From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bloom-beacon.mit.edu!eecs-usenet-02.mit.edu!newsswitch.lcs.mit.edu!test01.netnews.com!netnews.com!europa.clark.net!194.159.255.21!dispose.news.demon.net!demon!delos.dra.hmg.gb!server1.netnews.ja.net!warwick!bris.ac.uk!usenet From: Harry Witchel Subject: What is GPT? X-Nntp-Posting-Host: rwm4.pys.bris.ac.uk Content-Type: text/plain; charset=us-ascii Message-ID: <3433636A.30C0@Bristol.ac.Uk> Sender: usenet@fsa.bris.ac.uk (Usenet) Content-Transfer-Encoding: 7bit Cc: Harry.Witchel@Bristol.ac.uk Organization: Medical School University of Bristol Mime-Version: 1.0 Date: Thu, 2 Oct 1997 09:03:38 GMT X-Mailer: Mozilla 2.01KIT (Win95; U) Lines: 6 Hi folks -- I recently received an expression construct which has the GPT gene on it; unfortunately I don't know what GPT stands for or what it does for my transfections. Any ideas? Thanks, Harry From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!thetimes.pixel.kodak.com!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.4!news-pen-4.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-dc-26.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!news.maxwell.syr.edu!news-was.dfn.de!news-fra1.dfn.de!news-ge.switch.ch!c-1996!news.lth.se!merkurius.lu.se!not-for-mail From: pbio-cpi@pop.lu.se (C Pical) Newsgroups: bionet.molbio.methds-reagnts Subject: pb with concentrating a protein for antibody production Date: 2 Oct 1997 07:55:55 GMT Organization: Lund University Lines: 12 Message-ID: <60vk2b$2p3$1@merkurius.lu.se> NNTP-Posting-Host: pmac7600.plantbio.lu.se X-Newsreader: InterNews 2.0.1@pmac7600.plantbio.lu.se[U] XDisclaimer: User not authenticated Hi all, I am expressing a protein as a GST fusion, cutting my protein with thrombin while it is bound to the resin. Of course I get thrombin in my sample. I would like to get rid of thrombin and also concentrate my purified protein before injecting rabbits. I tried Centricon-50 (my protein is about 66 kD, I know it was a bit risky to try Centricon-50) but lost a lot of my protein which actually seems to be stuck on the membrane. So I would very much appreciate any suggestion. Thanks. C Pical From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!COUGAR.VUT.EDU.AU!s9401501 From: s9401501@COUGAR.VUT.EDU.AU (Matthew Knight) Newsgroups: bionet.molbio.methds-reagnts Subject: restriction digestion of PCR product for cloning Date: 2 Oct 1997 00:37:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I am having problems digesting a PCR product which has restriction enzyme sites placed into the PCR primers, thus they are very close to the end of the PCR product. Does anyone have any suggestions on how I can be sure that the restriction enzyme is going to cut the PCR product. And will the restriction enzyme cut with the sequence being so close to the end of the product (i.e. about 5 or 6 bases) Thanks in advance Matthew Knight From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!howland.erols.net!math.ohio-state.edu!news.physics.uiowa.edu!newsrelay.iastate.edu!news.iastate.edu!not-for-mail From: lmarek@iastate.edu (Laura F Marek) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Help on BAC DNA prep! Date: 1 Oct 1997 13:05:55 GMT Organization: Iowa State University, Ames, Iowa (USA) Lines: 38 Message-ID: <60thrj$eg9$1@news.iastate.edu> References: <19970929221400.SAA20314@ladder01.news.aol.com> NNTP-Posting-Host: marek.agron.iastate.edu Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.7 In article <19970929221400.SAA20314@ladder01.news.aol.com>, wzhao68@aol.com says... > >Hello there, I am having problem with BAC DNA preparation. I've tried >miniprep protocol from Research Genetics and Qiagen's Midiprep kit. The >miniprep never worked and Qiagen kit worked sometimes, but most of the time >gave me genomic DNA. >Could someone who has experience on this help me? Please e-mail me. >Thanks in advance! >David >Children's Hospital > David, We've done thousands of BAC minipreps and a "standard" alkaline lysis with a few modifications works best for us (soybean DNA inserts). Use 2mL of a 5 mL overnight culture, 100 ul of Maniatis etc soln I to resuspend cell pellet, 200 uL Maniatis etc soln II freshly mixed just before use to lyse (lyse by inverting 6-8 times and leave on ice no longer than 5 min). Neutralize with 150 uL 7.5M NH4OAc (soln not older than 4 weeks), mix as with soln II, stand on ice 5 min, invert just before centrifuging (room temp ok) not more than 8000 rpm (microcentrifuge) for 6-7 min. If you just want to do digests to size inserts etc, respin super to clarify and then precipitate nucleic acids with ethanol and resuspend in TE plus RNAase. If you want to do sequecing with the DNA, treat the super (from spinning after neutralizing) with RNAase, then extract with phenol cholorform 1x and chloroform 1x (1-200uL each depending on how cloudy the super was) and precipitate the nucleic acids with ethanol. I like to let the preps sit overnight at room temp and then spin 10-12 min at room temp to pellet DNA. Can be scaled up for 50 mL preps. If you need really clean DNA for a minilibrary or whatever then a CsCl gradient is necessary. Because the BAC vector is single copy there is always a significant carryover of bacterial DNA in a miniprep. Laura Marek lmarek@iastate.edu Agronomy Department Iowa State University From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!vixen.cso.uiuc.edu!ais.net!news.idt.net!dispose.news.demon.net!demon!news.demon.co.uk!genesys.demon.co.uk!demon.co.uk!duncan From: "Dr. Duncan Clark" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Trying to clone DIMERS Date: Thu, 2 Oct 1997 11:25:47 +0100 Organization: GeneSys Ltd Distribution: world Message-ID: References: NNTP-Posting-Host: genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk [158.152.17.110] MIME-Version: 1.0 X-Newsreader: Turnpike Version 3.04 Lines: 21 In article , Marcos De La Pena Del Rivero writes > -When the monomers (eluted from a gel) are ligated, I always obtain a >nice ladder (monomers, dimers, trimers... even hexamers), but the >eluted dimers cannot be cloned in any vector :( Could it be that the E.coli you are cloning into just doesn't handle these types of multimers. Does your PCR product express and could therefore be lethal with multiple copies? What about trying a lower copy number vector. I assume you are using a recA(-) (and the rest) host. Duncan -- The problem with being on the cutting edge is that you occasionally get sliced from time to time.... Duncan Clark DNAmp Ltd. TEl/FAX 01252376288 http://www.dnamp.com http://www.genesys.demon.co.uk From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!howland.erols.net!news-peer.gsl.net!news.gsl.net!gip.net!news.idt.net!dispose.news.demon.net!demon!news.demon.co.uk!genesys.demon.co.uk!demon.co.uk!duncan From: "Dr. Duncan Clark" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Long PCR Date: Thu, 2 Oct 1997 11:54:34 +0100 Organization: GeneSys Ltd Distribution: world Message-ID: References: <5jgt7m$orh@cardinal1.Stanford.EDU> NNTP-Posting-Host: genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk [158.152.17.110] MIME-Version: 1.0 X-Newsreader: Turnpike Version 3.04 Lines: 27 In article <5jgt7m$orh@cardinal1.Stanford.EDU>, John Ladasky writes >I have found that two things have greatly increased my ability to >do long PCR. First, use HPLC-purified primers. Also use very high quality nucleotides. The presence of trace levels of dUTP is enough to stop long PCR above say 10kb on lambda by inhibiting the proof-reading component. We find that Taq on its own will amplify 10kb of lambda, albeit poorly, and that a 10kb PCR of lambda is equivalent to PCRing 2kb of human genomic. Beoyond that you have to use mixes. Mg optimum for anything longer is very narrow so use 0.1mM steps in the optimisation. We've found that even unpurified primers will work up to 35kb so that may not be that critical. Duncan -- The problem with being on the cutting edge is that you occasionally get sliced from time to time.... Duncan Clark DNAmp Ltd. TEl/FAX 01252376288 http://www.dnamp.com http://www.genesys.demon.co.uk From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!cam-news-feed3.bbnplanet.com!news.bbnplanet.com!das-news2.harvard.edu!news.dfci.harvard.edu!usenet From: "MP Frosch, MD, PhD" Newsgroups: bionet.molbio.methds-reagnts Subject: FMP3: finding empty records Date: Thu, 02 Oct 1997 15:12:00 -0400 Organization: Neuropathology, Brigham and Women's Hospital Lines: 22 Message-ID: <3433F200.1524@dsg.harvard.edu> Reply-To: frosch@dsg.harvard.edu NNTP-Posting-Host: 134.174.175.37 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Macintosh; I; PPC) I have a database which keeps track of my animal colony. One of the files consists of the individual cage names. This is connected via a portal to a database of animals in which the current cage location appears. In the cage database I perform a calculation to find out how many animals are in a cage. Within the case database, I can do a FIND and get cages with any different number of animals except zero. Why can't I find my empty cages? TIA. -- M.P. Frosch, MD, PhD Neuropathology | Center for Neurologic Diseases Brigham & Women's Hosp. | Brigham & Women's Hosp. 75 Francis Street | 77 Avenue Louis Pasteur (HIM 764) Boston, MA 02115 | Boston, MA 02115 ***** | ***** 617 732-7532 (secretary) | 617 525-5240 (lab/office) 617 975-0944 (fax) | 617 525-5305 (fax) mpfrosch@bics.bwh.harvard.edu | frosch@dsg.harvard.edu From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!nature.berkeley.edu!lhom From: lhom@nature.berkeley.edu (Louis Hom) Newsgroups: bionet.molbio.methds-reagnts Subject: Magenta-Gal experiences Date: 2 Oct 1997 18:49:53 GMT Organization: University of California, Berkeley Lines: 7 Message-ID: <610qch$8ve@agate.berkeley.edu> NNTP-Posting-Host: nature.berkeley.edu I'm thinking of buying some of that magenta-colored analog of X-Gal but I was wondering if folks out there have anything to say about it first. -- _______________________________________________________________________________ Lou Hom >K '93 lhom@nature.berkeley.edu http://www.ocf.berkeley.edu/~lhom From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!rutgers!nntp.upenn.edu!news.misty.com!www.nntp.primenet.com!globalcenter0!news.primenet.com!nntp.primenet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!logbridge.uoregon.edu!news.uoregon.edu!newsfeed.orst.edu!128.193.4.3.MISMATCH!news.orst.edu!news From: Aaron Liston Newsgroups: bionet.plants,bionet.molbio.methds-reagnts Subject: Re: pcr on plant genomic Date: Thu, 02 Oct 1997 11:26:00 -0700 Organization: Oregon State University Lines: 21 Message-ID: <3433E737.665FB9A4@bcc.orst.edu> References: <60oco5$kd$1@infa.central.susx.ac.uk> Reply-To: listona@bcc.orst.edu NNTP-Posting-Host: liston-laptop-4086-b.cordley.orst.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) Xref: biosci bionet.plants:16753 bionet.molbio.methds-reagnts:61673 I have encountered a PCR problem that is new to me: A student is having difficulty with amplification of a plastid DNA locus in the plant Cimicifuga (Ranunculaceae). I suggested she add a recalcitrant DNA sample [that had never worked before] to her positive control to test if the Cimicifuga DNA extraction had a PCR inhibitor. To our surprise, both the control AND the Cimicifuga locus amplified! We don't want to add the control DNA to each sample - any thoughts on how to replicate its effect? All ideas appreciated. Aaron Liston Oregon State University Martin Goodson wrote: > hello all, > > I've heard that PCR on plant genomic DNA has specific diffulties not > encountered with the DNA of other organisms. From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.algonet.se!news.maxwell.syr.edu!Supernews60!supernews.com!news.he.net!news.pagesat.net!news.itis.com!news.doit.wisc.edu!svetlov From: svetlov@oncology.wisc.edu (Vladimir Svetlov) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: QuikChange kit Date: Thu, 02 Oct 1997 12:31:33 -0500 Organization: UW-Madison, McArdle Lab Lines: 11 Message-ID: References: NNTP-Posting-Host: alpha.oncology.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 In article , uwe@itsa.ucsf.edu (Uwe Klein) wrote: > Has anyone experience using the new QuikChange site-directed mutagenesis > kit from Stratagene? It sounds great, is it? It works OK for point mutations (up to three at a time) and for deletions of up to 70 bases (that's what I tried myself). Have not done any larger loop-outs or insertions with it though. Regards, V. From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!thetimes.pixel.kodak.com!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.4!news-pen-4.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-dc-26.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!199.60.229.5!newsfeed.direct.ca!news.he.net!news.pagesat.net!news.itis.com!news.doit.wisc.edu!svetlov From: svetlov@oncology.wisc.edu (Vladimir Svetlov) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: cloning by www? Date: Thu, 02 Oct 1997 12:28:00 -0500 Organization: UW-Madison, McArdle Lab Lines: 22 Message-ID: References: <343028FD.7578@whoccr.oulu.fi> NNTP-Posting-Host: alpha.oncology.wisc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.4.0 In article <343028FD.7578@whoccr.oulu.fi>, PN wrote: > Hi, > Nowadays there should be companies having the whole human genome in > pieces of cDNA. Can I run a mouse cDNA against a database of any of > them to get the corresponding human sequence? Or do they even clone it > to me? > Could you name any company and give a possible web-site? Human genome is not yet entirely sequenced so you can not do such thing. You can do it with yeast though - pick up a gene or a sequence through text/blast search and through yeast genomic database you can get a phage or cosmid clone, that contains the fragment you are interested in. I'm not sure if genomic clones for sequenced prokaryotes are available commercially. You still can clone it from the genomic/cDNA library (by hybridization) and Clontech is one of the most comprehensive collection of various cDNA/genomic libraries. I won't be surprised if one can outsource this kinda work, since it's tedious but rather trivial job. Regards, V. From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.algonet.se!newsfeed.direct.ca!news.he.net!news.pagesat.net!news.itis.com!not-for-mail From: Petr Kuzmic Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Graph analysis programs suitable for FACS Date: Thu, 02 Oct 1997 12:00:22 -0500 Organization: BioKin Consulting Lines: 57 Message-ID: <3433D326.8AD7E422@biokin.com> References: <60u42d$no2@panix.com> NNTP-Posting-Host: r4.itis.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) Ian A. York wrote: > > I have FACS tracings (from a Becton-Dickinson FACScalibur machine, with > CellQuest for the analysis) and I'd like to do some slightly more > sophisticated analysis of them. Here's my problem: My cells are > biphasic (the "High" and "Low" populations can't be distinguished by other > antibodies); the curves of the two populations are overlapping; but I'd > like to get some reasonably accurate stats on the two populations. If you think that your histogram ### ######## ## ############# ###### ################ ######### ################### ########### ########################################## ############################################# ############################################### ################################################## ###################################################### ######################################################### ############################################################# ################################################################## is an overlap of two NORMALLY distributed populations (or other such well-defined populations) you can use any standard curve-fitting program to deconvolute the two peaks. I recommend the program SigmaPlot from Jandel Scientific. If the disctribution was Gaussian, in SigmaPlot you would write the following transform file (replacing '...' with the initial estimate relevant to your data): -------------- 8< ------------- 8< -------------- [Parameters] k1=... ; height of peak 1 m1=... ; center of peak 1 s1=... ; width of peak 1 k2=... ; height of peak 2 m2=... ; center of peak 2 s2=... ; width of peak 2 [Variables] ; these are your data x=col(1) ; 'x' lives in column 1 y=col(2) ; 'y' lives in column 2 [Equations] f= (k1/s1)*exp(-0.5*((x-m1)/s1)^2)+(k2/s2)*exp(-0.5*((x-m2)/s2)^2) fit f to y -------------- 8< ------------- 8< -------------- Good luck... _____________________________________________________________ Petr Kuzmic Ph.D. * BioKin Ltd. * Madison, WI 53708-8336, USA pkuzmic@biokin.com * http://www.biokin.com * 608.256.1269 fax From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!aol.com!DOCTORHIM From: DOCTORHIM@aol.com Newsgroups: bionet.molbio.methds-reagnts Subject: Deglycosylation of N-linked and O-linked Oligosaccharides Date: 2 Oct 1997 22:12:09 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <971003011139_-793159976@emout15.mail.aol.com> NNTP-Posting-Host: net.bio.net A number of you have enquired about deglycosylating proteins of both N and O linked oligosaccharides. I think the kit that you are looking for is made by Prozyme. It has five enzymes that will remove virtually all N and O linked oligosaccharides. The instruction book is a good reference on techniques and specificities of the enzymes and comparison with chemical methods. You can download the Instructions for this kit at: http://www.prozyme.com/glycoenzymes/ You will need Adobe Acrobat 3 reader to view it. It is also available for download at this site but it may take 30-60 minutes depending on your modem speed. Good luck! From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!chem.pg.gda.pl!slawek From: slawek@chem.pg.gda.pl (Slawek Dabrowski 18-22) Newsgroups: bionet.molbio.methds-reagnts Subject: IPTG and B-galactosidase Date: 2 Oct 1997 21:39:14 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi everyone, I have simple question. Is IPTG desintegrated by B-galactosidase like lactose ? Best regards, SLawek From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!MOLECULE.BIO.UTS.EDU.AU!michelle From: michelle@MOLECULE.BIO.UTS.EDU.AU (Michelle Gleeson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: How to make a non-sticky glass plate Date: 2 Oct 1997 21:11:04 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net Hi, You can either try coating one plate with bind silane and leaving the other uncoated, or treat the other plate with a product such as Sigmacote or Coatasil (or even RainX) I can't remember the exact name of the sticky silane, but we got it from Sigma. AATAGGCAATGGGCCCCATATAGGAACACAGAGCTGCATGCGTATTGCATGCCAGGCTATTCATTCCAGGGAAA Michelle Gleeson Molecular Parasitology Unit Ph: (02) 9514 4043 University of Technology Fax:(02) 9514 4003 Westbourne St Gore Hill, NSW 2065 michelle.gleeson@uts.edu.au When you get to the end of your rope, tie a knot and hang on - FDR TTATCCGTTACCCGGGGTATATCCTTGTGTCTCGACGTACGCATAACGTACGGTCCGATAAGTAAGGTCCCTTT On Thu, 2 Oct 1997, C.K. Chen wrote: > Hi, > Could someone tell me how to make a sequencing gel glass plate > non-sticky? I use silver stain for sequencing gels and would like the gel > stick on one of the glass plates. I have tried Silan treatment for one of > the plates. However, the gel sticked on both plates. I am confident that > the plates were clean. > > C.K Chen > > From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!internet!biosci!not-for-mail From: mulder@saturn.med.nyu, edu@mcbi-34.med.nyu.edu Newsgroups: bionet.molbio.methds-reagnts Subject: re: How to make a non-sticky glass plate Date: 2 Oct 1997 20:28:59 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <199710030328.XAA10321@saturn.med.nyu.edu> NNTP-Posting-Host: net.bio.net To avoid the gel from stick on both of the glass plates, I was used to treat both the plates with silan , one 2 times and the other four or five times, meaning treat-dry-treat again etc. I know it sounds a waste of material but it always worked for me. Make sure to wash yor plates with detergent and rins them with ethanol before appling the silan. Hope it helps Ben From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!howland.erols.net!vixen.cso.uiuc.edu!news.uoregon.edu!newsfeed.orst.edu!news.orst.edu!news From: chend@ucs.orst.edu (Don Chen) Newsgroups: bionet.molbio.methds-reagnts Subject: RFI: vector with selection for hygromycin B resistance Date: Fri, 03 Oct 1997 03:20:10 GMT Organization: Oregon State University Lines: 17 Message-ID: <343463b3.7527581@news.orst.edu> NNTP-Posting-Host: slip188.ucs.orst.edu X-Newsreader: Forte Free Agent 1.11/16.235 Hi: I am looking for a vector that carries a gene for hygromycin B resistance. Does anyone know of either commercial or academic source(s) for such a vector? Thanks for any help you can offer. don ********************************************************* Don Chen * Standard disclaimers apply here. USDA-ARS-NFSPRC * 3450 SW Campus Way * Corvallis, OR 97331 * * 541-750-8741 * ******************************************************** From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 From: pjf Newsgroups: bionet.molbio.methds-reagnts Subject: Re: restriction digestion of PCR product for cloning Date: 02 Oct 1997 11:52:34 -0600 Organization: planet of the monkey boys Lines: 18 Sender: zinc@zifi.genetics.utah.edu Message-ID: References: NNTP-Posting-Host: zifi.genetics.utah.edu Mime-Version: 1.0 (generated by tm-edit 7.106) Content-Type: text/plain; charset=US-ASCII To: s9401501@COUGAR.VUT.EDU.AU (Matthew Knight) X-Newsreader: Quassia Gnus v0.9/XEmacs 20.2 X-Face: &dh1aHu*v\s24A9~2qU[ln~;(Cz#X]d2r-@X>#j9U@vx!v8%IYU''n-H1D)}U(-0Te~ LD|Cyg6Gh-aFN{B*VSKhs}en{nXo3xkveE#BxR9 I am having problems digesting a PCR product which has restriction enzyme > sites placed into the PCR primers, thus they are very close to the end of > the PCR product. -- "There is only one aim in life and that is to live it." Karl Shapiro,(1959) from an essay on Henry Miller's Tropic of Cancer finger zinc-pgp@zifi.genetics.utah.edu for PGP key zifi runs Linux 2.1.56 http://zifi.genetics.utah.edu From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!news-peer.sprintlink.net!news.sprintlink.net!Sprint!vixen.cso.uiuc.edu!asl4c407a.foods.uiuc.edu!user From: cchen2@uiuc.edu (C.K. Chen) Newsgroups: bionet.molbio.methds-reagnts Subject: How to make a non-sticky glass plate Date: Thu, 02 Oct 1997 18:01:06 -0500 Organization: UIUC Lines: 8 Message-ID: NNTP-Posting-Host: asl4c407a.foods.uiuc.edu Hi, Could someone tell me how to make a sequencing gel glass plate non-sticky? I use silver stain for sequencing gels and would like the gel stick on one of the glass plates. I have tried Silan treatment for one of the plates. However, the gel sticked on both plates. I am confident that the plates were clean. C.K Chen From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!rutgers!nntp.upenn.edu!news.misty.com!news-xfer.netaxs.com!europa.clark.net!164.67.42.145!nntp.info.ucla.edu!128.120.8.185!mark.ucdavis.edu!dogbert.ucdavis.edu!not-for-mail From: ez049617@dogbert.ucdavis.edu (Anne Gillen) Newsgroups: bionet.molbio.methds-reagnts Subject: AFLP adapters, help Date: 2 Oct 1997 21:59:53 GMT Organization: University of California, Davis Lines: 11 Message-ID: <6115gp$an6$2@mark.ucdavis.edu> NNTP-Posting-Host: dogbert.ucdavis.edu X-Trace: mark.ucdavis.edu 875829593 10982 (None) 128.120.8.191 X-Complaints-To: usenet@ucdavis.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] I would like to make my own adapters for AFLPs. According to GIBCO, one should use PAGE purified oligos to do this. Is this really necessary? Would HPLC purification work as well? Could I just use standard desalted and deprotected oligos? Thanks, Anne ******************************************************************************* Anne M. Gillen E-mail: amgillen@ucdavis.edu Pomology Department Phone: (916) 752-1462 University of California at Davis ******************************************************************************* From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!usenet.INS.CWRU.Edu!HSNX.wco.com!newsfeed.dacom.co.kr!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!164.67.42.145!nntp.info.ucla.edu!128.120.8.185!mark.ucdavis.edu!dogbert.ucdavis.edu!not-for-mail From: ez049617@dogbert.ucdavis.edu (Anne Gillen) Newsgroups: bionet.plants,bionet.molbio.methds-reagnts Subject: Re: pcr on plant genomic Followup-To: bionet.plants,bionet.molbio.methds-reagnts Date: 2 Oct 1997 21:55:05 GMT Organization: University of California, Davis Lines: 3 Message-ID: <61157p$an6$1@mark.ucdavis.edu> References: <60oco5$kd$1@infa.central.susx.ac.uk> <34318E71.88096C69@trishul.sci.gu.edu.au> NNTP-Posting-Host: dogbert.ucdavis.edu X-Trace: mark.ucdavis.edu 875829305 10982 (None) 128.120.8.191 X-Complaints-To: usenet@ucdavis.edu X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Xref: biosci bionet.plants:16763 bionet.molbio.methds-reagnts:61680 The problem with plant DNA is the sometimes huge amounts of contaminants. We have no problem with digestion and PCR of peach DNA if the DNA if cesium chloride banded. Otherwise, nothing works. From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!howland.erols.net!europa.clark.net!128.223.220.30!logbridge.uoregon.edu!news.bc.net!rover.ucs.ualberta.ca!tyr-2 From: tyr-2@bones.biochem.ualberta.ca (Karl Fischer) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Biotech student study group needs assistance Date: Thu, 02 Oct 1997 16:13:20 -0700 Organization: Med. Microbiology, Univ. of Alberta Lines: 35 Distribution: world Message-ID: References: NNTP-Posting-Host: duckter.glaxo.med.ualberta.ca Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Trace: pulp.ucs.ualberta.ca 875828937 12128 (None) 129.128.118.146 X-Complaints-To: usenet@pulp.ucs.ualberta.ca X-Newsreader: Yet Another NewsWatcher 2.2.0 In article , diane wrote: > My name is Diane and I am a 4th year university student in Biology. I am > part of a "Biotechnology student study group" which is working together > to answer a set of questions given to us by our professor. Our > professor has STRONGLY suggested that we seek answers to 11 specific > questions from various sources (eg. class notes, texts, the primary > literature, friends, colleagues, professors, internet and company > catalogues). I find it very difficult to believe that a professor would advocate the use of professors, scientific colleagues, and the internet community for providing ANSWERS to these questions (please note the capitals). I could, however, believe that said professor would instruct students to derive information from class notes, texts, information resources like catalogues/web and THEN formulate a suitable answer for each question which they THEN, if they so choose, run these answers by individuals who might have more practical/theoretical experience with the topics in question. Getting the answers without doing the mental legwork diminishes the learning experience and is, unfortunately, becoming a bit too common (IMHO). Karl the hepB guy -- Karl Fischer tyr-2@bones.biochem.ualberta.ca From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.algonet.se!news.maxwell.syr.edu!news.bc.net!torn!hone!informer1.cis.McMaster.CA!fhs.csu.McMaster.CA!millmanj From: Jon Millman Newsgroups: bionet.molbio.methds-reagnts,bionet.microbiology Subject: polA mutant strain? Date: Thu, 2 Oct 1997 12:00:52 -0400 Organization: McMaster University, Hamilton, Ontario, Canada (NewServer) Lines: 16 Message-ID: References: <3428D6D5.44DB@sunmail.lrz-muenchen.de> <34292F49.4DD3@ucdavis.edu> NNTP-Posting-Host: fhs.csu.mcmaster.ca Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <34292F49.4DD3@ucdavis.edu> Xref: biosci bionet.molbio.methds-reagnts:61678 bionet.microbiology:11180 I'm looking for a polA mutant strain of E. coli to perform a gene knockout, and would appreciate any assistance in finding such a strain. I've looked in the ATCC catalogue, but without any cross-reference for genotypes it would take forever to look through all the strains. If anyone has such a strain, or knows the ATCC (or other bank) number for a strain with a polA knockout I would very much like to hear from you. Thanks, Jon Millman Dept of Biochemistry McMaster University Hamilton. ON -- CANADA millmanj@fhs.csu.mcmaster.ca From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!rutgers!gatech!howland.erols.net!news-peer.sprintlink.net!news-peer-east.sprintlink.net!news.sprintlink.net!Sprint!news.idt.net!news.voicenet.com!gsl-penn-ns.gsl.net!news-dc.gsl.net!news.gsl.net!gip.net!news.pt.lu!news.restena.lu!not-for-mail From: Diederich Marc Newsgroups: bionet.molbio.methds-reagnts Subject: 1ST CALL : TRANSCRIPTION '98 Date: Thu, 02 Oct 1997 18:13:40 +0000 Organization: RCMS/CUL Lines: 395 Message-ID: <3433E454.4DF7@cu.lu> Reply-To: diederic@cu.lu NNTP-Posting-Host: ggt.labext.cu.lu Mime-Version: 1.0 Content-Type: multipart/mixed; boundary="------------5ADB1CBD5631" X-Mailer: Mozilla 3.01 (Macintosh; I; 68K) This is a multi-part message in MIME format. --------------5ADB1CBD5631 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit ****************************************************** 1st ANNOUNCEMENT Transcription Factors and Gene Regulation as a Therapeutic Target January 29-31, 1998 University Center, Luxembourg The latest update of this announcement, together with an online registration form, can also found at the URL : http://www.cu.lu/labext/rcms/colloque/index.html ****************************************************** Registration informations may be obtained by contacting : Marc Diederich, PhD Laboratoire RCMS Centre Universitaire de Luxembourg 162A, avenue de la Faiencerie L-1511 Luxembourg Luxembourg Tel : +352.46 66 44 434 Fax : +352 46 66 44 436 E-mail : diederic@cu.lu --------------5ADB1CBD5631 Content-Type: multipart/appledouble; boundary="----------ad7827F787762"; x-mac-type="5744424E"; x-mac-creator="4D535744"; name="ANNONCE.DOC 1" Content-Transfer-Encoding: 7bit Content-Description: Microsoft Word Document Content-Disposition: inline; filename="ANNONCE.DOC 1" ------------ad7827F787762 Content-Type: application/applefile Content-Transfer-Encoding: base64 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AAAAAAAAAAAAAAABAAAAAQAAAAAAAAAAEAAAAgAAAAEAAAD+////AAAAAAAAAAD///////// //////////////////////////////////////////////////////////////////////// //////////////////////////////////////////////////////////////////////// //////////////////////////////////////////////////////////////////////// /////////////////////////////////////////////////////////////w== ------------ad7827F787762-- --------------5ADB1CBD5631-- From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!chugaibio.com!dspinella From: dspinella@chugaibio.com (Dom Spinella) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RNA isolation from whole blood Date: 2 Oct 1997 12:49:40 -0700 Organization: Chugai Biopharmaceuticals, Incorporated Lines: 45 Sender: daemon@net.bio.net Distribution: world Message-ID: <34340A8C.745@chugaibio.com> Reply-To: dspinella@chugaibio.com, @chugaibio.com NNTP-Posting-Host: net.bio.net > We're trying to analyze RNA expression patterns of human mixed leukocyte > populations immediately after drawing the blood sample (i.e., we don't have > time to let the samples sit, lyse the RBCs, separate out individual > populations by adherence, etc.) > > Using reagents such as Trizol LS (with or without added acetic acid), > TriReagent BD, yields from either whole blood (directly added to the > reagent or from EDTA tubes) or from buffy coats (sucked out of EDTA tubes > after a quick spin) have been uniformly terrible (RNA isolation from > cultured lymphoblasts performed in parallel give good yields, so it's not a > simple problem of lab klutziness). > > Any tips? I know there's a protocol using Catrimox, but the amount of > Catrimox used relative to the volume of whole blood is huge. > > A long time ago I used to mix up my own guanidinium isothiocyanate-acid > phenol solution (a la the Chomczynksi & Sacchi original recipe), drop the > buffy coats into this, layer it all on a CsCl/EDTA cushion and spin it down > in an ultracentrifuge -- got fair yields of good quality RNA at the bottom > of the tube. I note that there was 10% sarcosyl in this GIT/acid phenol > solution -- could this be making the difference (anyone know if Trizol and > TriReagent include this stuff?) > > Thanks for your insights. > > Jon and Daina > > Jon Nakamoto, MD,PhD > Assistant Professor, Pediatric Endocrinology > UCLA > > Daina Dreimane, MD > Fellow, Pediatric Endocrinology > UCLA Well, I know you don't want to hear this, but I would at least lyse the red cells first -- they have lots of crud that appears to interfere with subsequent isolation and manipulation of nucleic acid. I bet if you just resuspend your buffy coat in a few ml of Tris-buffered ammonium chloride for a few minutes, (e-mail me if you need a recipe) then wash the leukocyte pellet a few times in a microfuge (at low speed) with PBS or TBS, any procedure you use for RNA isolation will work much better. It really doesn't take much time and its well worth the effort. Just a thought. -- Dom Spinella From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!S1.CXWMS.AC.UK!rabc010 From: rabc010@S1.CXWMS.AC.UK Newsgroups: bionet.molbio.methds-reagnts Subject: Re: High Speed Thermocyclers. Date: 2 Oct 1997 09:09:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <3433D70E.5407@cxwms.ac.uk> Reply-To: rabc010@s1.cxwms.ac.uk NNTP-Posting-Host: net.bio.net There are two types of rapid thermocycler - A. Based on Peletier effect. B. Light thermocycler (capillary thermocycler). The choice of thermocycler depends on your requirements and set up of the experiments. What is important, the way you use your cycler. From my own exprience use of refrigerated temperature decrseases the life of the cycler. I never use 4oC temperature to hold my PCR after the reaction is finished. OK, if you say that PCR product will be degraded, it is not true I never had that problem. If it happens, it happens before PCR due to the contamination. If you are not going to use radioactivity in your PCR, I will suggest cycler from Idaho Biotechnology, or for radioactivity, Peletier system based from MJ Reasearch. For more details visit their Web site. Hope this helps. I don,t represent any company. narendra From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!aol.com!WSchick From: WSchick@aol.com Newsgroups: bionet.molbio.methds-reagnts Subject: Re: High Speed Thermocyclers Date: 2 Oct 1997 08:49:52 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <971002114701_-898894713@emout18.mail.aol.com> NNTP-Posting-Host: net.bio.net The Corbett 960G has a 30 cycle time of one hour, and it includes an 8 step temperature gradient function with 96 wells for optimization at under $5000. The Idaho Rapid Cycler has a 30 cycle time of 10 minutes with 48 capillary tubes at about $4000. Biometra has a fast cycler with 96 wells with a cycling time of about 1 hour. I used to sell these systems as a manufacturers rep, and customers liked them. Techne has a fast cycler for under $4000. I have no experience with this one. Walter Schick BioSepCo From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!204.59.152.222!news-peer.gsl.net!gsl-penn-ns.gsl.net!news.gsl.net!gip.net!news.belnet.be!news.fundp.ac.be!immuno-poos-xa-ayman.sciences.fundp.ac.be!user From: Pascal.Mertens@fundp.ac.be (Pascal Mertens) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Primer Degradation? Date: 1 Oct 1997 08:41:49 GMT Organization: Lab. Immunologie, FUNDP Lines: 22 Message-ID: References: NNTP-Posting-Host: immuno-poos-xa-ayman.sciences.fundp.ac.be In article (Dans l'article) , gilbride@bu.edu (Kevin Gilbride) wrote (écrivait) : > I need some advise regarding the stabilty of primers. Not directly linked to your problem: long term (in)stability: a (previously good) primer stored 6 years at -20 as a 100X stock and reused in sequencing does not work anymore. It seems to be the same for a plasmid (maxiprep, 6 years old): no more readable sequence (tested of course with a new primer that works on a new DNA). Pascal -- Pascal Mertens Laboratoire d'Immunologie et Microbiologie URBM-FUNDP 61 Rue de Bruxelles 5000 Namur, BELGIUM Phone: 32 81 724438 Fax: 32 81 724420 From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Newsgroups: bionet.molbio.methds-reagnts Subject: long primers From: franck.laurent@medew.fyto.wau.nl (Franck Laurent) Organization: Wageningen Agricultural University, Fytopathology X-Newsreader: WinVN 0.99.7 MIME-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII NNTP-Posting-Host: flex187.dbh.wau.nl Message-ID: <3433b68a.0@wau.nl> Date: 2 Oct 97 14:58:18 GMT Lines: 5 Path: biosci!bloom-beacon.mit.edu!panix!howland.erols.net!surfnet.nl!wau.nl!flex187.dbh.wau.nl Hi netters, has anyone used PCR primers of 45 bases with approximatelly 30 bases overhang. Thanks for help Franck From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!server1.netnews.ja.net!server5.netnews.ja.net!server6.netnews.ja.net!server4.netnews.ja.net!news.cc.ic.ac.uk!not-for-mail From: Koen De Smet Newsgroups: bionet.molbio.methds-reagnts Subject: Re: restriction digestion of PCR product for cloning Date: Thu, 02 Oct 1997 15:40:23 +0100 Organization: Department of Medical Microbiology Lines: 33 Message-ID: <3433B257.4EDB@nospam.ic.ac.uk> References: Reply-To: k.desmet@nospam.ic.ac.uk NNTP-Posting-Host: cb-22.sm.med.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; PPC) Matthew Knight wrote: > > I am having problems digesting a PCR product which has restriction enzyme > sites placed into the PCR primers, thus they are very close to the end of > the PCR product. > > Does anyone have any suggestions on how I can be sure that the > restriction enzyme is going to cut the PCR product. And will the > restriction enzyme cut with the sequence being so close to the end of the > product (i.e. about 5 or 6 bases) > > Thanks in advance > > Matthew Knight The appendix of the New England Biolabs catalogue contains relevant info on cutting near the end of PCR products. If you cannot cut, you can first clone the PCR product (without digesting) in a vector with blunt ends (or T-A sticky ends for a Taq product), and cut the insert out. Koen De Smet _______________________________________________________________ To reply by email, remove "nospam." from the above address _______________________________________________________________ ___ ___ ___ Department of Medical Microbiology ___ ___ Imperial College School of Medicine at St Mary's ___ ___ London W2 1PG ___ ___ http://www.sm.ic.ac.uk/medmicro/home. ___ _______________________________________________________________ From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!uottawa.ca!s1081448 From: s1081448@uottawa.ca (diane) Newsgroups: bionet.molbio.methds-reagnts Subject: Biotech student study group needs assistance Date: 2 Oct 1997 07:12:52 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 130 Sender: daemon@net.bio.net Distribution: world Message-ID: Reply-To: diane NNTP-Posting-Host: net.bio.net Hello, My name is Diane and I am a 4th year university student in Biology. I am part of a "Biotechnology student study group" which is working together to answer a set of questions given to us by our professor. Our professor has STRONGLY suggested that we seek answers to 11 specific questions from various sources (eg. class notes, texts, the primary literature, friends, colleagues, professors, internet and company catalogues). It was suggested to me, by Mary McCarty (Associate Scientific Editor, BioTechniques), that the methods-reagents BioSci group would be the best newsgroups to address these questions. The 10 questions follow, 1) While travelling through the highlands of Borneo, you become aware of a plant natural product(an alkaloid) which induces a state of higher consciousness during exam writing. You have synthesized a large amount of this prodcut and investigated its mode of action. It appears that it affects on brain cells is mediated by a receptor, probably a surface glycoprotein. You have been given the task of isolating the human gene. How would you do it (detailed procedure/techniques)? How would you know you had done it (correctly)? 2)The expression of a foreign protein from a eukaryotic organism in a bacterium such as E.coli can be complicated by the differences in the biology of the two organisms. Discuss this problem and the potential solutions for the cloning of an EXPORTED ENZYME. If you were faced with these problems, what possible solutions would you propose and why? If it was necessary to express the gene in a previously unknown organism, how would you accomplish it? How would you know you had accomplished it? 3)Molecular genetics provides powerful tools for the study of biological phenomena. However, the key limiting factor(assuming no limit to MRC or NSERC funding) in this approach is our ability to isolate the underlying genes. Describe ways in which we can oovercome this problem. In your answer provide a description of and rationale for the techniques you describe. Be clear to state what types of genes can be identified by each technique. 4)RFLP analysis holds many advantages over traditional approaches for gene mapping. Describe thses advantages and how they can be used (must state the traditional techniques and their disadvantages). In many cases PCR technology can now be employed to replace RFLP analysis. Compare the advantages and disadvantages of each technique for gene mapping. Also, explain the use of both in DNA fingerprinting (forensic work). 5)Recently a new parasitic disease, due to infection by the organism Yudo canadiensis (fictional organism) has been discovered in the Ottawa area. Disease seems to result from degeneration of brain cells. In a major breakthrough, you discover genetic evidence that resistance to the parasite can be found in a model animal suystem, the mouse strain U0 which is inherited as a single locus, udo (autosomal,dominant allele is udo1) WHEN mated to strain CU. You have been given the task of isolating the human gene. How would you do it? How would you know you'd done it? 6)You are in the transgenic plant business, producing corn plants which are resistant to insects eg. corn borer. Various resistance genes in other plants can be identified be genetic studies and some of these might prove useful in corn. How would you determine which are likely to be useful and which are not? Once you have done this and identified the best candidate, how would you produce a resistant corn plant? 7)You have isolated and characterized a cDNA clone. It is 1.4kb long but by Northern analysis, an mRNA of 2.3kb is detected. Since one of the objectives of your grant application was the characterization of signals controlling expression, you need a strategy to isolate the full lenght cDNA. Listed belaow are several possible strategies. For each one describe how it could be employed to reach your objective. a. cDNA library b. PCR and a cDNA library c. mRNA and 5'RACE d. mRNA and 3'RACE e. oligonucleotides and mRNA 8)As part of your genome project oyu have sequenced ten overlapping PAC clones encompassing 500kb from a typical eukaryote. There are several possible routes to continue the analysis. For each one describe how it could be employed to reach your objective. a. Northern analysis b. chromosome walking c. Zoo blots d. exon trapping e. transformation into mutant yeast lines 9)You have cloned a full lenght cDNA from your typical eukaryote and wish to investigate some aspects of the protein encoded by it eg. subcellular distribution,interactions with other proteins in the cell etc. You decide to express it in E.coli in order to get lots of the protein. There are several possible routes to continue the analysis. For each one describe how it could be employed to reach your objective. a. codon usage b. fusion proteins c. affinity chromatography d. regulatory sequence e. protein TAGs 10)The isolation of DNA markers linked to the phenotype of interest is a major step forward in the genetic dissection of the phenotype. Listed below are several possible strategies. For each one describe how it could be employed to reach your objective. a. RFLPs b. microsattelites c. RAPDs d. AFLPs e. ESTs 11)In a continuation of question 10, you have used the RAPD approach and identified a polymorphism linked to rust resistance (R=resistance,r=sensitivity) in wheat. You clone the band, intending to use the cloned DNA as a probe for Southern analysis prior to positional cloning. So you isolate DNA from several generations of plants (F1 to F3) and probe. However, when you have finished the molecular analysis. it seems that linkage to two different chromosomes is observed. Offer several explanations for this result and attempt to devise a strategy to test your hypotheses. Any help provided would be GREATLY appreciated. Thank you. Sincerely, Diane email: s1081448@mailbox.uottawa.ca From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.algonet.se!newsfeed.direct.ca!ais.net!uunet!in1.uu.net!netnews.sbphrd.com!news From: Lynne_M_Roxbee_Cox Newsgroups: bionet.molbio.methds-reagnts Subject: Re: QuikChange kit Date: Thu, 02 Oct 1997 16:59:58 -0700 Organization: SmithKline Beecham Lines: 13 Message-ID: <3434357E.29F2@sbphrd.com> References: Reply-To: "Lynne_M_Roxbee_cox@sbphrd.com"@nospam NNTP-Posting-Host: hhd902.ha.uk.sbphrd.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) To: Uwe Klein Uwe Klein wrote: > > Has anyone experience using the new QuikChange site-directed mutagenesis > kit from Stratagene? It sounds great, is it? > In my experience, yes it is - at least 80% mutants Lynne -- The opinions expressed in this communication are my own, and do not necessarily reflect those of my employer. From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!rutgers!gatech!4.1.16.34.MISMATCH!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!164.67.42.145!nntp.info.ucla.edu!164.67.80.81!nnrp.info.ucla.edu!jnakamot From: jnakamot@ucla.edu (Jon Nakamoto) Newsgroups: bionet.molbio.methds-reagnts Subject: RNA isolation from whole blood Date: Thu, 02 Oct 1997 09:46:37 -0700 Organization: UCLA Lines: 35 Message-ID: NNTP-Posting-Host: 149.142.47.19 Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.6 We're trying to analyze RNA expression patterns of human mixed leukocyte populations immediately after drawing the blood sample (i.e., we don't have time to let the samples sit, lyse the RBCs, separate out individual populations by adherence, etc.) Using reagents such as Trizol LS (with or without added acetic acid), TriReagent BD, yields from either whole blood (directly added to the reagent or from EDTA tubes) or from buffy coats (sucked out of EDTA tubes after a quick spin) have been uniformly terrible (RNA isolation from cultured lymphoblasts performed in parallel give good yields, so it's not a simple problem of lab klutziness). Any tips? I know there's a protocol using Catrimox, but the amount of Catrimox used relative to the volume of whole blood is huge. A long time ago I used to mix up my own guanidinium isothiocyanate-acid phenol solution (a la the Chomczynksi & Sacchi original recipe), drop the buffy coats into this, layer it all on a CsCl/EDTA cushion and spin it down in an ultracentrifuge -- got fair yields of good quality RNA at the bottom of the tube. I note that there was 10% sarcosyl in this GIT/acid phenol solution -- could this be making the difference (anyone know if Trizol and TriReagent include this stuff?) Thanks for your insights. Jon and Daina Jon Nakamoto, MD,PhD Assistant Professor, Pediatric Endocrinology UCLA Daina Dreimane, MD Fellow, Pediatric Endocrinology UCLA From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!usenet.INS.CWRU.Edu!HSNX.wco.com!newsfeed.direct.ca!europa.clark.net!164.67.42.145!nntp.info.ucla.edu!164.67.80.81!nnrp.info.ucla.edu!jnakamot From: jnakamot@ucla.edu (Jon Nakamoto) Newsgroups: bionet.molbio.methds-reagnts Subject: delete this (just a test) Date: Thu, 02 Oct 1997 09:32:20 -0700 Organization: UCLA Lines: 1 Message-ID: NNTP-Posting-Host: 149.142.47.19 Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Yet Another NewsWatcher 2.3.6 this is just a test From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!howland.erols.net!news-peer.gsl.net!news.gsl.net!gip.net!news.idt.net!dispose.news.demon.net!demon!news.demon.co.uk!genesys.demon.co.uk!demon.co.uk!duncan From: "Dr. Duncan Clark" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Long PCR Date: Thu, 2 Oct 1997 11:56:58 +0100 Organization: GeneSys Ltd Distribution: world Message-ID: References: <3432715B.72BA@virginia.edu> NNTP-Posting-Host: genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk [158.152.17.110] MIME-Version: 1.0 X-Newsreader: Turnpike Version 3.04 Lines: 19 In article <3432715B.72BA@virginia.edu>, Rajesh Kumar writes >I need to do a long PCR (~9 kb). Can somebody suggest me a suitable >polymerase and a source of it ? 9kb of what, human xsomal, bacterial xsomal or lambda. It makes a lot of difference as to how difficult it will be. Lambda is possible with just Taq the others need a mix and possibly a lot of playing. Duncan -- The problem with being on the cutting edge is that you occasionally get sliced from time to time.... Duncan Clark DNAmp Ltd. TEl/FAX 01252376288 http://www.dnamp.com http://www.genesys.demon.co.uk From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!news.maxwell.syr.edu!howland.erols.net!winter.news.erols.com!news From: Ulrich Tillmann Newsgroups: bionet.molbio.methds-reagnts Subject: Research Associate Date: Thu, 02 Oct 1997 08:46:54 -0400 Organization: Antex Biologics Lines: 6 Message-ID: <343397BE.4D80@erols.com> Reply-To: mcarb@erols.com NNTP-Posting-Host: rkv-as3s52.erols.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Received-On: 2 Oct 1997 12:50:29 GMT X-Mailer: Mozilla 3.01C-KIT (Win95; I) Research Associate- Biopharmaceutical company seeks to hire BS/MS level molecular biologist to assist in the identification and cloning/ expression of novel antigen candidates. Applicants will have hands-on experience (2-3 years) in the cloning and sequencing of genes using contemporary recombinant DNA methods. Send resume to HR, Code RSA, Antex Biologics, 300 Professional Dr., Gaithersburg, MD 20879 From owner-methds-reagnts@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!eru.mt.luth.se!news.algonet.se!newsfeed.direct.ca!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!baron.netcom.net.uk!netcom.net.uk!server3.netnews.ja.net!is.bbsrc.ac.uk!news From: Andrei Popov Newsgroups: bionet.molbio.methds-reagnts Subject: Re: restriction digestion of PCR product for cloning Date: Thu, 02 Oct 1997 14:43:19 -0300 Organization: The Babraham Institute, Cambridge, UK Lines: 19 Message-ID: <61086o$md7@is.bbsrc.ac.uk> References: Reply-To: andrei.popov@SPAM.bbsrc.ac.uk NNTP-Posting-Host: pc0743.iapc.bbsrc.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; PPC) Matthew Knight wrote: > > Does anyone have any suggestions on how I can be sure that the > restriction enzyme is going to cut the PCR product. And will the > restriction enzyme cut with the sequence being so close to the end of the > product (i.e. about 5 or 6 bases) > > Thanks in advance > > Matthew Knight Blunt/kinase, concatemerize, cut, clone. -- Andrei Popov to reply remove SPAM. from return address From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!howland.erols.net!rill.news.pipex.net!pipex!tank.news.pipex.net!pipex!newshost.zeneca.co.uk!usenet From: John Brennand Newsgroups: bionet.molbio.methds-reagnts Subject: Re: What is GPT? Date: Fri, 03 Oct 1997 10:56:51 +0100 Organization: Zeneca Pharmaceuticals Lines: 13 Message-ID: <3434C161.254F@alderley.zeneca.com> References: <3433636A.30C0@Bristol.ac.Uk> NNTP-Posting-Host: 156.71.144.158 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Macintosh; I; PPC) To: Harry Witchel harry back in the old days.. when i was a lad.. etc... gpt was one of the first genes used for selection in mammalian cells (before neo/g418, hyg, etc, came along) - gpt is an e coli gene that can be selected for in mycophenolic acid. it is still available in pharmacia's pMSG vector. see their '97 catalogue pg 93 which will also direct you to the sequence. cheers john From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!iagnet.net!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!nih.gov!not-for-mail From: Chris Baumann Newsgroups: bionet.molbio.methds-reagnts Subject: Drosophilia expression Date: Fri, 03 Oct 1997 06:20:06 -0400 Organization: National Institutes of Health Lines: 11 Message-ID: <3434C6D5.226CDBA2@dino.nci.nih.gov> NNTP-Posting-Host: 137.187.208.35 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) Hi folks, Does anyone out there know of a commercially available vector that supports transient expression in Drosophilia Schneider 2 cells. I know that invitrogen sells a kit but I only need the vector and they will not sell it alone. Thanks, Chris From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!thetimes.pixel.kodak.com!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.4!news-pen-4.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-dc-26.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsxfer3.itd.umich.edu!iagnet.net!EU.net!newsfeed.internetmci.com!139.130.235.93!news.telstra.net!harbinger.cc.monash.edu.au!bunyip.cc.uq.edu.au!not-for-mail From: Paul Rohde Newsgroups: bionet.molbio.methds-reagnts Subject: mRNA extractions with diatoms/celite ** mRNA? & Celite Grade? ** Date: 3 Oct 1997 07:03:03 GMT Organization: University of Queensland Lines: 32 Message-ID: <6125b7$17e$1@nargun.cc.uq.edu.au> NNTP-Posting-Host: sgrbh16.herston.uq.edu.au Hi, I'm trialling the the N.A. extraction method of Boom et al [1] based on diatamaceous earth/celite Is this method Ok for mRNA extractions? On a dummy trial, extracting total RNA as starting material (obtained from cell lines with TRI reagent) the method yeilds rRNA beatifully, but background mRNA smaller than the rRNA bands are much decreased, and gets worse with smaller RNA until there is no tRNA at all. (The starting total RNA has tRNA as the brightest part on a gel!) I will be using a lot of celite for each extraction (on ejaculate) so I opted to buy the much cheaper 95% Celite(R) 521 (Aldrich) (muddy looking colour) instead of the white analytical grade. ** Is this a problem or is celite not good for mRNA extraction? ** Any advances on Booms et al method for the laboratory? [1] Boom et al "Rapid and Simple Method for Purification of Nucleic Acids" J. Clin. Micro. (1990) 28(3) p495. Thanks in advance Paul R. Rohde Dep of Surgery, Univ. Qld., Royal Brisbane Hosp. Australia. aus.net@usa.net From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!vixen.cso.uiuc.edu!ais.net!news.idt.net!dispose.news.demon.net!demon!news.demon.co.uk!genesys.demon.co.uk!demon.co.uk!duncan From: "Dr. Duncan Clark" Newsgroups: bionet.molbio.methds-reagnts Subject: mRNA extractions with diatoms/celite ** mRNA? & Celite Grade? ** Date: Fri, 3 Oct 1997 09:51:26 +0100 Organization: GeneSys Ltd Distribution: world Message-ID: References: <6125b7$17e$1@nargun.cc.uq.edu.au> NNTP-Posting-Host: genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk [158.152.17.110] MIME-Version: 1.0 X-Newsreader: Turnpike Version 3.04 Lines: 27 In article <6125b7$17e$1@nargun.cc.uq.edu.au>, Paul Rohde writes >On a dummy trial, extracting total RNA as starting material (obtained >from cell lines with TRI reagent) the method yeilds rRNA beatifully, >but background mRNA smaller than the rRNA bands are much decreased, >and gets worse with smaller RNA until there is no tRNA at all. >(The starting total RNA has tRNA as the brightest part on a gel!) Having just started using the Merlin recipes, I would hazard a guess that the small products are either binding too tightly to be eluted with TE or water or they aren't binding at all. From memory the original Glass Milk recipes had the same problem and it was due to non-elution. With DNA one can take the elution component temperature up to 70C but that might not be a good idea with RNA. Duncan -- The problem with being on the cutting edge is that you occasionally get sliced from time to time.... Duncan Clark DNAmp Ltd. TEl/FAX 01252376288 http://www.dnamp.com http://www.genesys.demon.co.uk From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!howland.erols.net!news.maxwell.syr.edu!logbridge.uoregon.edu!news.uoregon.edu!newsfeed.orst.edu!news.orst.edu!news From: "Bryan L. Ford" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Long PCR Date: Thu, 02 Oct 1997 18:01:10 -0700 Organization: Marine/Freshwater Biomedical Sciences Center Lines: 18 Message-ID: <343443D6.7888@bcc.orst.edu> References: <3432715B.72BA@virginia.edu> Reply-To: fordb@bcc.orst.edu NNTP-Posting-Host: loveland-228-f.fst.orst.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (WinNT; I) Dr. Duncan Clark wrote: > > In article <3432715B.72BA@virginia.edu>, Rajesh Kumar > writes > >I need to do a long PCR (~9 kb). Can somebody suggest me a suitable > >polymerase and a source of it ? > > 9kb of what, human xsomal, bacterial xsomal or lambda. It makes a lot of > difference as to how difficult it will be. Lambda is possible with just > Taq the others need a mix and possibly a lot of playing. Duncan: Yes, good point. Our own successful 9.6 Kb work was with trout total genomic DNA, which has about the same complexity as human genomic DNA. -Bryan From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!thetimes.pixel.kodak.com!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.4!news-pen-4.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-dc-26.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!news.maxwell.syr.edu!eerie.fr!jussieu.fr!ravel.ijm.jussieu.fr!user From: bregeon@ijm.jussieu.fr (Bregeon Damien) Newsgroups: bionet.molbio.methds-reagnts,bionet.microbiology Subject: Re: polA mutant strain? Date: Fri, 03 Oct 1997 09:57:56 +0100 Organization: IJM Lines: 22 Message-ID: References: <3428D6D5.44DB@sunmail.lrz-muenchen.de> <34292F49.4DD3@ucdavis.edu> NNTP-Posting-Host: ravel.ijm.jussieu.fr Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Newsreader: Value-Added NewsWatcher 2.0b24.0+ Xref: biosci bionet.molbio.methds-reagnts:61690 bionet.microbiology:11185 > I'm looking for a polA mutant strain of E. coli to perform a gene > knockout, and would appreciate any assistance in finding such a strain. > I've looked in the ATCC catalogue, but without any cross-reference for > genotypes it would take forever to look through all the strains. In order to find a polA mutant strain of E.coli, i suggest you to consult the "E. coli genetic stock center" web server (http://cgsc.biology.yale.edu/cgsc.html) and to look for polA in the category "Gene Symbol Nameserver: Check for use as either Symbol or Synonym". In this case you cill have the choice between 15 different strains carying a polA mutation. In order to have the genotype of those strain choose anyone and "click" on it. I hope you will find the strain of your interest. -- BREGEON Damien Institut Jacques MONOD 2, Place Jussieu 75251 Paris cedex05 e-mail : bregeon@ijm.jussieu.fr Tel : +33 (0)1 44 27 47 31 Fax : +33(0)1 44 27 57 16 From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!206.229.87.25!news-peer.sprintlink.net!news-sea-19.sprintlink.net!news-in-west.sprintlink.net!news.sprintlink.net!Sprint!129.98.1.7!moonbeam.aecom.yu.edu!not-for-mail From: George Karkanias Newsgroups: bionet.molbio.methds-reagnts Subject: PCR with only one known primer? Date: Fri, 03 Oct 1997 09:04:53 -0400 Organization: Albert Einstein College of Medicine Lines: 8 Message-ID: <3434ED75.5D1D9188@aecom.yu.edu> NNTP-Posting-Host: aegean.aecom.yu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) Is there anyway to do a PCR rxn and get a product when you only know the sequence for one of the primers? Thanks in advance. George Karkanias Assistant Professor of Neuroscience From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!fcs280s.ncifcrf.gov!not-for-mail From: pnh@ncifcrf.gov (Paul N Hengen) Newsgroups: bionet.molbio.methds-reagnts Subject: Pointer to Methods FAQ list Date: 3 Oct 1997 21:18:30 GMT Organization: NCI-FCRDC Frederick Biomedical Supercomputing Center Lines: 90 Message-ID: <613nf6$9h41@ncisun1-nf0.ncifcrf.gov> NNTP-Posting-Host: cockleberry.ncifcrf.gov Summary: How to find the Methods FAQ list Keywords: FAQlist, FAQ list, FAQ X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] -- ************************************************************* * MINI-FAQ for bionet.molbio.methds-reagnts * * version 01.06.08.1997 (version.day.month.year) * ************************************************************* *** POINTER TO FAQ LISTS *** A hypertext version of the Frequently Asked Questions (FAQ) list for bionet.molbio.methds-reagnts in HTML format is now available at http://www-lmmb.ncifcrf.gov/~pnh/FAQlist.html The ASCII text version is available by anonymous FTP from ftp.ncifcrf.gov as the file pub/methods/FAQlist and can be accessed through anonymous FTP from net.bio.net as pub/BIOSCI/METHDS-REAGNTS/METHODS.FAQ If you don't know how to use FTP to obtain the FAQ list, you may request a copy by e-mail to pnh@ncifcrf.gov New users of BIOSCI/bionet may want to read the BIOSCI FAQ list. This list provides details on how to participate in these forums and is available by anonymous FTP from net.bio.net in pub/BIOSCI/biosci.FAQ. It may also be requested by sending e-mail to biosci-help@net.bio.net The FAQ sheet is posted on the first of each month to the newsgroup BIONEWS/bionet.announce immediately following the posting of the BIOSCI information sheet. Both of these FAQ lists are available through http://www.bio.net or through my Homepage at http://www-lmmb.ncifcrf.gov/~pnh/ *** SEARCHING FOR PAST ARTICLES *** The best way to search for past articles is by using the new WWW service from BIONET. The latest messages posted to the bionet as well as all past archived messages are located at net.bio.net and all you will need to do in order to read and/or post to any of the newsgroups is point your World Wide Web browser to the URL http://www.bio.net and then click on the "Access the BIOSCI/bionet Newsgroups" hyperlink. A new hypermail archiving system now gives you the advantages of USENET without requiring a local news server. The message headers are threaded by default, but messages can also be displayed chronologically or sorted by author or subject line. This capability gives you, in effect, a threaded newsreader through the Web. In addition, the FAQ list for bionet.molbio.methds-reagnts is directly accessible through that URL. Make your way down to and click on the "METHDS-REAGNTS" hyperlink. You will then see the link to the FAQ list. If you have any questions or encounter any problems with the new server, or you need further help with the archives please e-mail to biosci-help@net.bio.net *** POSTING TEST MESSAGES *** Please DO NOT send a message to the newsgroup which allows you to see a test posting you made without saying anything. This is a tremendous waste of time for readers and can be extremely annoying. A test message should be posted to misc.test where receipt of your message by various machines around the world will be acknowledged through e-mail. Also, if you do not specify a subject heading, your message will be threaded with all those others without one. Your chance of a specific response will therefore be limited. Try to make the subject as specific as possible, so people who read the postings through E-mail can decide if they want to read your message. THINK about how you would react to getting lots of JUNK messages through the newsgroup or by e-mail every day! *** HOW TO UNSUBSCRIBE *** Please DO NOT post a message to the newsgroup asking to be subscribed or unsubscribed! Send an e-mail message to biosci@net.bio.net if you are within the Americas or Pacific Rim, or to biosci@daresbury.ac.uk if you are in Europe, Africa, or Central Asia. In the body of the message, request that you be removed from the newsgroup. For example, to unsubscribe to the `Methods and Reagents' newsgroup, send an e-mail message to biosci@net.bio.net with no subject, and the body as: unsubscribe methods There is a file at ftp://ftp.ncifcrf.gov/pub/methods/how.to.subscribe which explains in a little more detail how to subscribe and unsubscribe to the methods newsgroup by e-mail if you are still having problems. If all else fails, write to biosci-help@net.bio.net and ask how to do it. -- ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/FAQlist.html - - - * * - TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - - * * - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/ * ******************************************************************************* From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.algonet.se!news.maxwell.syr.edu!news.cis.ohio-state.edu!magnus.acs.ohio-state.edu!ts6-8.homenet.ohio-state.edu!user From: snyder.9@osu.edu (Pam Snyder) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Primer Degradation? Date: Fri, 03 Oct 1997 17:14:07 -0500 Organization: Just me Lines: 34 Message-ID: References: NNTP-Posting-Host: ts6-8.homenet.ohio-state.edu In article , gilbride@bu.edu (Kevin Gilbride) wrote: > I need some advise regarding the stabilty of primers. I am using a primer > set which consists of a 25 and 22 mer oligos with a balanced AT / CG > content. My main concern is a laddering of bands appearing in the dH2O > control lanes. It is not a contamination, my other primers are free of > this problem, including Beta Globin and Actin sets. My water source > remains constant and when I have tried other sources the problem persists. > I have tried storage at 4º, -20º and -80º avoiding multiple freeze thaw. > The primers were synthisized by Oligo's Etc. they work initially upon > arrival but soon after the waters show these bands, it wouldn't be that > bad but they are in right in the range of which my positive bands appear. > Any advise or comiseration would be welcome. > > > Thanks > > KJ Gilbride > Mallory Institute of Path. > BUSM > gilbride@bu.edu "clean" primers are stable. Do you store master (concentrated) stocks separate from your working stocks? Then anytime you see something odd you go back to you masters and see where the problem is starting. It also greatly limits the # of times primers master stocks are frozen/thawed/pipeted. We make multiple working stocks from the masters in 1 ml vols, and further aliquots these to 200ul aliquots and use them one at a time. If we have any reason to suspect an aliquot, we pitch it. We have masters that have remained clean and intact for 8 years. Why do you think that contamination is not a problem? From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!news.maxwell.syr.edu!newsxfer3.itd.umich.edu!news1.best.com!uninett.no!sn.no!not-for-mail From: "Bjørn K. Pedersen" Newsgroups: bionet.molbio.methds-reagnts Subject: CD34 Direct on the Net Date: Fri, 03 Oct 1997 23:14:51 +0200 Organization: SN Internett Lines: 12 Message-ID: <3435604B.32C3@online.no> Reply-To: eped@online.no NNTP-Posting-Host: ti01a02-0061.dialup.online.no Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 3.0C-SNINETT (Win95; I) You can now get anti-human CD34 Mabs (clone 581) at a very good price direct on the Net. The company also supply other CD Mabs at a good price. The company also gives you direct researcher to researcher communication via e-mail. Check them out at: http://www.diatec.com -- - Bjørn Pedersen Oslo Norway From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!xfer.kren.nm.kr!su-news-hub1.bbnplanet.com!cam-news-feed1.bbnplanet.com!news.bbnplanet.com!nutmeg.uchc.edu!not-for-mail From: "Heinz J. Schaefers" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: MmeI Date: Fri, 03 Oct 1997 17:01:16 -0400 Organization: Univ of CT Health Center Lines: 46 Message-ID: <34355D1A.43422EBC@sun.XuchcX.edu> References: <61399d$r9h$1@bignews.shef.ac.uk> NNTP-Posting-Host: 155.37.2.192 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 (Macintosh; I; PPC) To: M Safaie X-Priority: 3 (Normal) M Safaie wrote: > I am looking for MmeI restriction enzyme.I am wandering if anybody > could tell me which companies supply the enzyme. > > thank a lot > > Mehran Safaie > department of molecular biology > university of sheffield > uk > E-mail;mbq95ms@sheffield.ac.uk Hi! MmeI Source: W.J. Brammer NOT COMMERCIALLY AVAILABLE Related References: Boyd, A.C., Charles, I.G., Keyte, J.W., Brammar, W.J. Isolation and computer-aided characterization of MmeI, a Type II restriction endonuclease from Methylophilus methylotrophus. Nucleic Acids Res. v14 p.5255-5274 1986. Tucholski, J., Skowron, P.M., Podhajska, A.J. MmeI, a class-IIS restriction endonuclease: purification and characterization. Gene v157 p.87-92 1995. source: http://www.neb.com/cgi-bin/reb_enz.pl bye Heinz -- Heinz J. Schaefers, Ph.D. University of Connecticut Health Center Department of Physiology Farmington, CT email: schaefers@sun.XuchcX.edu (remove both X to get valid email address) From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!rutgers!nntp.upenn.edu!dsinc!spool.mu.edu!uwm.edu!vixen.cso.uiuc.edu!howland.erols.net!news.maxwell.syr.edu!news.algonet.se!uninett.no!sn.no!not-for-mail From: "Bjørn K. Pedersen" Newsgroups: bionet.molbio.methds-reagnts Subject: CD34 Direct on the Net Date: Fri, 03 Oct 1997 21:19:55 +0200 Organization: SN Internett Lines: 12 Message-ID: <3435455B.C39@online.no> Reply-To: eped@online.no NNTP-Posting-Host: ti01a04-0006.dialup.online.no Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 3.0C-SNINETT (Win95; I) You can now get anti-human CD34 Mabs (clone 581) at a very good price direct on the Net. The company also supply other CD Mabs at a good price. The company also gives you direct researcher to researcher communication via e-mail. Check them out at: http://www.diatec.com -- - Bjørn Pedersen Oslo Norway From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!GPU.SRV.UALBERTA.CA!jkennie From: jkennie@GPU.SRV.UALBERTA.CA (J Kennie) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: help PCR Date: 3 Oct 1997 11:12:39 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 34 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <833B3E5B4C@trans.plants.ox.ac.uk> NNTP-Posting-Host: net.bio.net On 1 Oct 1997, Julian Robinson wrote: > Iam trying to amplify a ~450bp fragment with two 20bp primers. > It was working perfectly before I went on holiday. However when I > came back I begtan to have problems. The problem was smears in all > the lanes, including the control lane. It wasn't the template DNA > that was the problem (it oocured in the -ve control), and it is'nt > contamination (new everything has been used). > We are leaning towards primer dimers as the answer at the moment (as > there is some complementarity? between the primers), but the real > question is why has it just started to occur, when it was perfect > (i.e. no smears and clean bright product) before I went on holiday? > > many thanks > julian > --- > julian.robinson@plant-sciences.oxford.ac.uk > > Julian, I have been having the same problem. My template should be fine, the primers I use are kept at -20, I've tried new buffers, enzyme, water, dNTPs, etc. and I still get smears in all the lanes. Incidentally, this started happening a few days before I went on holiday and is still a problem now that I'm back. Does anyone have any suggestions on how to fix this problem?? I am amplifying a 850 bp fragment using primers that are about 30 nt long. I have previously had it working fine. Help. Jan University of Alberta From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!rutgers!news.cis.ohio-state.edu!magnus.acs.ohio-state.edu!usenet.INS.CWRU.Edu!HSNX.wco.com!newsfeed.dacom.co.kr!europa.clark.net!128.230.129.106!news.maxwell.syr.edu!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!server1.netnews.ja.net!server5.netnews.ja.net!news.shef.ac.uk!not-for-mail From: MBQ95MS@shef.ac.uk (M Safaie) Newsgroups: bionet.molbio.methds-reagnts Subject: MmeI Date: 3 Oct 1997 17:16:29 GMT Organization: Your Organization Lines: 13 Message-ID: <61399d$r9h$1@bignews.shef.ac.uk> NNTP-Posting-Host: pc065085.shef.ac.uk Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII X-Newsreader: WinVN 0.99.7 I am looking for MmeI restriction enzyme.I am wandering if anybody could tell me which companies supply the enzyme. thank a lot Mehran Safaie department of molecular biology university of sheffield uk E-mail;mbq95ms@sheffield.ac.uk From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!news.maxwell.syr.edu!cs.utexas.edu!news.uh.edu!not-for-mail From: benedik@uh.edu (Michael Benedik) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: IPTG and B-galactosidase Date: 3 Oct 1997 16:57:33 GMT Organization: University of Houston Lines: 17 Distribution: world Message-ID: <61385t$6t6$1@Masala.CC.UH.EDU> References: NNTP-Posting-Host: moose.bchs.uh.edu X-Newsreader: InterNews 2.0.2@moose.bchs.uh.edu.[R] X-Authenticated: benedik on POP host ns2.uh.edu. In article slawek@chem.pg.gda.pl (Slawek Dabrowski 18-22) writes: > > Hi everyone, > I have simple question. Is IPTG desintegrated by > B-galactosidase like lactose ? > > Best regards, > SLawek No. It is not metabolized. Michael Benedik Department of Biochemical Sciences University of Houston benedik@uh.edu From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!eecs-usenet-02.mit.edu!newsfeed.gte.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gsl.net!news.gsl.net!gip.net!fu-berlin.de!news.belwue.de!news.uni-mannheim.de!news.rhein-neckar.de!birdland.rhein-neckar.de!ibis.rhein-neckar.de!steffen.roth From: steffen.roth@rhein-neckar.de (Steffen Roth) Newsgroups: bionet.molbio.methds-reagnts Subject: mAb A1.4 from Olympus Biochemicals ? Date: Fri, 3 Oct 1997 17:55:04 Organization: RNI Lines: 12 Message-ID: NNTP-Posting-Host: ibis.rhein-neckar.de X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Hi, I'm looking for the Olympus Biochemicals company, formerly located in Lake Success, NY. This company was established by Dr. Chang Yi Wang. Product range: A lot of different antibodies. The company seems not to exist anymore. Does anyone know, whether there is a successor company, or whether there is a different source for the mAb A1.4, which recognizes the a3 domain of the HLA-A molecule ? Thanks Steffen From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!abdn.ac.uk!gen191 From: gen191@abdn.ac.uk (Andrew Hayhurst) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Hygromycin resistance Date: 3 Oct 1997 23:29:55 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net There is a group at Dupont that have engineered a hygR gene to make it more user friendly. Their details are at http://www.kumc.edu/res...fgsc/fgn41/carroll.html I guess it must be available after signing a clone agreement perhaps. Hope this is of use, Andrew From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!mindspring!news.mindspring.com!usenet From: Rick Bright Newsgroups: bionet.molbio.methds-reagnts Subject: Re: How to make a non-sticky glass plate Date: Sat, 04 Oct 1997 02:21:39 -0400 Organization: Emory University, Molecular Therapeutics & Toxicology Lines: 6 Message-ID: <3435E073.5C109949@emory.edu> References: NNTP-Posting-Host: user-2k7i807.dialup.mindspring.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Server-Date: 4 Oct 1997 06:22:13 GMT X-Mailer: Mozilla 4.03 [en] (Win95; U) To: "C.K. Chen" I use something called acrylease, I believe. I put on only one plate and always that plate. It ensure the gel to stick to the other plate consistently. Quick spray and wipe down with a kim wipe. Rick Bright From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!abdn.ac.uk!gen191 From: gen191@abdn.ac.uk (Andrew Hayhurst) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: cloning dimers Date: 3 Oct 1997 22:49:53 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net It is possible to clone multimers of fragments as head to tail units. The bugs such as XL1-Blue and SURE tolerate upto a certain number and then begin to delete and rearrange things! My own case was multimerising the HCMV enhancer borne on a 520bp fragment and I got up to 8 (within a pUC vector) before problems arose. However, I ensured only head to tails were being formed by judicious use of terminal restriction sites. What you may have is head to head and tail to tail (inverted repeats) forming which, if they are over a certain size, are extremely difficult to stabilise within the common strains. There was a Gene paper that employed destruction of a large palindrome as a positive selection in standard hosts. They used a special strain to propagate the unmodified vector which may be of use to you. The paper is by Elhai and Wolk, Gene 68:119-138 (1988)- A class of positive selection vectors based on the non-viability of palindrome containing plasmids that allows cloning into long polylinkers. Hope this is of use, goodluck. From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!164.67.42.145!nntp.info.ucla.edu!132.239.254.208!ihnp4.ucsd.edu!munnari.OZ.AU!metro!metro!unsw.edu.au!NewsWatcher!user From: d.jacobs@unsw.edu.au (daniel jacobs) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Long PCR Date: Fri, 03 Oct 1997 12:15:16 +1000 Organization: unsw Lines: 18 Message-ID: References: <3432715B.72BA@virginia.edu> NNTP-Posting-Host: 149.171.168.40 In article <3432715B.72BA@virginia.edu>, Rajesh Kumar wrote: > Hi, > > I need to do a long PCR (~9 kb). Can somebody suggest me a suitable > polymerase and a source of it ? How about using a mixture of two > polymerases, say, Taq and Pfu ? > > Any suggestion will be highly appreciated. > > Rajesh Try using a mixture of taq/pfu (10/1 unit) in your standard PCR buffer. I use it all the time and it works, at least upto 12 kB. Good luck Daniel From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.algonet.se!news.maxwell.syr.edu!Supernews60!supernews.com!news.he.net!news.pagesat.net!news1.good.net!news.goodnet.com!news From: Stephen & Helen Newsgroups: bionet.molbio.methds-reagnts Subject: Re: IPTG and B-galactosidase Date: Fri, 03 Oct 1997 16:51:26 -0700 Organization: GoodNet Lines: 13 Message-ID: <343584FE.A132BC2A@c2i2.com> References: NNTP-Posting-Host: tuc-ns4-9.goodnet.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) Slawek Dabrowski 18-22 wrote: > > Hi everyone, > I have simple question. Is IPTG desintegrated by > B-galactosidase like lactose ? > > Best regards, > SLawek IPTG is a non metabilizable analogue of lactose and is non broken down by B-gal. sjb From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!chugaibio.com!dspinella From: dspinella@chugaibio.com (Dom Spinella) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: 5' protruding ends Date: 3 Oct 1997 17:04:14 -0700 Organization: Chugai Biopharmaceuticals, Incorporated Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <343597AE.2DEE@chugaibio.com> Reply-To: dspinella@chugaibio.com, @chugaibio.com NNTP-Posting-Host: net.bio.net > Hello > > Just wanted a quick opinion regarding converting protruding termini > to blunt ends from those out there who do this more regularly than I. > I have used Mung-bean nuclease for this in the past, but perhaps there > is something better available. Also, for my general knowledge, I recall > that Mung-bean is often considered a better choice for this than > nuclease S1 but don't remember the exact reason. > > Thanks for your help, > > Craig Garen Craig: Actually, I think most folks just fill in 5' overhangs with Klenow or T4 polymerase and dNTPs. These enzymes also have 3'->5' exonuclease activity and can therefore also be used to remove 3' overhangs. Mung bean and especially S1 nuclease can be a little "agressive" and sometimes chew back even the double stranded DNA, so a lot of people don't use them for this purpose anymore. Hope this helps. -- Dom Spinella From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!HOTMAIL.COM!craig_garen From: craig_garen@HOTMAIL.COM (Craig Garen) Newsgroups: bionet.molbio.methds-reagnts Subject: 5' protruding ends Date: 3 Oct 1997 15:25:49 -0700 Organization: University of Alberta Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <34357396.A48273FC@hotmail.com> NNTP-Posting-Host: net.bio.net Hello Just wanted a quick opinion regarding converting protruding termini to blunt ends from those out there who do this more regularly than I. I have used Mung-bean nuclease for this in the past, but perhaps there is something better available. Also, for my general knowledge, I recall that Mung-bean is often considered a better choice for this than nuclease S1 but don't remember the exact reason. Thanks for your help, Craig Garen From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!gsl-penn-ns.gsl.net!news.gsl.net!gip.net!sunqbc.risq.qc.ca!news.uow.edu.au!metro!metro!news From: Heather Medbury Newsgroups: bionet.molbio.methds-reagnts Subject: which PCR machine? Date: Fri, 03 Oct 1997 08:48:24 -0700 Organization: Information Technology Services, The University of Sydney, NSW, Australia Lines: 20 Distribution: inet Message-ID: <343513C8.2974@renal.wsahs.nsw.gov.au> Reply-To: medbury@renal.wsahs.nsw.gov.au NNTP-Posting-Host: mars.wsahs.nsw.gov.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win16; I) HI, We are looking at buying one of those PCR machines which has fully automated real time detection of specific PCR products. They work by detecting the fluorescence of a target specific probe. I know that there is a machine put out by Perkin Elmer and one put out by Idaho Technology. Does anyone have any experience with either of these machines? WHat do you think of them? Are there any other alternatives available at the moment, or soon to come onto the market? THankyou in advance Heather Medbury SYDNEY AUSTRALIA medbury@renal.wsahs.nsw.gov.au From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!not-for-mail From: pnh@ncifcrf.gov (Paul N Hengen) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR with only one known primer? Date: 3 Oct 1997 21:27:14 GMT Organization: NCI-FCRDC Frederick Biomedical Supercomputing Center Lines: 37 Message-ID: <613nvi$9h42@ncisun1-nf0.ncifcrf.gov> References: <3434ED75.5D1D9188@aecom.yu.edu> NNTP-Posting-Host: cockleberry.ncifcrf.gov X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] George Karkanias (karkania@aecom.yu.edu) wrote: > Is there anyway to do a PCR rxn and get a product when you only know the > sequence for one of the primers? > > Thanks in advance. > > George Karkanias > Assistant Professor of Neuroscience Yes. I know of a couple ways to do it. You should look at this review for details about boomeranging... @article{Hengen1995Septibs, author = "P. N. Hengen", title = "Methods and reagents - Vectorette, splinkerette, and boomerang {DNA} amplification", journal = "Trends in Biochemical Sciences", volume = "20", number = "9", pages = "372-373", month = "September", year = "1995"} ftp://ftp.ncifcrf.gov/pub/methods/TIBS/sep95.txt -- ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/FAQlist.html - - - * * - TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - - * * - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/ * ******************************************************************************* From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!not-for-mail From: levenson@uic.edu (Victor Levenson) Newsgroups: bionet.molbio.methds-reagnts Subject: US/IL Research Specialist, U of I at Chicago Date: Fri, 03 Oct 1997 21:03:59 GMT Organization: University of Illinois at Chicago Lines: 42 Message-ID: <34355d8e.9623608@news.cc.uic.edu> NNTP-Posting-Host: e128.gene.uic.edu X-Newsreader: Forte Free Agent 1.11/32.235 I have an opening for a Research Specialist in my group. Primary duties include: * tissue culture maintenance (human cell lines); * transfection-infection-selection; * bacterial mini and maxipreps; * thorough record keeping; * slight ordering and general lab maintenance; I look for a highly motivated person who can learn fast, solve problems on his/her own and would require minimum supervision after the initial training period. BS is required; MS is preferred, although I will teach everything, so the desire to learn is essential. Two year commitment will be required. Position is funded through the NIH grant for at least two years. Additional funding is expected. NB: I cannot invite foreign specialists (sorry!), so this announcement is directed ONLY to people in the US. I will answer all the e-mails; cannot promise the same for phone calls. Victor Levenson MD/PhD Research Assistant Professor levenson@uic.edu Dept of Genetics Tel 312-413-3887 U of IL at Chicago Fax 312-413-3128 Victor Levenson MD/PhD Res Asst Prof UIC, Dept of Genetics M/C 669 900 S Ashland Ave The one who wants finds HOW Chicago IL 60607 The one who doesn't finds WHY levenson@uic.edu 312-413-3887 312-413-3128 FAX From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!ibm.net!lsu0640 From: lsu0640@ibm.net (Philip Cheng) Newsgroups: bionet.molbio.methds-reagnts Subject: why BSA is need ed in some resriction enzymes reaction Date: 3 Oct 1997 09:30:46 -0700 Organization: VMP-LSU Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <34351DD9.17B7@ibm.net> Reply-To: lsu0640@ibm.net NNTP-Posting-Host: net.bio.net Hi, there, Does anyone know why BSA is needed in some restriction enzymes reaction? Thank you. Philip From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!sigsmtp.sial.com!CSK1 From: CSK1@sigsmtp.sial.com (CHANDRA KRISHNAN) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Biotech student study group needs assistance Date: 3 Oct 1997 09:07:59 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 63 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Actually this not very uncommon. Term papers and open book exams have always been in vogue. On completion of these papers or exams there is always an oral exam where in the professor can check the amount of knowledge assimilated by the student in the process, irrespective of the source. In this case I am really impressed that the student chose the internet to seek his answers. After all is'nt one of the purposes of the internet is to spread the information around . krishnan To: methods@net.bio.net From: tyr-2@bones.biochem.ualberta.ca (Karl Fischer) Subject: Re: Biotech student study group needs assistance Date: Thu, 02 Oct 1997 16:13:20 -0700 In article , diane wrote: > My name is Diane and I am a 4th year university student in Biology. I am > part of a "Biotechnology student study group" which is working together > to answer a set of questions given to us by our professor. Our > professor has STRONGLY suggested that we seek answers to 11 specific > questions from various sources (eg. class notes, texts, the primary > literature, friends, colleagues, professors, internet and company > catalogues). I find it very difficult to believe that a professor would advocate the use of professors, scientific colleagues, and the internet community for providing ANSWERS to these questions (please note the capitals). I could, however, believe that said professor would instruct students to derive information from class notes, texts, information resources like catalogues/web and THEN formulate a suitable answer for each question which they THEN, if they so choose, run these answers by individuals who might have more practical/theoretical experience with the topics in question. Getting the answers without doing the mental legwork diminishes the learning experience and is, unfortunately, becoming a bit too common (IMHO). Karl the hepB guy -- Karl Fischer tyr-2@bones.biochem.ualberta.ca From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!MAD.SCIENTIST.COM!stuart From: stuart@MAD.SCIENTIST.COM ("Stuart A. Ali") Newsgroups: bionet.molbio.methds-reagnts Subject: re:isolation of RNA from whole blood. Date: 3 Oct 1997 09:04:38 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <199710031604.SAA15956@mailbox.univie.ac.at> NNTP-Posting-Host: net.bio.net Your problem is RNAse degradtion. One alternative method you might want to try uses Catrimox-14 (from Sigma). This is described in J. of Infectious Diseases 176:20-6 (and refs cited therein). We use this for isolating cellular and viral RNA from whole blood. Regards, Stuart Ali stuart@mad.scientist.com From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!vixen.cso.uiuc.edu!ais.net!news.idt.net!dispose.news.demon.net!demon!delos.dra.hmg.gb!server1.netnews.ja.net!server5.netnews.ja.net!server6.netnews.ja.net!server4.netnews.ja.net!news.cc.ic.ac.uk!not-for-mail From: Koen De Smet Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RFI: vector with selection for hygromycin B resistance Date: Fri, 03 Oct 1997 16:05:14 +0100 Organization: Department of Medical Microbiology Message-ID: <343509AA.7579@nospam.ic.ac.uk> References: <343463b3.7527581@news.orst.edu> Reply-To: k.desmet@nospam.ic.ac.uk NNTP-Posting-Host: cb-22.sm.med.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; PPC) To: Don Chen Lines: 32 Don Chen wrote: > > Hi: > > I am looking for a vector that carries a gene for hygromycin B > resistance. Does anyone know of either commercial or academic > source(s) for such a vector? Thanks for any help you can offer. > > don > We use hygromycin B resistance markers on our shuttle vectors for cloning and expression in mycobacteria. Our vectors are all based on p16R1, described in Microbiology 140, 133-138 (1994). Selection is with 250 ug/ml hyg in E.coli, 50-100 ug/ml in mycobacteria. I am not sure if there are any suitable sites to cut out the hyg gene only. Email me if you want more info, or if you want me to send you some plasmid. Koen De Smet _______________________________________________________________ To reply by email, remove "nospam." from the above address _______________________________________________________________ ___ ___ ___ Department of Medical Microbiology ___ ___ Imperial College School of Medicine at St Mary's ___ ___ London W2 1PG ___ ___ http://www.sm.ic.ac.uk/medmicro/home. ___ _______________________________________________________________ From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!daresbury!not-for-mail From: "Alexander N. Kukushkin" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: What is GPT? Date: 3 Oct 1997 15:27:29 +0100 Organization: l mmcd i cytology ras Lines: 11 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <612vch$ruf@mserv1.dl.ac.uk> References: <9710210390.~INN-YMCa01336.bionet-news@dl.ac.uk> MIME-Version: 1.0 Original-To: methods@dl.ac.uk > Hi folks -- > I recently received an expression construct which has the GPT > gene on it; unfortunately I don't know what GPT stands for or what it > does for my transfections. Any ideas? > Thanks, > Harry > Guanine phosphoribosyl transferase (GPT) gene of E.coli is a selective marker. Mammalian cells transfected by the GPT construct will grow in media containing mycophenolic acid. Alex From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!eru.mt.luth.se!www.nntp.primenet.com!globalcenter0!news.primenet.com!nntp.primenet.com!news.maxwell.syr.edu!newsfeed.acns.nwu.edu!news.acns.nwu.edu!merle!gbe392 From: gbe392@merle.acns.nwu.edu (Gina Berardesco) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: How to make a non-sticky glass plate Date: 3 Oct 1997 13:56:26 GMT Organization: Northwestern University, Evanston, IL Lines: 14 Message-ID: <612tia$rso@news.acns.nwu.edu> References: Reply-To: g-berardesco@nwu.edu (Gina Berardesco) NNTP-Posting-Host: merle.acns.nwu.edu X-Newsreader: TIN [version 1.2 PL2] C.K. Chen (cchen2@uiuc.edu) wrote: > Hi, > Could someone tell me how to make a sequencing gel glass plate > non-sticky? I use silver stain for sequencing gels and would like the gel > stick on one of the glass plates. I have tried Silan treatment for one of > the plates. However, the gel sticked on both plates. I am confident that > the plates were clean. Hi - I used to do the silver stain sequencing thing myself. I always used RainX on my non-sticky plate, it worked great! -gina From owner-methds-reagnts@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!MAIL.MCG.EDU!KMANGOLD From: KMANGOLD@MAIL.MCG.EDU (Kathy Mangold) Newsgroups: bionet.molbio.methds-reagnts Subject: RFI: vector with selection for hygromycin B resistance -Reply Date: 3 Oct 1997 07:0