) Newsgroups: bionet.molbio.methds-reagnts Subject: Cracked/wrinkled sequencing gel. Date: 2 Feb 1995 15:31:35 GMT Organization: University of Western Ontario, London, Ont. Canada Lines: 15 Message-ID: <3gqtsn$cbn@falcon.ccs.uwo.ca> NNTP-Posting-Host: julian.uwo.ca I've just prepared an 8% sequencing gel using the bio rad Sequi-Gen system. The gel was poured yesterday and left over night covered with seran wrap. It was made using a sharks tooth comb and wedged shaped spacers, and was clamped at the top (to keep the comb tight). This morning I noticed some wrinkles/fractures in one corner of the gel, near the top. As I'm pre-warming the gel, prior to loading, the fractures are spreading across the top of the gel and down into it. I'll try running it nonetheless, but would hope to prevent this from happening again. If anyone has had this happen, and been able to correct the problem, please post the solution, or e-mail me directly at: ovanham@uwo.ca Thanks Oded Van-Ham From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!pipex!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: "k.d.preuss" Newsgroups: bionet.molbio.methds-reagnts Subject: Poblem with extended PCR Date: 2 Feb 1995 15:46:24 -0000 Lines: 39 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3gquog$e7r@mserv1.dl.ac.uk> Reply-To: tm12kpkp@rz.uni-sb.de X-NUPop-Charset: English Original-To: methods@dl.ac.uk Hi netters, I have a problem making extended PCR. I am able to produce two PCR fragments each by using specific primers. Both fragments (each around 1 kb) have an overlapping region of about 80 nt and I want to fuse them by PCR. P1-> f1: ====================================== <-P2 P3-> f2: ================================= <-P4 I made asymetric PCR using f1 with P1 and f2 with P4. After purifying the products by agarose gels I mixed f1asym and f2asym. After denaturation and reannealing I made 10 cycles 94!C 1 min, 72!C 2 min. Then I added P1 and P4 and made 30 cycles (94!C 30sec, 55!C 30 sec, 72!C 2 min). No full length product is detectable even by hybridization. Questions: - Why isn't it working? - Does anybody have a real working protocol? Please answer to tm12kpkp@rz.uni-sb.de Thanks in advance Dieter ****************************************************************** * Dr.K.D.Preuss E-MAIL: tm12kpkp@rz.uni-sb.de * * Physiologie II Phone: +6841-166123 * * Universitaet d. Saarlandes FAX: +6841-166655 * * 66421 Homburg/Saar * * Germany * ****************************************************************** From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!MED.UNC.EDU!syc From: syc@MED.UNC.EDU (Shao-Yu Chen) Newsgroups: bionet.molbio.methds-reagnts Subject: DNA fragment Date: 2 Feb 1995 07:29:19 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502021529.AA01220@earl.med.unc.edu> NNTP-Posting-Host: net.bio.net We have tried to assay DNA fragment from kidney using following protocol: 1. Tissue homogenate was lysed with hypotonic lysing buffer(10mMTris,1 mMEDTA) containing 0.2% Triton X-100 for 20 min. 2. The lysates were centrifuged at 13,000XG for 10min to separeate intact from fragmented chromatin . 3. Both pellet and supernatant were precipitated overnight at 4C in 12.5% trichloroacetic acid. The precipitates were sedimented at 13,000 XG for 4min. 4. The DNA in the precipitates was hydrolyzed by heating to 90C for 10min in 5% trichloroacetic acid and was quantitated by the diphenylamine method. However , the DNA in the precipitates can not be hydrolyed by heating to 90C(or boiling ) in 5% trichloroacetic acid. Can anyone out there give me some advice? Thank you in advance. Shaoyu Chen UNC- CH syc@med.unc.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!uunet!news.uiowa.edu!blue.weeg.uiowa.edu!blue.weeg.uiowa.edu!mdeshpan From: deshpande milind Newsgroups: bionet.molbio.methds-reagnts Subject: Ampicillin addition for plasmid Date: Thu, 2 Feb 1995 08:55:24 -0600 Organization: University of Iowa, Iowa City, IA, USA Lines: 6 Distribution: world Message-ID: NNTP-Posting-Host: blue.weeg.uiowa.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I am growing E. coli at 100 liter scale for plasmid preparation. Generally I add about 50 microgram ampicilline every hour. Does anybody know about the dose required to maintain plasmid, how fast amp. is depleted from the medium? At the end of 6 hours we are looking at 1.5 kg of E. coli, hence wonder about how much ampicilline should be added. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!gatech!newsxfer.itd.umich.edu!agate!cat.cis.Brown.EDU!cis-ts3-slip5.cis.brown.edu!user From: Stephen_Lasky@brown.edu (Stephen R. Lasky, Ph.D.) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: reference manager? Date: 2 Feb 1995 15:02:25 GMT Organization: Roger Williams Hospital/Brown U. Lines: 42 Message-ID: References: <3goj7f$1c7c@bigblue.oit.unc.edu> NNTP-Posting-Host: cis-ts3-slip5.cis.brown.edu In article <3goj7f$1c7c@bigblue.oit.unc.edu>, guest@halley.sph.unc.edu wrote: > I'm sorry if this is an inappropriate post, but I imagine that some of you must > have some experience with reference managers that are specifically formatted > for scientific journals. I'm looking for the best software package to manage > references, create bibliographies with different formats (depending on the > journal I'm submitting to), accept downloaded info from current content > searches, etc. We use IBMs in the lab, so the program has to be compatible with > DOS and Window. I've heard of EndNote, ProCite and Papyrus(?). Do any of you > use these? What do you think about them? > Thanks for your help. Camella > You can email me directly at cbailey@uncvx1.oit.unc.edu I used to use Reference Manager but have switched to Endnote Plus (Both on a mac). I think that Endnote is better than RefMan, but there is a problem, on Mac's anyway, in that it doesn't work with Word6 and probably won't for a while. Don't know if this is true on the IBM versions. From a Mac perspective, I would recommend Endnote. It does very fast searches and can use multiple boolian operators. This helps when you have a large database (mine is almost 7000 refs). The newest version has lots of journal formats (hundreds?) and you can make it import references in any format you get them in by creating a little filter file which is not hard. Usual disclaimers: I'm not associated with Endnote but I like it. SRLasky SRLasky -- ********************************************************************* Stephen R. Lasky, Ph.D. Roger Williams Medical Center/Brown University Phone: 401-456-6572 Fax: 401-456-6569 e-mail: Stephen_Lasky@brown.edu ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ "The speed of a computer is inversly proportional to the legnth of time it has been on your desk." Michael Yablonski, circa 1989 ********************************************************************* From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!daresbury!not-for-mail From: Clemens Suter-Crazzolara Newsgroups: bionet.molbio.methds-reagnts Subject: semi-quant pcr Date: 2 Feb 1995 15:07:39 -0000 Lines: 18 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3gqsfr$ccu@mserv1.dl.ac.uk> MIME-Version: 1.0 Original-To: methods@dl.ac.uk Hello, a question about semi-quantitative PCR: I would like to have a control primer pair of a gene that is constitutively expressed in the eukaryotic (rat) cell. Up to now I have been using S12: a very abundant ribosomal protein. Disadvantage; S12 is so abundant, that all cDNAs must be diluted 1000-fold before anything can be concluded. Do you know of a rather rare RNA that is constitutively expressed in the cell ? Also, would it be theoretically possible to mix the standard primers with the target primers in one reaction... and still be semi-quantitative ? (somebody in our lab is trying this, however I can imagine both primer pairs me interfere with eachother...) clemens, Heidelberg From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!TCPLINK.NREL.GOV!roesslep From: roesslep@TCPLINK.NREL.GOV Newsgroups: bionet.molbio.methds-reagnts Subject: luciferase clone wanted Date: 30 Jan 1995 16:15:13 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <9500307915.AA791514934@tcplink.nrel.gov> NNTP-Posting-Host: net.bio.net Has anyone by chance constructed a luciferase gene that has an NcoI (or compatible) site on the 5' end? Cheers, Paul Roessler National Renewable Energy Lab Golden, Colorado USA paulr@nrel.gov From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!rutgers!uwm.edu!lll-winken.llnl.gov!enews.sgi.com!decwrl!tribune.usask.ca!quartz.ucs.ualberta.ca!unixg.ubc.ca!sidky From: sidky@unixg.ubc.ca (adam omar sidky) Newsgroups: bionet.molbio.methds-reagnts Subject: in situ probes-HELP Date: 30 Jan 1995 21:03:37 GMT Organization: University of British Columbia, Vancouver, B.C., Canada Lines: 6 Message-ID: <3gjk79$9st@nnrp.ucs.ubc.ca> NNTP-Posting-Host: interchg.ubc.ca X-Newsreader: TIN [version 1.2 PL2] I am trying to find an in situ probe and I don't want it to hybridize with anything else, is there any program available on internet to help with this or if you know anything about this please reply to (or post): sidky@unixg.ubc.ca thanks From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!agate!newsxfer.itd.umich.edu!zip.eecs.umich.edu!panix!cmcl2!yale.edu!news.ycc.yale.edu!primer!stephen From: stephen@primer.med.yale.edu (stephen marshalko) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: KlenTaq/Pfu for LA-PCR? Date: 2 Feb 1995 13:33:26 GMT Organization: Yale University Lines: 31 Message-ID: <3gqmv6$12r@news.ycc.yale.edu> References: <3gocdv$dr7@quartz.ucs.ualberta.ca> NNTP-Posting-Host: primer.med.yale.edu X-Newsreader: TIN [version 1.2 PL2] John Spafford (jspaffor@gpu2.srv.ualberta.ca) wrote: : Who sells KlenTaq/Pfu for W. Barne's LA-PCR? : Thanks in advance, : David Spafford : University of Albert As far as I am aware, no one sells the combination of the two thermostable polymerases. However, each can be purchased separately from the following vendors. Klentaq: Ab Peptides, Inc 6272 Marmaduke Ave St. Louis, MO 63139 1-800-383-3362 Pfu: Stratagene, Inc 11011 North Torrey Pines Road La Jolla, CA 92037 1-800-424-5444 I used this combination, with good success, for my long PCR needs. Hope this info in helpful. Stephen From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!agate!howland.reston.ans.net!news2.near.net!cat.cis.Brown.EDU!cis-ts3-slip5.cis.brown.edu!user From: Stephen_Lasky@brown.edu (Stephen R. Lasky, Ph.D.) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RNA stat-60? Date: 2 Feb 1995 14:52:49 GMT Organization: Roger Williams Hospital/Brown U. Lines: 17 Message-ID: References: NNTP-Posting-Host: cis-ts3-slip5.cis.brown.edu In article , pdzah@pdn1.gene.nottingham.ac.uk (Alan Hair) wrote: > Could anyone tell me who manufacture an RNA extraction kit called RNA > stat-60? > Thanks Al Tel-Test "B" Inc in Friendsville TX -- ********************************************************************* Stephen R. Lasky, Ph.D. Roger Williams Medical Center/Brown University Phone: 401-456-6572 Fax: 401-456-6569 e-mail: Stephen_Lasky@brown.edu ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ "The speed of a computer is inversly proportional to the legnth of time it has been on your desk." Michael Yablonski, circa 1989 ********************************************************************* From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!howland.reston.ans.net!news2.near.net!cat.cis.Brown.EDU!cis-ts3-slip5.cis.brown.edu!user From: Stephen_Lasky@brown.edu (Stephen R. Lasky, Ph.D.) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Does Schleicher & Schuell BA-S 85 go off???? Date: 2 Feb 1995 14:49:09 GMT Organization: Roger Williams Hospital/Brown U. Lines: 36 Distribution: world Message-ID: References: <950201132834.22488f@thorin.uthscsa.edu> NNTP-Posting-Host: cis-ts3-slip5.cis.brown.edu In article <950201132834.22488f@thorin.uthscsa.edu>, HARDIES@THORIN.UTHSCSA.EDU wrote: > jpcd0@mole.bio.cam.ac.uk (John Dixon) asked if S&S nitrocellulose goes > off and how you can tell if it has. > > We see it go off if the role has been opened and kept around a year or > so. You can tell if you wet it in water before the transfer buffer. If > it doesn't wet immediately and uniformly, it's no good. Typically, the > outer layer of the role and the edges go first and the rest can be salvaged. > So it's obviously exposure to something in the air. Humidity sounds as > good as any. I think the problem did get worse when I moved to a more > humid environment, but I can't swear to it. > I assume this would affect nitrocellulose from other vendors > as well, but not nylon based filters. Do any manufacturers of NC pretreat > their membrane so it stores better? > > Cheers, > Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio > Hardies@uthscsa.edu I believe that I have had the same problem with old S&S nytran filters, so I'm not sure that it doesn't apply to nylon. You are right though about the wetting. If it doesn't wet evenly as you lay it into the water, the membrane doesn't seem to give even transfers. SRLasky -- ********************************************************************* Stephen R. Lasky, Ph.D. Roger Williams Medical Center/Brown University Phone: 401-456-6572 Fax: 401-456-6569 e-mail: Stephen_Lasky@brown.edu ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ "The speed of a computer is inversly proportional to the legnth of time it has been on your desk." Michael Yablonski, circa 1989 ********************************************************************* From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!CSHL.ORG!lodhi From: lodhi@CSHL.ORG (Muhammad Lodhi) Newsgroups: bionet.molbio.methds-reagnts Subject: ABI 48cm strech problem Date: 2 Feb 1995 06:33:11 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502021431.AA00258@phage.cshl.org> NNTP-Posting-Host: net.bio.net Dear Fellows: Lately we are having lots of trouble with our 48 cm strech. We have tried running gels with Taq terminator and dye primer rxns on it and most of the sequences, even though look good on gel, are pretty bad. Sequences are noisy, base spacing is -12 and peaks tend to overlap. This is mostly but not entirely true for rxns with forward primers. I am wondering if anyone has any idea how to correct it or what are we doing wrong. Is there any problem with the sequencer matrix of filter set A. Any help/advice will be appreciated. Muhammad Lodhi Cold Spring Harbor Lab From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!newshub.sdsu.edu!nic-nac.CSU.net!usc!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!msunews!harbinger.cc.monash.edu.au!news.cs.su.oz.au!metro!OzEmail!mcmillan From: mcmillan@ozemail.com.au (Nigel McMillan) Newsgroups: bionet.molbio.methds-reagnts Subject: Single Copy Mouse Gene Date: 31 Jan 1995 11:21:31 GMT Organization: OzEmail Pty Ltd - Australia Lines: 9 Message-ID: <3gl6fs$j23@oznet03.ozemail.com.au> NNTP-Posting-Host: shell01.ozemail.com.au X-Newsreader: TIN [version 1.2 PL2] I am looking for a single copy mouse gene to confirm copy number in a transgeneic mouse. There must be lots available but can someone point me in the right direction. A nice single band on an EcoR1 digest would be good Thanks Nigel McMillan University of Queensland Australia From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!quagga.ru.ac.za!caesar.wits.ac.za!pc17.biol.wits.ac.za!theano From: theano@gecko.biol.wits.ac.za (Theano Markoulides) Newsgroups: bionet.molbio.methds-reagnts Subject: In situ staining with x-gal Date: Thu, 2 Feb 1995 13:15:45 GMT Organization: University of the Witwatersrand, Life-Sciences Lines: 37 Message-ID: NNTP-Posting-Host: pc17.biol.wits.ac.za Hi folks, Can anybody out there enlighten my boggled mind ??? We have transfected chick cells (hopefully successfully) with a plasmid construct containing the Lac Z gene. The lac gene however has a human -B- actin promoter so we are holding our fingers crossed and hope to see if it will `switch on' in the chick cell. To see if it is working, we would like to stain cells with x-gal. Now this is my problem.... The x-gal staining solution (if I may call it that ), has the following components : Phosphate buffered saline containing ; 5mM Potassium ferricyanide 5mM Potassium ferrocyanide 2mM Magnesium chloride 3% DMSO and 1mg x-gal (added freshly ) Knowing that x-gal is a pain in the gall bladder to dissolve, I assumed that one of the reasons why DMSO is added is to aid in dissolving this compound. However when I add the (X-gal/DMSO) to the remaining solution I immediately get a fine white precipitate. I also tried adding the xgal without dissolving it first and I still get this precipitate. Should I, very merrily, filter the prcipitate off and apply the solution to my cells ? Or would that be a moronically stupid thing to do. At the risk of advertising my chemical ignorance to the entire network, does anybody know what on earth this precipitate is ? Any advice, criticism etc. etc. would be greatly appreciated. Thanking you all in advance and waiting anxiously for ANY reply, Theano. Theano Markoulides ( Theano@gecko.biol.wits.ac.za ) Zoology dept Wits University Johannesberg From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!pipex!uknet!strath-cs!st-and!Aberdeen!gen124 From: gen124@nof.abdn.ac.uk (d.a.bailey) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: A GOOD KIT FOR PCR CLONING Date: 1 Feb 1995 17:26:14 GMT Organization: University of Aberdeen, Scotland Lines: 41 Distribution: world Message-ID: <3gog7m$bli@nof.abdn.ac.uk> References: NNTP-Posting-Host: bio.if2.abdn.ac.uk X-Newsreader: TIN [version 1.2 PL2] : > : > > I am looking for a good kit for PCR cloning. Can you give me some ideas : > > , based on your EXPERENCE? : > : > The best so far for me is the TA cloning kit from Invitrogen. With a 25 : > microliter PCR I use 2 to 4 microliters directly in 25 microliters of : > competent cells. Follow their transformation protocol, plate the entire : > transformation on two plates and voila!- Bunches of white colonies and a : > few blue ones. : Er. Maybe I'm missing something here. Don't you need a ligation in there : somewhere between the PCR and the transformation? If not, I'd be very : interested to hear the protocol! I recently had a go with the Promega T-vector system which worked pretty well, roughly 50/50 whites to blues. Although only about half of the whites contained an insert. I sequenced a couple of the whites with no insert and found deletions in the polylinker which is presumably due to the a bit of residual exonuclease activity in the ligase (yes this protocol does involve a ligation step ;-) The kit comes with 50ul of pre-cut vector, a test insert and ligase. I think you can also buy in the comp. cells if you have more money than sense. I have used it it clone fragments from 0.4 to 3kb following the standard protocol and it's worked ok, hope this is of use to you. -- ______________________________________________________________________ bye bye! | David A. Bailey, | | Molecular and Cell Biology, | 0 | Marischal College, | "..root toot tooty, /O\/ | University of Aberdeen. | Oh what a beauty..!" \\ | Tel. (0224) 273188. | | d.a.bailey@abdn.ac.uk | ---------------------------------------------------------------------- From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!newshub.sdsu.edu!nic-nac.CSU.net!usc!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!pipex!uknet!comlab.ox.ac.uk!ivpc038.nerc-oxford.ac.uk!igp From: igp@mail.nerc-oxford.ac.uk Subject: Re: Request for Baculovirus Transfer Vectors Message-ID: Keywords: baculovirus; multiple expression Lines: 49 Organization: Natural Environment Reseach Council X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] References: <3gbder$9b2@mserv1.dl.ac.uk> Distribution: bionet Date: Tue, 31 Jan 1995 13:03:20 In article <3gbder$9b2@mserv1.dl.ac.uk> r-mehta@nimr.mrc.ac.uk (Raj Mehta) writes: >I am looking for anyone who has any double (or triple or more) promoter >vectors for baculovirus expression - such as pAcUW31 (Clontech) or pAcUW51 >(Pharmigen) etc. I would be grateful if anyone can provide me with the >above. Also, at the moment I am co-expressing two sub-units of a >heterodimeric protein by co-infecting the cells with two different >recombinant viruses. Has anyone got any info on the merits of this approach >as opposed to the double promoter transfer vector? Dear Raj, I'm sorry that I cannot offer to send you the dual promoter baculovirus transfer vectors but I can provide some information on their use. In our lab, we have expressed up to 5 foreign genes using a single recombinant baculovirus. It will be quicker and easier to clone your 2 genes into seperate transfer vectors to generate single expression recombinant baculoviruses. With only two recombinant viruses you should get most cells infected with both types of viruses (with a moi of 5 to 10 pfu per cell each) and hence both genes coexpressed within one cell. However, theoretical and practical studies here have shown that the ratios of the two recombinant proteins synthesised within each cell will vary significantly, depending on the kinetics of infection. The use of a single dual expression recombinant baculovirus would provide more consistant levels of each protein produced within cells. Another point to consider is that the amount of foreign protein synthesised varies greatly depending on the gene being expressed. The use of 2 single expression recombinant baculoviruses will, to some extent, allow you to vary the moi of each recombinant baculovirus to vary the average ratio of expressed products. The final choice will depend on the specifics of your research. Hope this has been of some help. Please contact me by email if you require further information. NOTE: the above information represents my own thoughts and opinions only, and not those of NERC , Oxford University or the QDPI. with regards Ian Polkinghorne (POLK@molbiol.ox.ac.uk) NERC Institute of Virology and Environmental Microbiology Mansfield Rd Oxford OX1 3SR England From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!psgrain!charnel.ecst.csuchico.edu!olivea!nntp-hub.barrnet.net!nntp-ucb.barrnet.net!noc.usfca.edu!Dr.Chihara From: Chihara@alm.admin.usfca.edu (C. Chihara) Newsgroups: bionet.molbio.methds-reagnts Subject: charon clone mapping Date: 3 Feb 1995 01:26:57 GMT Organization: USF Lines: 11 Sender: -Not-Authenticated-[5160] Message-ID: <3gs0p1$552@noc.usfca.edu> NNTP-Posting-Host: 138.202.6.144 X-Posted-From: InterNews 1.0@noc.usfca.edu. Xdisclaimer: No attempt was made to authenticate the sender's name. We have an old Charon 4 library of D.m. My students have isolated single clones and are mapping them. They are having a hard time getting restrictions maps that are consistent. Most often the problem seems to be either some incomplete cutting or maps that are not consistent with the arms we find in the _Current Protocols_ map. Does anyone have any info that might help sort out the problems? Chihara Chihara@compserv.usfca.edu USF __________________________________ From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!MSDISK.WUSTL.EDU!Troianovsk_s From: Troianovsk_s@MSDISK.WUSTL.EDU (Sergey Troianovsky) Newsgroups: bionet.molbio.methds-reagnts Subject: RE: Detergents micellar MW Date: 2 Feb 1995 21:29:42 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >>Date: 28-JAN-1995 02:23 >>From: >>Description: Detergents micellar MW >> >>Hi! >>Does anybody knows the micellar MW of Triton X-100 and SDS? Sorry to ask, but >this is my last resource to know. >> >>Giorgio. Hi, Giorgio In accordance with Sigma catalog (p.1692) Triton X-100 and SDS have the following features: __________________________________________________________________ Compound CMC (mM) Aggregation number MW ------------------------------------------------------------------ Triton X-100 0.24 140 624.9 SDS 8.27 62 288.4 ------------------------------------------------------------------ The catalog climes that Micellar Molecular Weight (Micellar Mr) = the average size of one micelle of pure detergent. Where one could recalculate it as follows: Micellar Mr = Monomer Mr x Aggregation number (i.e average number of monomers in one micelle). While I personally don`t understand it to much sins always believed that at CMC all detergent`s molecules will fall into micelle formation so that there will be no two similar micelle, while I expect some Gaussian distribution of their MW, nevertheless there are some numbers you probably was looking for: Micellar Mr for SDS - 36 x 10(-3); for Triton X-100 - 90 x 10(-3). The numbers were taken from Boehringer Mannheim catalog of bio.detergents. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!NETCOM.COM!quincicc From: quincicc@NETCOM.COM (quin chou) Newsgroups: bionet.molbio.methds-reagnts Subject: help for probe labelling Date: 2 Feb 1995 20:21:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear bionetters: I am having some problems labelling probes with biotin, either labelling DNA template by nick-translation or random priming with biotined dNTPs; or 5'- or 3'-end labelling through oligo synthesis. My problem is to get consistent results with the sensitivity I wanted. The probe is used for in situ hybridization. All suggestions or inputs are welcome! Thanks in advance for helps! Please contact me through email address: quincicc@netcom.com. quinn. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!uunet!ftpbox!news.acns.nwu.edu!kschlage From: kschlage@unseen2.acns.nwu.edu (Kurt Schlageter) Newsgroups: bionet.molbio.methds-reagnts Subject: RECOVERING OLIGOS FROM BRAIN TISSUE Date: 31 Jan 1995 21:25:14 GMT Organization: Northwestern University, Evanston, IL, US Lines: 1 Message-ID: <3gm9rq$hik@news.acns.nwu.edu> NNTP-Posting-Host: unseen1.acns.nwu.edu X-Newsreader: TIN [version 1.2 PL2] From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!uunet!ftpbox!news.acns.nwu.edu!kschlage From: kschlage@unseen2.acns.nwu.edu (Kurt Schlageter) Newsgroups: bionet.molbio.methds-reagnts Subject: RECOVERING OLIGOS FROM BRAIN TISSUE Date: 31 Jan 1995 21:23:59 GMT Organization: Northwestern University, Evanston, IL, US Lines: 1 Message-ID: <3gm9pf$hik@news.acns.nwu.edu> NNTP-Posting-Host: unseen1.acns.nwu.edu X-Newsreader: TIN [version 1.2 PL2] From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!rutgers!uwm.edu!news.moneng.mei.com!howland.reston.ans.net!news2.near.net!das-news2.harvard.edu!fas-news.harvard.edu!fas!remeans From: remeans@fas.harvard.edu (Robert Means) Newsgroups: bionet.molbio.methds-reagnts Subject: shortened GFP? Date: 31 Jan 1995 02:20:26 GMT Organization: Harvard University, Cambridge, Massachusetts Lines: 9 Message-ID: <3gk6pa$9lt@decaxp.harvard.edu> NNTP-Posting-Host: fas-2.harvard.edu X-Newsreader: TIN [version 1.2 PL2] Hello, I'm interested in using GFP as a reporter and I would like to know if there is any info out there on what sequence is required for flourescence. Is the whole sequence required or is there a central chromophore that can be isolated and still have activity, either alone or as a fusion protein. Thanks for any info or pointers. Robert Means From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!sl16s From: sl16s@cc.usu.edu (NAME RAVI PRAKASH .D) Newsgroups: bionet.molbio.methds-reagnts Subject: genbak comparison Message-ID: <1995Feb2.135959.40272@cc.usu.edu> Date: 2 Feb 95 13:59:59 MDT Organization: Utah State University Lines: 4 Dear Netters, I compared DNA andslated sequences of the same to sequences in the genbank. . The comparison shows a column "High Score" and another column "N". I would be happy if anyone can tell me what they mean. Thanks in adavnce. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!dog.ee.lbl.gov!news.cs.utah.edu!cc.usu.edu!sl16s From: sl16s@cc.usu.edu (NAME RAVI PRAKASH .D) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: prestaining prot.M.W. markers Message-ID: <1995Feb2.135237.40271@cc.usu.edu> Date: 2 Feb 95 13:52:37 MDT Organization: Utah State University Lines: 3 Biorad markets prestained molecular weight markers at almost the same price as regular unstained markers. Hope this helps. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!psgrain!nntp.ski.mskcc.org!mac-96.k-g2.ski.mskcc.org!user From: h-petrie@ski.mskcc.org (Howard T. Petrie) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Shearing DNA to ~2Kb Date: Thu, 02 Feb 1995 18:18:39 -0500 Organization: Memorial Sloan-Kettering Cancer Center Lines: 13 Message-ID: References: <3gp2jj$1bk@pith.uoregon.edu> NNTP-Posting-Host: mac-96.k-g2.ski.mskcc.org In article <3gp2jj$1bk@pith.uoregon.edu>, margolin@darkwing.uoregon.edu (Brian Margolin) wrote: > I would like to shear a sample of genomic DNA down to about 2 Kb and > don't know of any shearing method that will get it down that small. Can > anybody recommend a shearing method that will give me the desired > results? Sonication, as in the method for shearing of fish sperm DNA as a blocking reagent for Southern and Northern blotting (see standard methods texts), results in fragments which average 300-500 bp (you can check it out on a gel). From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!sfu.ca!junz From: junz@sfu.ca (Jun Zhang) Newsgroups: bionet.molbio.methds-reagnts Subject: nuclei isolation Date: 2 Feb 1995 18:13:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502030213.AA12569@fraser.sfu.ca> NNTP-Posting-Host: net.bio.net to whom it may concern: sorry, i lost your message. there is a lab guide talking about this: "Gene Transcription a practical approach" edited by B.D. Hames and S.J. Higgins, IRL PRESS 1993 hope this has some help. Mark Jun Zhang Dept. of Biological Sciences Simon Fraser University Burnaby, BC V5A 1S6 604-291-4595 junz@sfu.ca From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!rutgers!netnews.upenn.edu!dsinc!newsfeed.pitt.edu!gatech!howland.reston.ans.net!news.sprintlink.net!news.onramp.net!dal41.onramp.net!user From: joe1@onramp.net (j milburn) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: About the ECL Western blot kit Date: Thu, 02 Feb 1995 19:29:15 -0500 Organization: na Lines: 5 Message-ID: References: NNTP-Posting-Host: dal41.onramp.net I use ECL exclusively for Westerns. I use Amersham's ECL film for generating autorads. So far it is great. I take the blot to medical illustration and have them do glossy prints. joe milburn From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!rutgers!netnews.upenn.edu!dsinc!spool.mu.edu!torn!mcshub!informer1.cis.McMaster.CA!mcmail.cis.McMaster.CA!not-for-mail From: g9013118@mcmail.cis.mcmaster.ca (K. Legate) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: precipitate in NEB T4 Ligase buffer? Date: 2 Feb 1995 13:55:50 -0500 Organization: McMaster University, Hamilton, Ontario, Canada Lines: 12 Distribution: bionet Message-ID: <3gr9rm$jon@mcmail.CIS.McMaster.CA> References: NNTP-Posting-Host: mcmail.cis.mcmaster.ca In article , Colin Rasmussen wrote: > >It's the DTT. However, repeated freeze-thawing of ATP does have effects, >at least according to one supplier of T4 ligase. The efficiency of my ligations goes way down after repeated freeze-thaw cycles of the buffer. Adding a small amount of ATP to the mix restores original efficiency. kyle From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!rutgers!netnews.upenn.edu!msunews!uwm.edu!news.moneng.mei.com!howland.reston.ans.net!torn!mcshub!informer1.cis.McMaster.CA!mcmail.cis.McMaster.CA!not-for-mail From: g9013118@mcmail.cis.mcmaster.ca (K. Legate) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Temperature to pour plates Date: 2 Feb 1995 13:53:53 -0500 Organization: McMaster University, Hamilton, Ontario, Canada Lines: 12 Message-ID: <3gr9o1$j67@mcmail.CIS.McMaster.CA> References: <3gp7hp$oko@newsbf02.news.aol.com> NNTP-Posting-Host: mcmail.cis.mcmaster.ca In article <3gp7hp$oko@newsbf02.news.aol.com>, WmsFan wrote: >sterilize the milk if we pour the agar too hot. What temperature should we >cool the agar to before we pour it? > I pour agar plates when the solution is cool enough to hold in my bare hand. That would be aroud 55-60 C. Would it be better to pour the plates before adding the milk, then smearing a small amount of milk on them when the agar is solid? That way you can be sure the milk hasn't been sterilized. kyle From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!daresbury!bioftp.unibas.ch!citi2.fr!jussieu.fr!news.oleane.net!oleane!pipex!news.sprintlink.net!ixc.ixc.net!news.ecn.bgu.edu!newspump.wustl.edu!fas-news.harvard.edu!fas!remeans From: remeans@fas.harvard.edu (Robert Means) Newsgroups: bionet.molbio.methds-reagnts Subject: Cytoplasmic protease? Date: 3 Feb 1995 00:12:37 GMT Organization: Harvard University, Cambridge, Massachusetts Lines: 13 Message-ID: <3grsdl$jqo@decaxp.harvard.edu> NNTP-Posting-Host: fas-2.harvard.edu X-Newsreader: TIN [version 1.2 PL2] Hello, Possibly somebody out there working with proteases can help. I'm trying to produce a protein which will be cleaved at a specific site. If the protein was targeted to the golgi I could use fusin or something like that, but this protein is produced on membrane-free ribosomes. Is there any protease that occurs within the cytoplasm that the recognition site is known and has been used for such an experiment? Thanks for any pointers. Robert Means P.S. I know about Ubiquitin, but I think I would have to put about 72 a.a. in for it to work. If someone knows differently I'd like to know. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uunet!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: pngarrison@aol.com (PNGarrison) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR mutagenesis Date: 2 Feb 1995 19:27:27 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 7 Sender: root@newsbf02.news.aol.com Message-ID: <3grt9f$9tt@newsbf02.news.aol.com> References: <9502011533.AA12490@umailsrv1.UMD.EDU> Reply-To: pngarrison@aol.com (PNGarrison) NNTP-Posting-Host: newsbf02.mail.aol.com See Leung, D.W. et al. Technique 1 (1) 11-15 1989. I haven't used this method - don't know if it's any good. Good luck, P. Garrison garrisonp@uthscsa.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uunet!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: pngarrison@aol.com (PNGarrison) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Shearing DNA to ~2Kb Date: 2 Feb 1995 19:22:10 -0500 Organization: America Online, Inc. (1-800-827-6364) Lines: 4 Sender: root@newsbf02.news.aol.com Message-ID: <3grsvi$9sd@newsbf02.news.aol.com> References: <3gp2jj$1bk@pith.uoregon.edu> Reply-To: pngarrison@aol.com (PNGarrison) NNTP-Posting-Host: newsbf02.mail.aol.com I've used a probe sonicator to shear DNA to about 300 bp. P. Garrison garrisonp@uthscsa.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!news.starnet.net!wupost!kuhub.cc.ukans.edu!mbcf!heath Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Distinguishing stringent from relaxed? Message-ID: <1995Feb1.151237.8824@mbcf> From: heath@mbcf.stjude.org (Richard_Heath) Date: 1 Feb 95 15:12:37 -600 References: <3g66v4$a6v@mserv1.dl.ac.uk> Distribution: bionet Organization: St. Jude Children's Research Hospital Lines: 46 SMG plates can be used to screen for relaxed mutants: rel+ cells grow on this medium, and rel- cells die, so make sure you make duplicate streaks of each colony onto normal plates! Whether this is actually the *best* method...? It is probably worth doing however, unless you are expecting *very* high co-transduction frequencies if the Tn10 and relA allele. I would also demonstrate that the mutants you do isolate are in fact defficient in the stringent response by labelling with 32P, starving for an amino acid, and running the extracts on PEI-cellulose plates - mutants won't accumulate ppGpp. Any reveiwer will want to see this kind of demonstration...(at least, I would). You may need to also show these mutants still accumulate stable RNA after starvation - this depends on what you are planning on doing with the cells! Good luck Richard Heath In article <3g66v4$a6v@mserv1.dl.ac.uk>, mbpln@s-crim1.dl.ac.uk (M.J. Pallen) writes: > I am in the process of transduing a Tn10 relA mutation from one strain of > Salmonella into another. Does anyone have a handy way of scoring the > relA-negative phenotype, so that I can check that I really do have a relA > mutation in the second strain? Cahel & Rudd in Neidhardt's E. coli & S. > typhimurium book mention SMG medium. Is this the best way of > distinguishing stringent from relaxed strains? > > Mark > ****************************************************************************** > Dr Mark Pallen, Senior Lecturer in Medical Microbiology, > St Bartholomew's Hospital Medical College, London, EC1A 7BE > currently at the Dept of Biochemistry, Imperial College, London, SW7 2AY > email:m.pallen@ic.ac.uk > phone: day ++44(0)1715945254, eves ++44(0)1815057937, FAX ++44(0)1715945255 > ****************************************************************************** > lag ellum > f e lag > ecoliecoliecoliecoliecoli l mf > cecoliecoliecoliecoliecolii l u > eecoliecoliecoliecoliecolic umf ll > coecoliecoliecoliecoliecoli l ge > ecoliecoliecoliecoliecoli a > p f fl g > i l lum el > l agel lu > u mflagellum > s > ****************************************************************************** From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!darwin.sura.net!gatech!newsfeed.pitt.edu!hudson.lm.com!godot.cc.duq.edu!news.duke.edu!solaris.cc.vt.edu!news.alpha.net!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!ihnp4.ucsd.edu!munnari.oz.au!news.unimelb.EDU.AU!ucsvc.ucs.unimelb.edu.au!u2605163 From: u2605163@ucsvc.ucs.unimelb.edu.au (Jill Maddox) Newsgroups: bionet.molbio.methds-reagnts Subject: Long PCR - merits of different kits Message-ID: <1995Jan31.124340.7609@ucsvc.ucs.unimelb.edu.au> Date: 31 Jan 95 12:43:40 +1100 Organization: The University of Melbourne Lines: 9 We have just started doing long PCR and are interested in finding out people's opinions on the merits of the various kits (Perkin Elmer, Takara, Boehringer Mannheim etc). Please mail responses to me and I will post a summary. Jill Maddox Centre for Animal Biotechnology University of Melbourne Parkville Australia From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!spool.mu.edu!uwm.edu!caen!kuhub.cc.ukans.edu!mbcf!neale Newsgroups: bionet.molbio.methds-reagnts Subject: best polyA+ from total RNA method?? Message-ID: <1995Feb1.174632.8825@mbcf> From: neale@mbcf.stjude.org Date: 1 Feb 95 17:46:32 -600 Organization: St. Jude Children's Research Hospital Lines: 22 I was wondering if anyone had a strong recommendation of method for purification of poly A+ RNA from total RNA. The samples of total RNA that we have are about 300 ug each. We could pool some if necessary. We could purify poly A+ directly from cells if that is thought superior to purifying from total RNA. It's just that we already have the total RNA, and know that our gene of interest is expressed in these samples. We wish to use the poly A+ RNA for cDNA library construction. It's been a while (5 years) since I've had to do this and so I thought there may be an improved method for selection of poly A+ other than oligo dT column chromatography (eg magnetic beads-dT) Thanks in advance for your help. Geoff Neale Dept. of Virology and Molecular Biology E-mail: neale@mbcf.stjude.org St. Jude Children's Research Hospital Phone: (901) 522-0400 Memphis, TN Fax: (901) 523-2622 From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!news.msfc.nasa.gov!sol.ctr.columbia.edu!howland.reston.ans.net!spool.mu.edu!olivea!decwrl!pa.dec.com!dibit.hsr.it!borsang From: borsang@dibit.hsr.it (Giuseppe Borsani) Message-ID: Subject: New Scientist Date: Tue, 31 Jan 1995 18:10:29 +0100 X-Received: by usenet.pa.dec.com; id AA12007; Tue, 31 Jan 95 09:12:22 -0800 X-Received: by pobox1.pa.dec.com; id AA17795; Tue, 31 Jan 95 09:12:19 -0800 X-Received: from dibit.hsr.it by inet-gw-3.pa.dec.com (5.65/10Aug94) id AA28887; Tue, 31 Jan 95 09:07:54 -0800 X-Received: from [192.167.193.54] by dibit.hsr.it (AIX 3.2/UCB 5.64/4.03) id AA08280; Tue, 31 Jan 1995 18:05:36 +0100 X-Mime-Version: 1.0 X-Content-Type: text/plain; charset="us-ascii" X-To: bionet.molbio.methds-reagnts.usenet@decwrl.dec.com Lines: 20 Does anybody know how to subscribe to the New Scientist journal. It would be helpful to get the phone number of the publisher in UK. Any info about the price in Europe? Thanks / } { / Giuseppe Borsani X Tigem - Telethon Institute for Genetics and Medicine { \ Via Olgettina 58 \ } 20132 Milano X Italy / } { / e-mail: borsang@dibit.hsr.it X tel 39-2-21560203 { \ fax 39-2-21560220 \ } From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!SHU.SACREDHEART.EDU!0059621 From: 0059621@SHU.SACREDHEART.EDU (Kevin Campbell) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Blut-ending ligation Date: 2 Feb 1995 15:57:51 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net It is very hard to do a blunt ended ligation but, you may have to change the enzyme or change the ends to a compatible one using a staggered cutting technique where the sequence of the gene can be recognized by finding the complenent of that Tc gene. This can be done by a reverse transcription enzyme. If this is complicated you may have to go with the route that you may have not liked to. On Fri, 27 Jan 1995, Mr. J. Membrillo-Hernandez wrote: > > I would like to clone Tc gene (from pBR322) into a fragment of > 500bp (to interrupt it), I have tried several times with out success, the > main problem is no compatible ends so I have to do a blunt-ended > ligation. Is it the enzyme I am using?. > This blunt-end ligation is preceed by a T4 DNA polymerase filling > of the vector in the site Sph1 (in the middle of my fragment) an the Tc > gene was cut with the Ava1 and Ssp1 (I think this fragment contains all > the fuctional Tc gene), then Tc fragment was blut ended with klenow. > > Could anybody help me to set: > > a) good conditions for klenow reaction > b) good conditions for T4 polymerase > c) good conditions for blunt end ligation using T4 DNA ligase. > > > Thanks in advance. > Sian > > From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!news.starnet.net!wupost!golf!newton2.biosci.missouri.edu!user From: newton_lab@biosci.mbp.missouri.edu (Newton Lab) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Need an expression vector for E. coli Date: 2 Feb 1995 23:10:09 GMT Organization: Div. Biol. Sci., Univ. Missouri-Columbia Lines: 33 Message-ID: References: NNTP-Posting-Host: newton2.biosci.missouri.edu In article , rb690253@bcm.tmc.edu (Rob Britton) wrote: > I need a vector with which I can control the level of protein in the cell > ie. using lac or something. I cannot use the pET vectors or other > commercially available vectors because they are mainly for purifying > proteins from the cell and the expression is titratable. I am doing a > genetic experiment in which overexpression of my protein is being used to > test for suppression of another mutation. > > What I need is: > > 1. A vector that has a regulatable promoter > 2. A NdeI site so that I can clone the gene in frame > 3. A ribosome binding site to drive translation of the cloned gene > > Any help would be greatly appreciated. Thanks > > Rob Britton Hi Rob. i highly recommend pQE series from QIAGEN. Believe me, I tried a lot of vectors for the regulated expression of somehow deleterious proteins. these pQE guys are pretty silent without IPTG, some constructs may need 1-2 mM IPTG for the complete induction, some much less. there are pQE vectors for fusion and just for authentic product, with 6xHis tag or without. please look in their blue booklet ("QIAexpressionist") - it also contains very reasonable advice about expression in E.c. i believe there is no NdeI in frame there but such a great variety of polylinkers. Luck, eugene kuzmin From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!panix!zip.eecs.umich.edu!newsxfer.itd.umich.edu!gatech!howland.reston.ans.net!news.starnet.net!wupost!golf!newton2.biosci.missouri.edu!user From: newton_lab@biosci.mbp.missouri.edu (Newton Lab) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Alkaline vs. High-salt transfer of Southerns Date: 2 Feb 1995 22:56:31 GMT Organization: Div. Biol. Sci., Univ. Missouri-Columbia Lines: 15 Message-ID: References: NNTP-Posting-Host: newton2.biosci.missouri.edu In article , stauth@crunch.usgmrl.ksu.edu (Darren M. Stauth) wrote: > Just wondering if anyone had any experience with transfering DNA by the > alkaline method for Southern Hybridization and how it compares to > high-salt(SSC) transfer. Through various lab manuals I've learned that > alkaline transfer onto charged nylon takes a shorter period of time(2hrs > vs. overnight), binds DNA better(no need for UV crosslink), has higher > background. It seems superior to SSC method. Any comments appreciated. quite right! all above is true and add tolerance to numerous reprobings. it seems like best blotting routine in combination with Hybond N+ and like. do have no doubt. eugene kuzmin From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bloom-beacon.mit.edu!panix!zip.eecs.umich.edu!newsxfer.itd.umich.edu!gatech!howland.reston.ans.net!pipex!uunet!dziuxsolim.rutgers.edu!gandalf.rutgers.edu!not-for-mail From: dak@gandalf.rutgers.edu (Dorothy Klein) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: reference manager? Date: 2 Feb 1995 17:41:49 -0500 Organization: Rutgers University Lines: 58 Distribution: world Message-ID: <3grn3d$qjl@gandalf.rutgers.edu> References: <3goj7f$1c7c@bigblue.oit.unc.edu> NNTP-Posting-Host: gandalf.rutgers.edu I use Papyrus, and haven't had any problems with it. Matching the Papyrus import formats with the CCOD export formats can be a pain, but as long as you write down what you did that worked (knew I forgot to do something!), an hour of trial and error solves the problem. It's possible to write your own import scripts, but I've never thought it worth the trouble (YMMV if you had a few hundred references in a word-processed bibliography!). The papyrus manuals are some of the best I've seen -- very usable. The workbook will walk you through citing references in a paper, then creating the bibliography and the final properly-formatted cited text, for an amazing variety of journal formats -- and the ones I've tried look _exactly_ like the printed journal's references. The entire program, including the manuals, shows a sense of humor (if you're using the program when your computer clock hits midnight, it pops up a message telling you you're working too hard, go get coffee, for example). It's been a few years since we bought Papyrus, but it was about $100 for the full program, which has a generous licence for four distinct databases and as many program installations as those databases require. That means no guilt over copying the program so you/your secretary/your grad student can enter references both at home and at work, for example. A trial program is also available. Papyrus is available from Research Software Design, 503-796-1368, or RSD@applelink.apple.com. No connection beyond being a satisfied user. Dorothy Klein grad student, Microbiology and Molecular Genetics Rutgers University, NJ From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!PHARMDEC.WUSTL.EDU!anja From: anja@PHARMDEC.WUSTL.EDU (Anja Thilenius) Newsgroups: bionet.molbio.methds-reagnts Subject: dotap Date: 2 Feb 1995 15:21:49 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502022320.AA19469@pharmdec.wustl.edu> NNTP-Posting-Host: net.bio.net I want to do stable transfections into EL4 cells (murine T lymphoma) using= =20 DOTAP (from Boehringer Mannheim) Does anyone have a protocol in which they= =20 have optimized for these conditions? Thanks From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!newsfeed.pitt.edu!godot.cc.duq.edu!news.duke.edu!byrd.biochem.duke.edu!user From: bedal001@mc.duke.edu (Wendy Bedale) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Acrylamide gels sitcks to plates Date: 2 Feb 1995 22:39:25 GMT Organization: Duke University, Durham, NC, USA Lines: 4 Message-ID: References: NNTP-Posting-Host: byrd.biochem.duke.edu Yet another non-toxic and cheap alternative to silanizing your sequencing plates: try a nonstick cooking spray like Pam. I usually use a couple of very fast squirts on one plate, and rub it in with a kimwipe. I have used Rainex on my plates with succes as well. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!THORIN.UTHSCSA.EDU!HARDIES From: HARDIES@THORIN.UTHSCSA.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: re: Is pcr derived sequence acceptable for publication? Date: 30 Jan 1995 08:53:00 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 61 Sender: daemon@net.bio.net Distribution: world Message-ID: <950130105506.21d717@thorin.uthscsa.edu> M.E. Hudson writes: > I am planning to isolate cDNA sequence which may at some point be used in transforming plants. It would be necessary to publish the sequence if the work was ever published. Consequently, I have been warned off using pcr methods such as inverse pcr or amplifying cDNA by using homopolymer tailing and oligo dT + dC primers. Sure you can publish seq. obtained by PCR. What you don't want to do is risk blowing a chunk of your life trying to transform a plant with a mutant cDNA. Or publish work that your collegues regard as sloppy. You eliminate this vulnerability by sequencing multiple independent isolates (from different PCR rxns). You should be doing this anyway, because just the obligatory RT step in making the cDNA puts you at serious risk that both the sequence and the clone are wrong. > Is it true that pcr sequence is regarded as dubious, does this apply to pcr using polymerases with proofreading activity and to conventionally cloned fragments sequenced by pcr, Things like using a proofreading enzyme and using optimal rxn conditions can greatly reduce the amount of trouble you encounter. For example, it might save you from trying to splice a functional clone together from multiple mutated copies for your transformation experiment. Also remember that you can get heterogeneity from a multi gene family, or from allelic differences in your starting material. So you'd like to limit the amount of artificial heterogeneity in the system because you're going to eventually have to examine the heterogeneity that you see. > and is the taq error rate significant compared to that of dideoxy sequencing? Accuracy in Dideoxy sequencing is also a function of redundancy. The reviewer's normal expectation is that the sequence is not submitted for publication until the author is sure that it is correct. A good target to shoot for is that errors from both all sources will have been reduced to < 10^-4 by adequate use of redundancy and controls. In terms of the sequencing step, this normally means sequencing both strands to completion and resequencing trouble spots however it is necessary to clear up the ambiguity. Reviewers will accept a lower standard of accuracy if it is justified. For example, no one fusses much about short single stranded gaps in noncritical regions. Also, some projects intentionally trade accuracy for bulk of data, as in EST compilation, for example. But in these cases, the paper should clearly state the lower standard of accuracy. Since reviewers don't actually see the data, they largely have to trust the authors. Some authors violate this trust. Consequently, if you recover sequence from a database, you almost have to resequence it yourself if your project is critically dependent on the accuracy of the sequence. As a further consequence, people who submit bad sequence become known to their collegues and then the entire body of their work is treated with greater skepticism. Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio Hardies@uthscsa.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!hubcap.clemson.edu!biosci!SELWAY.UMT.EDU!bi__mvw From: bi__mvw@SELWAY.UMT.EDU ("Michael van Waes") Newsgroups: bionet.molbio.methds-reagnts Subject: Re: rRNA isolation Date: 30 Jan 1995 08:03:55 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <9501301602.AA07328@selway.umt.edu> References: > >I would like to isolate just the ribosomal RNA (the 5S could be missing!) from >my fungus, an ascomycete. All the method books tell you how to get >total RNA or how to hold on to the messenger RNA with poly T devices. As >those poly T devices are plus minus expensive and time consuming, I am curious >if there is another possibility besides using those poly T devices and >keep what one normally would dispose off which is the rRNA. >Being appreciative for any good answers and ideas!!! > Birgitt Oeser > oser@uni-muenster.de > > I don't want to get into too much detail, but have you thought of isolating the ribosomes (sucrose gradients of cell extracts) followed by phenol extraction of the rRNA? Cheers! MvW. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!internex.net!usenet From: hanzel@mdyn.com Newsgroups: bionet.molbio.methds-reagnts Subject: Re: shortened GFP? Date: 2 Feb 1995 22:25:27 GMT Organization: Molecular Dynamics Lines: 17 Message-ID: <3grm4n$3el@news.internex.net> References: NNTP-Posting-Host: 198.68.101.23 X-Newsreader: AIR News 3.X (SPRY, Inc.) > I'm interested in using GFP as a reporter and I would like to >know if there is any info out there on what sequence is required for >flourescence. Is the whole sequence required or is there a central >chromophore that can be isolated and still have activity, either alone or >as a fusion protein. Thanks for any info or pointers. > > Robert Means To paraphase Roger Tsien, always a dangerous proposition, sequence alone is not enough. The protein must be modified [reduced?] to fluoresce. So although the fluorescing sequence is known cells need all or most of the GFP protein for correct processing. The field is HOT and more colors are on the way. david hanzel hanzel@mdyn.com From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!adam.cc.sunysb.edu!news.sprintlink.net!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!psgrain!nntp.ski.mskcc.org!mac-93.k-g2.ski.mskcc.org!user From: d-burtrum@ski.mskcc.org,mrtour@stud.med.cornell.edu (michelle or doug......) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: barely molecular question... Date: Thu, 02 Feb 1995 11:02:32 -0500 Organization: Memorial Sloan-Kettering Cancer Center Lines: 31 Message-ID: References: <3g765f$6qi@vixen.cso.uiuc.edu> NNTP-Posting-Host: mac-93.k-g2.ski.mskcc.org In article <3g765f$6qi@vixen.cso.uiuc.edu>, "Michael R. Frank" wrote: > We need some new quartz spectrophotometer cells for use in DNA and > especially mRNA quantitation (see, it IS a molecular biology question > almost!). At my previous postion we had catalogs for several companies > which specialized in cuvettes and other glassware, but I don't have their > names or numbers with me here, and the people who do the ordering here > tend to just grab the first big thick catalog off the shelf without > looking for the best source.... > > Any suggestions? Phone and FAX numbers for sources in the U.S. > would be greatly appreciated! > > Thanks, > Michael R. Frank Hey, Michael: Our lab purchased quartz cuvettes from a company in Brooklyn call Scientific Cell. They have worked very well; the company also sells matched sets of cuvettes. Phone: 718 268 6887. sorry, i don't know the fax number. doug -- db or Michelle From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!RBSE.Mountain.Net!slip-2.mountain.net!jbradb From: J Brad Bellotte Newsgroups: bionet.molbio.methds-reagnts Subject: HELP: Random peptide phage display library Date: 2 Feb 1995 22:05:44 GMT Organization: . Lines: 10 Distribution: world Message-ID: <3grkvo$d46@rbse.Mountain.Net> NNTP-Posting-Host: adanet.wvnet.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Nuntius 1.3b28_68K X-XXMessage-ID: X-XXDate: Thu, 2 Feb 1995 17:07:48 GMT I was wondering if anyone could give me a basic textbook description (or point me to one) of random peptide phage display libraries. For example, what are they, how are they made? Thanks for any help. Please mail replies to: aguappon@wvumbrcc1.hsc.wvu.edu Brad. From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!olivea!nntp-hub.barrnet.net!nntp-ucb.barrnet.net!noc.usfca.edu!Dr.Chihara From: Chihara@alm.admin.usfca.edu (C. Chihara) Newsgroups: bionet.molbio.methds-reagnts Subject: lambda charon clones Date: 2 Feb 1995 21:10:23 GMT Organization: USF Lines: 10 Sender: -Not-Authenticated-[5160] Message-ID: <3grhnv$157@noc.usfca.edu> NNTP-Posting-Host: 138.202.6.144 X-Posted-From: InterNews 1.0@noc.usfca.edu. Xdisclaimer: No attempt was made to authenticate the sender's name. We have an old Charon 4 library of D.m. My students have isolated single clones and are mapping them. They are having a hard time getting restrictions maps that are consistent. Most often the problem seems to be either some incomplete cutting or maps that are not consistent with the arms we find in the _Current Protocols_ map. Does anyone have any info that might help sort out the problems? Chihara Chihara@compserv.usfca.edu USF __________________________________ From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!CBDA9.APGEA.ARMY.MIL!rxyoung From: rxyoung@CBDA9.APGEA.ARMY.MIL (Ronald Young) Newsgroups: bionet.molbio.methds-reagnts Subject: ADP-ribose transferase Date: 2 Feb 1995 13:35:32 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502021634.aa13513@cbdcom.apgea.army.mil> NNTP-Posting-Host: net.bio.net Studies with NAD glycohyrolase and ADP-ribose tranferase are uaually carried out with radiolabled NAD. Is thee another assay method of equal or near sensitivity that does not use radiolabled NAD? Thanks Ron Young From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!CBDA9.APGEA.ARMY.MIL!rxyoung From: rxyoung@CBDA9.APGEA.ARMY.MIL (Ronald Young) Newsgroups: bionet.molbio.methds-reagnts Subject: ADP-transferase Date: 2 Feb 1995 13:32:47 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 4 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502021627.aa11964@cbdcom.apgea.army.mil> NNTP-Posting-Host: net.bio.net Studies of ADP-ribosylation reactions or NAD glycohydrolase commonly usr radiolabled NAD. Does anyone know another method to follow reactions without using radiolabeled NAD and is also reasonably sensitive? Thanks Ron Young From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!MED.UNC.EDU!syc From: syc@MED.UNC.EDU (Shao-Yu Chen) Newsgroups: bionet.molbio.methds-reagnts Subject: free radicals, embryo Date: 2 Feb 1995 12:48:36 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502022048.AA02622@earl.med.unc.edu> NNTP-Posting-Host: net.bio.net Does anyone know the mechanism for the generation of free radicals in embryo exposed to ethanol? Are there evidence in rodent embryos for the activity of cutochrome P450 2E1? Thank you for your help. Shaoyu Chen UNC-CH syc@med.unc.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!MED.UNC.EDU!syc From: syc@MED.UNC.EDU (Shao-Yu Chen) Newsgroups: bionet.molbio.methds-reagnts Subject: SOD , embryo Date: 2 Feb 1995 12:43:54 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502022043.AA02612@earl.med.unc.edu> NNTP-Posting-Host: net.bio.net Does anyone out there know if SOD in culture medium could transfer across yolk sac in whole embryo culture system? Thank you for your help. Shaoyu Chen UNC-CH syc@med.unc.edu From owner-methds-reagnts@net.bio.net Wed Feb 01 22:00:00 1995 Path: biosci!sfu.ca!junz From: junz@sfu.ca (Jun Zhang) Newsgroups: bionet.molbio.methds-reagnts Subject: tubes Date: 2 Feb 1995 12:21:22 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502022021.AA18088@fraser.sfu.ca> NNTP-Posting-Host: net.bio.net Hi, there, I need tubes. They must be: 2.0ml or more in volume, RNase free, pre-siliconized polypropylene microtubes. They must fit the desk-top microcentrifuge. Could you please tell me the information if you know? Thanx. Mark Jun Zhang Dept. of Biological Sciences Simon Fraser University Burnaby, BC, V5A 1S6 604-291-4595 junz@sfu.ca From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!OFFICE.MMC.ORG!varyc.mmcri From: varyc.mmcri@OFFICE.MMC.ORG (Varyc) Newsgroups: bionet.molbio.methds-reagnts Subject: quant gel retard software Date: 3 Feb 1995 03:32:48 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <43EE302F01B938D9@office.mmc.org> Hi all, Does anyone know of any programs designed to handle electrophoretic mobility shift analysis of multiple DNA species binding one protein, or recent papers, theoretical or otherwise mathematical?? thanx in advance, Cal Vary From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!Germany.EU.net!zib-berlin.de!informatik.tu-muenchen.de!lrz-muenchen.de!ipp-garching.mpg.de!genmac28.biochem.mpg.de!user From: spang@vms.biochem.mpg.de (Anne Spang) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: yeast transformations Date: 3 Feb 1995 21:59:37 GMT Organization: Rechenzentrum der Max-Planck-Gesellschaft in Garching Lines: 15 Distribution: world Message-ID: References: <950129111908.20c50682@molbio.uoregon.edu> NNTP-Posting-Host: genmac28.biochem.mpg.de Hi John! We are currently using a lithium acetate-DMSO method - grow O/N culture in 2 ml - spin down in a 2 ml-Eppi - remove supernatant, add 0.01 ml carrier DNA (= 0.1 mg) and transforming DNA in minimal volume, vortex briefly - add 0.5 ml PEG (40% 3350, 0.05 ml 10xLiOAc, 0.05 ml 10xTE, H2O ad 0.5 ml) - add 0.051 ml DMSO - shake 15 min RT - shock cells for 15 min at 42C - spin down, wash the pellet with TE, resuspend in TE and plate on selective plates Hope this helps Anne From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!news.service.uci.edu!kornberg.bio.uci.edu!rmauk From: rmauk@kornberg.bio.uci.edu (Rob Mauk) Newsgroups: bionet.molbio.methds-reagnts Subject: Best Enzymes for 5' RACE? Date: 4 Feb 1995 01:02:20 GMT Organization: University of California, Irvine Lines: 1 Message-ID: <3gujms$7at@news.service.uci.edu> NNTP-Posting-Host: kornberg.bio.uci.edu From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!parc!barrnet.net!nntp-ucb.barrnet.net!agate!dog.ee.lbl.gov!ihnp4.ucsd.edu!news.service.uci.edu!hammer.biochem.uci.edu!user From: anon Newsgroups: bionet.molbio.methds-reagnts Subject: 3' race Date: Mon, 30 Jan 1995 23:58:01 -0800 Organization: University of California, Irvine Lines: 8 Message-ID: NNTP-Posting-Host: hammer.biochem.uci.edu I'm getting ready to try 3' race pcr. Im looking for valuable information that might make my life a little easier. Anyone who has experience with this technique and a few minutes, could you share your valuable wisdom by suggesting a few ideas that might improve my chanves of success. I have at my disposal total rna from my organism of choice and 200 bps of sequence somewhere in the vicinity of the 3' end (hopefully). Thanks for your time. my address is mmiller@darwin.bio.uci.edu. From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!daresbury!sunsite.doc.ic.ac.uk!lyra.csx.cam.ac.uk!bioc.cam.ac.uk!cdw1000 From: cdw1000@bioc.cam.ac.uk (Christopher Walentas) Newsgroups: bionet.molbio.methds-reagnts Subject: Minipreps and Automated Sequencing Date: 4 Feb 1995 01:05:06 GMT Organization: Somewhere in the University of Cambridge Lines: 32 Distribution: world Message-ID: <3gujs2$kov@lyra.csx.cam.ac.uk> NNTP-Posting-Host: bruce.bioc.cam.ac.uk Does anyone out there know what the best way to prepare low-copy number plasmid (mini-prep scale) for ABI automated sequencing? We've tried Promega Wizards (Magic uesd to work but that's another story), but they don't like to yield any sequence even with additional precipitations/ washed to remove excess salt. We tried QIAGEN 20 tips-- they work great down the hall where they use pUC and cosmids, but the yields for pKK223-3 and pGP1-2 derived vectors are deplorable. Folk here have even put CsCl banded DNA down a 20 tip to find that they are only able to recover 10% or so. What we've settled on is either doing larger minpreps (or rather a bunch of 'em), or chloramphenicol. Is there a better way? As an aside, if Promega was in patent conflict with their Magic resin, who DOES have the patent so that I can go back to what used to work. Please forward any and all suggestion to the email address below. Cheers in advance! Tripp - Christopher D. Walentas Centre for Molecular Recognition, Tel.: Sir William Hardy Bldg., within the U.K. (0223) 333662 Department of Biochemistry, outside +44 (223) 333662 University of Cambridge, FAX 333345 Tennis Court Road, Answerphone & alt. FAX 333661 Cambridge, England CB2 1QW e-mail: c.d.walentas@bioc.cam.ac.uk under the supervision of Professor R. N. Perham From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!bcm!mg690769 From: mg690769@bcm.tmc.edu (Michael Gerdes) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: fluorescence microscopy Date: 3 Feb 1995 21:00:47 GMT Organization: Baylor College of Medicine, Houston, Tx Lines: 19 Message-ID: <3gu5hv$mvg@gazette.bcm.tmc.edu> References: NNTP-Posting-Host: condor.mbcr.bcm.tmc.edu In article cgrunau@rkna50.riken.go.jp (Christoph Grunau) writes: >As a newcomer in fluorescence microscopy I would appreciate any advice >concerning the following problem: > >Does anyone has experience using DABCO (1,4-diazabicyclo [2.2.2] - octane >or p-Phenylenediamine in fluorescence microscopy. Which of both can you >recommend or in which special case is one better than the other one. > >thanks - Christoph Grunau from y experiences with these compounds, DABCO works best with FITC, while phenylenediamine works best with rhodamine. If doing double label experiments, the two compounds can be mixed (keeping concentration constant). Phenylenediamine will go brown with time, and I would suggest storing alliquots at -20C. hope this helps. michael From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!daresbury!sunsite.doc.ic.ac.uk!agate!news.Stanford.EDU!rutgers!gatech!howland.reston.ans.net!Germany.EU.net!zib-berlin.de!gs.dfn.de!fauern!news.th-darmstadt.de!news.uni-mainz.de!apppbg.vk.medizin.uni-mainz.de!user From: Jahnen@Mzdmza.zdv.uni-mainz.de (Willi Jahnen-Dechent) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: 3 block thermal cyclers? Date: 3 Feb 1995 08:39:45 GMT Organization: Institut fuer Physiologische Chemie Uni Mainz Lines: 12 Distribution: world Message-ID: References: <9501262254.AA02766@calvin.jci.tju.edu> NNTP-Posting-Host: apppbg.vk.medizin.uni-mainz.de In article , anderson@pharmdec.wustl.edu (Eric C. Anderson) wrote: > In article <9501262254.AA02766@calvin.jci.tju.edu>, > fgarbrec@CALVIN.JCI.TJU.EDU (Frederick Garbrecht) wrote: > > > Are there any PCR machines on the market with 3 independently programmable > > (oil free) blocks? Thanks for any info > We use a three block cycler by Biometra that is very popular around here. US distributor is Biometra Inc. Tampa, Florida Tel 813/2875132 Fax 813/2875163 From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!Germany.EU.net!zib-berlin.de!gs.dfn.de!fauern!news.th-darmstadt.de!news.uni-mainz.de!apppbg.vk.medizin.uni-mainz.de!user From: Jahnen@Mzdmza.zdv.uni-mainz.de (Willi Jahnen-Dechent) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: fluorescence microscopy Date: 3 Feb 1995 08:01:40 GMT Organization: Institut fuer Physiologische Chemie Uni Mainz Lines: 15 Message-ID: References: NNTP-Posting-Host: apppbg.vk.medizin.uni-mainz.de In article , cgrunau@rkna50.riken.go.jp (Christoph Grunau) wrote: > As a newcomer in fluorescence microscopy I would appreciate any advice > concerning the following problem: > > Does anyone has experience using DABCO (1,4-diazabicyclo [2.2.2] - octane > or p-Phenylenediamine in fluorescence microscopy. Which of both can you > recommend or in which special case is one better than the other one. > > thanks - Christoph Grunau give n-propyl gallate a thought. it is cheap and effective in reducing bleaching. So good, it made it to Science as a method paper. Giloh and Sedat Science 217,1252-1255 (1982) From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!caen!msunews!netnews.upenn.edu!aaron From: aaron@netnews.upenn.edu (Aaron Pawlyk) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RNAses Date: 3 Feb 1995 20:10:35 GMT Organization: University of Pennsylvania Lines: 24 Message-ID: <3gu2jr$50j@netnews.upenn.edu> References: NNTP-Posting-Host: ccat.sas.upenn.edu X-Newsreader: TIN [version 1.1 PL9] Tracy Aquilla (aquilla@salus.med.uvm.edu) wrote: : In Article , pootanak@hermws.bc.edu : (Kusol Pootanakit) wrote: : >Hi, : > : >Just out of curiosity, does anyone out there know if after one cross-linked : >RNA on to nylon membrane; can the RNases still get to them. : > : >thanks : Yes. : Tracy Yeah, if you expose the blot to a solution of RNAse you will loose a lot of signal, but blots are much more resistant to RNAse contamination floating around in a lab. I've never had any problems with RNAse contamination on a blot while I have had problems in RNA solutions. Adding RNAse to a hybridization mix will have more of an effect on the RNA and I also feel that the RNAse molecules will interfere with hybridization themselves. -- Aaron From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!uunet!news.u.washington.edu!news.u.washington.edu!homer10.u.washington.edu!rpierce From: Robert Pierce Newsgroups: bionet.molbio.methds-reagnts Subject: Help! Des.Seeking Tumors Date: Fri, 3 Feb 1995 11:37:36 -0800 Organization: University of Washington Lines: 7 Message-ID: NNTP-Posting-Host: homer10.u.washington.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Looking for small fragments of human tumor growing in immunoincompetent (nude) mice. Any tumor cell type, either from cell lines or transplanted tumor fragments welcome. Please contact Tony Lehr at University of Washington, Department of Pathology, Phone (206) 548-6400, Fax (206) 548-4928, or by e-mail c/o rpierce@u.washington.edu. Thank you very much. From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!daresbury!bioftp.unibas.ch!citi2.fr!jussieu.fr!news-rocq.inria.fr!news2.EUnet.fr!EU.net!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!newsrelay.iastate.edu!hobbes.physics.uiowa.edu!news.ksu.ksu.edu!darenmac.usgmrl.ksu.edu!user From: stauth@crunch.usgmrl.ksu.edu (Darren M. Stauth) Newsgroups: bionet.molbio.methds-reagnts Subject: multi-primer PCR screening protocol Followup-To: bionet.molbio.methds-reagnts Date: Fri, 03 Feb 1995 13:32:41 -0600 Organization: USGMRL Lines: 32 Message-ID: NNTP-Posting-Host: darenmac.usgmrl.ksu.edu I had several people request this protocol so here it is: This protocol involved the use of a hot-air thermo-cycler in a 10ul volume. I tested two multiprimer cocktails each containing six unique primers, then a common primer used in both cocktails (thus each had a total of 7 primers). In my templates (12 controls, one for each unique primer) only one of the unique primers and the common primer would anneal (I haven't tried amplifying multiple products with one mix). Also the product expected was less than 500bp. In theory the first cocktail should give amplification in the first six control templates and give no amplification in the second six controls. And vice versa for the second cocktail. To my amazement everything worked as expected and gave the proper size product. Negligible misannealing occurred giving very light bands in a couple negative control lanes. Reduction of the concentration of each primer may solve this problem. The following is my protocol: 1.Make cocktails (10ul reaction): 1ul 10X buffer/BSA/20mM MgCl 1ul six primer mix (10pmol/ul each) 1ul common primer (10pmol/ul) 1ul 2mM dNTP mix 4ul ddH2O 1ul Taq Pol (dilute-- ~1U) 2. Add cocktail to 1ul template (I used 1:1000 dilution of a miniprep) 3. Run on PCR program: 1. 94C 30sec 2. 30cycles of 94C 0sec 50C 0sec 72C 10sec 3. 72C 7min. e-mail me for any specific questions. From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!usc!news.cerf.net!NewsWatcher!user From: Scott_Stoughton@Sequana.com (Scott Stoughton) Newsgroups: bionet.molbio.methds-reagnts Subject: Subcloning/CIP Question. Date: Fri, 03 Feb 1995 11:14:57 -0800 Organization: Sequana Therapeutics Lines: 5 Message-ID: NNTP-Posting-Host: 198.207.137.146 Trying to shotgun subclone cosmids into pSPL3 splicing vector (exon trapping). Question: any advice on phosphotasing so the damn thing will ligate? Tx. s> Post here or email Scott_Stoughton@sequana.com From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!post.its.mcw.edu!not-for-mail From: skonings@post.its.mcw.edu (Steve Konings) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Red cell lysis with NH4Cl ? Date: 3 Feb 1995 12:43:08 -0600 Organization: Medical College of Wisconsin; Milwaukee Wisconsin Lines: 14 Message-ID: <3gttfs$g28@post.its.mcw.edu> References: <1995Feb1.065623.15619@news.unige.ch> NNTP-Posting-Host: post.its.mcw.edu X-Newsreader: TIN [version 1.2 PL2] Mike Morris (mike@medsun.unige.ch) wrote: : Can someone remind me of the protocol (ie the conc. of NH4Cl needed : to lyse RBC in human blood)? I haven't done it for years, and I : cannot find a recipe anywhere (I guess everyone except me knows : it by heart :( Mike, I use 0.8% NH4Cl and 0.1% EDTA-Na3 in distilled H20. Steve Konings Blood Research Lab Medical College of WI Milwaukee - A Great Place On A Great Lake From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!news.oleane.net!oleane!jussieu.fr!centre.univ-orleans.fr!univ-lyon1.fr!swidir.switch.ch!nedcu0!epjean From: Jean.Mariaux@zool.unine.ch Newsgroups: bionet.molbio.methds-reagnts Subject: PCR template prep from Pharmacia Message-ID: <1995Feb3.170248.3239@nedcu0> Date: 3 Feb 95 17:02:48 MET Organization: University of Neuchatel, Switzerland Lines: 16 I hesitate to try Pharmacia's "PCR template prep for ssDNA sequencing". I would appreciate any reports of your experiences with this kit. Did it significantly improve the quality of your PCR products direct-sequencing? Is it worth its price (plus the price of primer phosphorylations)? Thanks in advance. Jean Mariaux ************************************************************************ Jean Mariaux Phone: (+41) 38 233 051 Institute of Zoology (A115) Fax: (+41) 38 233 001 E.-Argand 11 E-mail: jean.mariaux@zool.unine.ch CH-2007 NEUCHATEL Switzerland ************************************************************************ From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!lll-winken.llnl.gov!decwrl!pagesat.net!news.cerf.net!class.class.org!kedziekm From: kedziekm@class.class.org (Allergan) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Anyone use Silver Sequencing? Does it work? Date: 3 Feb 1995 18:01:19 GMT Organization: C.L.A.S.S. Cooperative Library Agency for Systems and Services Lines: 45 Message-ID: <3gtr1f$1bo@news.cerf.net> References: NNTP-Posting-Host: class.class.org In article MURIANAP@FOODSCI.PURDUE.EDU (Pete Muriana) writes: >We're planning on doing some DNA sequencing but since we don't do much in my >lab I prefer not to get rigged up for radio-labelled sequencing (dedicated >area, disposal, etc.). The only other options include biotin-labelled >sequencing or "silver" sequencing (i.e., Promega). Just wondering if the >silver sequencing works as straight forward as the advertisements lead you to >believe. > >-Regards, Peter I've used the Promega Silver Sequencing system for over a year, and feel it is a good alternative to radioactive sequencing. But, it isn't quite as easy as they claim, although once the critical steps are identified, it works well. Things to consider: you need ultra-pure water (18 mohm, such that you would get from a MilliQ system), and a large, heavy duty orbital shaker. Also, I found that using large gels (such as with the BRL S2) was difficult, due to the size of the tray, and the weight of the solutions. It wasn't really too heavy, but very ackward to manipulate. Also, you will end up with silver waste, either in solution, or precipitated into a salt, that must be dealt with as hazardous waste. On the positive side, the system did work for me. And when I got through the ackward learning stage, the gels were beautiful, with nice banding patterns. The gel is dried on a glass plate, and a parmanent copy can be made with EDF film (Kodak) or a new duplicating paper. If you go for it, just keep hammering them with questions when things don't work. What turned it around for me was a workshop, where we ran and stained gels. Good luck. Karen Karen Kedzie, Ph.D. Allergan Pharmaceuticals, Inc Irvine CA 92715 (714) 752-4330 kedziekm@class.orf No affiliation with Promega. All opinions are my own and do not reflect those of Allergan Pharmaceuticals, Inc. From owner-methds-reagnts@net.bio.net Thu Feb 02 22:00:00 1995 Path: biosci!daresbury!bioftp.unibas.ch!citi2.fr!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!elna.ethz.ch!wawona!BCKRAEV From: bckraev@wawona ("Alexander Kraev" bckraev@aeolus.ethz.ch) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: ?plasmid flourometry standard Date: 3 Feb 1995 17:01:18 GMT Organization: ETH Zuerich, Switzerland Lines: 25 Message-ID: <3gtngu$qis@elna.ethz.ch> References: Reply-To: bckraev@wawona NNTP-Posting-Host: wawona.ethz.ch In article