From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!uunet!in2.uu.net!newsfeed.pitt.edu!bullwinkle.paccm.pitt.edu!user From: lalarcon+@pitt.edu (Lou Alarcon) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Is there a shareware plasmid drawing program? Date: Wed, 02 Aug 1995 17:18:13 -0500 Organization: University of Pittsburgh, Dept of Surgery Lines: 14 Message-ID: References: <3v9i9i$ej8@netserv.unmc.edu> NNTP-Posting-Host: bullwinkle.paccm.pitt.edu In article <3v9i9i$ej8@netserv.unmc.edu>, dchakrav@netserv.unmc.edu (Dhruba Chakravarti) wrote: > Anybody knows of a shareware or freeware plasmid drawing program? > > Regards, > > Dhruba. Try the link site: http://sun1.bham.ac.uk/s.m.williams.bcm/images/viewers.html They have several shareware-versions of molecular-bio software programs, including what you are looking for. From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!sgigate.sgi.com!spool.mu.edu!news.moneng.mei.com!news.ecn.bgu.edu!psuvax1!news.eecs.nwu.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!uicvm.uic.edu!u12201 Organization: University of Illinois at Chicago, ADN Computer Center Date: Wed, 2 Aug 1995 10:36:26 CDT From: Message-ID: <95214.103626U12201@uicvm.uic.edu> Newsgroups: bionet.molbio.methds-reagnts Subject: Re: microtiterplates for sequencing References: <1995Jul31.171504.22159@newsserver.rrzn.uni-hannover.de> Lines: 7 In article <1995Jul31.171504.22159@newsserver.rrzn.uni-hannover.de>, bmay says: > >I've heard of those microtiterplates for sequencing, but I dont know >where to obtain them. Does anybody out there know ? Is this simply the streptavidin coated ELISA plates? KeldS@uic.edu From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!uunet!in1.uu.net!spool.mu.edu!news.moneng.mei.com!news.ecn.bgu.edu!psuvax1!news.eecs.nwu.edu!newsfeed.acns.nwu.edu!news.cc.uic.edu!uicvm.uic.edu!u12201 Organization: University of Illinois at Chicago, ADN Computer Center Date: Wed, 2 Aug 1995 09:17:58 CDT From: Message-ID: <95214.091758U12201@uicvm.uic.edu> Newsgroups: bionet.molbio.methds-reagnts Subject: SuperSignal CL-HRP for Nothern & Southern Lines: 6 Pierce carries a new enhanced chemiluminescent substrate that is less expensive than ECL and in western blots show higher light emission & longer emission times (up to 6 hours and more). Has anybody experience with this product in Norhtern and Southern? KeldS@uic.edu From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!usenet From: David Clayton Newsgroups: bionet.molbio.methds-reagnts Subject: FLAG epitope - sources and advice Date: 2 Aug 1995 20:52:37 GMT Organization: University Of Illinois Lines: 10 Message-ID: <3vooil$e67@vixen.cso.uiuc.edu> NNTP-Posting-Host: claytond.life.uiuc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.methds-reagnts/29007-29092 I need to put a small epitope onto a protein to allow detection of it in cultured cells and perhaps tissues. I have the impression that the FLAG epitope might be a good choice for this. Questions: 1) is there a commercial source for FLAG-epitope related reagents? 2) Anybody want to offer opinions as to the utility of FLAG, vs. other candidates such as His, myc, HA, etc? Any advice appreciated! >>David dclayton@uiuc.edu 217-244-4525 From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-stuttgart.de!news.rhrz.uni-bonn.de!news.rwth-aachen.de!newsserver.rrzn.uni-hannover.de!news From: bmay Subject: Re: infor. on PCR machine Message-ID: <1995Aug2.163811.28728@newsserver.rrzn.uni-hannover.de> Sender: news@newsserver.rrzn.uni-hannover.de (News Service) Organization: RRZN References: Date: Wed, 2 Aug 1995 16:38:11 GMT Lines: 29 hfang@darwin.mbb.sfu.ca (Hung Fang) wrote: > > Hello there: > Our lab. is interested in buying a PCR machine. We'd like to buy >one like Biometra's Trio Thermoblock which one can run three >different program at the same time. The problem is Biometra's >machine are quite expensive, we are wondering whether any other >companies making tri-block or twin-block PCR machine. Any >information or personal experience on this will be very helpful >for us and will be very much appreciated. > We are also about buying a thermal cycler, at this moment my personal favourite is MJ researchs "DNA-engine", because it claims to cool/heat 3° per second !!! Further more it features two independent blocks for 0.2 or 0.5ml vials or slides . On the other hand i have not yet tested it (but I soon will). if anyone out there has, let me know Bernhard Mayr Medizinische Hochschule Hannover Dept. Clinical Endocrinology Hannover Germany email: ndxdendo@rrzn-user.uni-hannover.de From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!uunet!in1.uu.net!gwu.edu!sullivan From: sullivan@gwis2.circ.gwu.edu (Steven Sullivan) Newsgroups: bionet.molbio.methds-reagnts Subject: Library screening alternatives? Date: 2 Aug 1995 20:18:06 GMT Organization: The George Washington University, Washington DC Lines: 9 Message-ID: <3vomhu$65j@cronkite.seas.gwu.edu> NNTP-Posting-Host: 128.164.127.252 X-Newsreader: TIN [version 1.2 PL2] It's been some years since I screened a lambda gt11/cDNA library, using the tried-and-tru (but labor intensive and slow) plaque hybridization method -- I'm wondering if in the interim any cool new (e.g. PCR based?) methods have arisen to make library screening quicker, easier, etc. From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!news.sprintlink.net!newsfeed.internetmci.com!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!news.imag.fr!ciril.fr!univ-lille1.fr!cict.fr!news From: Laurent Seroude Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Is there a shareware plasmid drawing program? Date: 2 Aug 1995 21:13:40 GMT Organization: Centre de Biologie du Developpement Lines: 7 Message-ID: <3vopq4$qpd@news.cict.fr> References: <3v9i9i$ej8@netserv.unmc.edu> NNTP-Posting-Host: 130.120.104.2 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:3v9i9i$ej8@netserv.unmc.edu dchakrav@netserv.unmc.edu (Dhruba Chakravarti) wrote: >Anybody knows of a shareware or freeware plasmid drawing program? You can find the shareware "Bluegene" at : "ftp://src.doc.ic.ac.uk/packages/mac-umich/misc/biology/bluegenes.sit.hqx" From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!newsfeed.internetmci.com!news.sprintlink.net!cs.utexas.edu!news.unt.edu!hermes.oc.com!usenet From: Gordon Betts Newsgroups: bionet.molbio.methds-reagnts Subject: RFLP of birds Date: 2 Aug 1995 19:39:59 GMT Organization: OpenConnect Systems, Dallas, TX, USA Lines: 5 Message-ID: <3vokaf$2r3@hermes.oc.com> NNTP-Posting-Host: acme.etsu.edu I would like to try a project this fall involving RFLP or birds. Is there a probe available for birds? Any experience with existing probes and birds? Thanx Gordon Betts From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!news.sprintlink.net!cs.utexas.edu!news.unt.edu!hermes.oc.com!usenet From: Gordon Betts Newsgroups: bionet.molbio.methds-reagnts Subject: I have a student interested........ Date: 2 Aug 1995 19:38:10 GMT Organization: OpenConnect Systems, Dallas, TX, USA Lines: 13 Message-ID: <3vok72$2r3@hermes.oc.com> NNTP-Posting-Host: acme.etsu.edu I have a student interested in graduate school in the areas of neurobiology and behaviour. Shelley will be doing an undergraduate research project with me this fall and graduate with a BS in biology next May. If you know of possible programs to recommend, etc. she would like the info at "tacker@wizard.etsu.edu" or you can write to her at: Shelley Tacker c/o Dr. J. Gordon Betts Biology Dept. East Texas State University Commerce, TX 75429-3011 BTW- she made an A in my physiology course. From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!daresbury!sunsite.doc.ic.ac.uk!plug.news.pipex.net!pipex!tank.news.pipex.net!pipex!news.uoregon.edu!vixen.cso.uiuc.edu!newsrelay.iastate.edu!gw1.phibred.com!news From: Subject: Re: infor. on PCR machine Message-ID: Sender: news@phibred.com (USENET News System) Organization: Pioneer Hi-Bred International, Inc. References: Date: Wed, 2 Aug 1995 19:08:44 GMT Lines: 12 hfang@darwin.mbb.sfu.ca (Hung Fang) wrote: > > Hello there: > Our lab. is interested in buying a PCR machine. We'd like to buy one like Biometra's Trio Thermoblock which one can run three different program at the same time. The problem is Biometra's machine are quite expensive, we are wondering whether any other companies making tri-block or twin-block PCR machine. Any information or personal experience on this will be very helpful for us and will be very much appreciated. > Our lab has a Gradient Robocycler, which can be used to sample a gradient of annealing temperatures at the the same time. It cost a bit ($5800) but it is licensed for PCR. The gradient is hard to beat, it's worked quite well for me. John McElver mcelverja@phibred.com From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!NewsWatcher!user From: hdang@channel.neusc.bcm.tmc.edu (hong dang) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: pET vector cloning of hydrophobic protein gene Date: Wed, 02 Aug 1995 14:26:28 -0500 Organization: BCM, Neuroscience Lines: 54 Message-ID: References: <3vltmi$qn5@warp.cris.com> NNTP-Posting-Host: d.neusc.bcm.tmc.edu X-Newsreader: Yet Another NewsWatcher 2.0b30 > In article <3vltmi$qn5@warp.cris.com>, mike.holloway@stjude.org (Mike > Holloway) wrote: > > > I'm giving up on the pET system and wondered if anyone else has had > > similar experiences. Even with the thioredoxin fusion protein vector, > > I can not isolate a transformant with my sequence and an intact vector. > > I do get many rearrangements - things that are not whole pET vector or > > PCR template from the preparation of my insert. The transformants are of > > various sizes, with various restriction sites missing. Other cloning > > projects using the same bugs and reagents are working just fine, without > > any evidence of contamination. > > > > I have to conclude that some protein is being made, despite what anyone > > says about the T7 promoter not being used without the polymerase being > > pumped out, and that its toxic. This has been the previous experience > > in our lab with this protein using other bacterial expression vectors. > > Apparently this isn't an isolated experience, since several companies > > have recently come out with this thioredoxin fusion protein scheme that's > > supposed to solve everything. > > > > Anyone had experience with pET vectors? > > > > ========================================================================= > >============= There may be 1 way out: recently people in our lab has succeeded in cloning one of those "toxic" thing in a pET, which had failed before. Here's the trick: a PCR product was direction-cloned into both pET-20b and pET25b, 1st one, no colony, 2nd one, many. The relevant difference, there is a Lac-op following T7 promoter in 25b. So in addition to controlling T7 pol, 25b also has a lac-repression on your gene of interest that may further cut down on leaky expression of the thing and make it tolerable for the bug. So seems there is a dose effect, if your protein is really, really toxic to the bug, it still may not express. But try the T7-lac combo vector before giving it up, since you might hit the same problem with another system as well. Hong -- Hong Dang (hdang@channel.neusc.bcm.tmc.edu) O O O _/|\ _/|\ _/ | \ |/ |/ | / / \_ / \_ / \_ o | o | o | From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!cs.utexas.edu!swrinde!emory!darwin.sura.net!fconvx.ncifcrf.gov!fcsparc6!pnh From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) Subject: More on Boomerang DNA Amplification Message-ID: Summary: Upcoming TIBS Article Preview Sender: Paul N. Hengen Nntp-Posting-Host: fcsparc6.ncifcrf.gov Organization: Frederick Cancer Research and Development Center Distribution: bionet Date: Wed, 2 Aug 1995 17:12:38 GMT Lines: 36 A little while ago there was a brief discussion on this group concerning the Boomerang DNA Amplification (BDA) process that Kevin Ahern (ahernk@bcc.orst.edu) invented. [Kevin: If you read this, check your e-mail!] Since the technique is difficult to describe in words, Kevin allowed us to see a diagram of the process by sending a BinHexed version by e-mail, or by sending a fax to anyone interested. I then put the diagram as different formats at my ftp site for people to grab. I thought this was such an interesting topic that it should be described somewhere, so one of the next TIBS columns (September 1995 issue) is dedicated to this and similar amplification techniques: @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 = "???-???", month = "sep", year = "1995"} I've since made a new diagram (IN COLOR) to be included with the TIBS article and I am making it available to everyone through my ftp site. People who would like a sneak preview of it can use anonymous ftp to ftp.ncifcrf.gov and look in the directory pub/methods/boomerang. There you will find Figure 1 for my column as figure1.cdr (CorelDraw), figure1.gif, figure1.tif, and figure1.ps ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - - - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/ - - - - - - * * - - - Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - * ******************************************************************************* From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!THORIN.UTHSCSA.EDU!HARDIES From: HARDIES@THORIN.UTHSCSA.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: re: Gradients with automated DNA sequencers? Date: 2 Aug 1995 11:24:01 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 36 Sender: daemon@net.bio.net Distribution: world Message-ID: <950802132622.21c437@thorin.uthscsa.edu> NNTP-Posting-Host: net.bio.net In response to my inquirery about whether anyone uses gradient gels on an ABI sequencer, Tom Chappell writes: > > Why would you want to use a wedge gel in an ABI sequencer? The only point > of a wedge is to slow down the lower MW fragments in the wedge, so they > don't run off the bottom of the gel while you're resolving the higher MW > fragments. On an ABI sequencer you want the fragments to fall off the > bottom of the gel after they pass the sensor. Thanks for your response. Yes, all the applications of gradient gels to DNA sequencing that I have seen are exactly as you say; and hence irrelevant to the ABI sequencer. Theoretically, gradient gels can also deliver peak sharpening and enhanced resolution, which could be real helpful on an ABI sequencer, say out past 500 bases. However, it's not trivial to configure a gel to actually deliver this effect; and, as near as I can tell, it may not even be possible with currently available gradient methods, and within the practical limitations of the ABI sequencer. However, if my understanding is wrong and people know how to do this, I want to know about it before I commit a blunder in this grant proposal I'm writing. The one gradient method that I know of that would be technically doable on an ABI sequencer is this buisness of putting 0.5 x TBE in the upper chamber to create a stacking gradient to sharpen the initial peak width. It's not obvious to me that this actually has a discernable effect on autoradiographed sequencing gels. Maybe the bands stack so well to start with that there's nothing to be gained. Does anyone do that on an ABI sequencer; and if so, does it do much? Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio Hardies@uthscsa.edu For information on our graduate program in Biochemistry: http://bioc02.uthscsa.edu/biochem.html From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!uunet!in2.uu.net!pipeline!not-for-mail From: tmurray@nyc.pipeline.com (Tony Murray) Newsgroups: bionet.molbio.methds-reagnts Subject: Gradients with automated DNA sequencers? Date: 2 Aug 1995 13:47:49 -0400 Organization: The Pipeline Lines: 49 Message-ID: <3vodo5$b3i@pipe1.nyc.pipeline.com> References: <950802103414.21c437@thorin.uthscsa.edu> NNTP-Posting-Host: pipe1.nyc.pipeline.com Why would one wish to use a wedge when such a gel is primarily intended to compress the short fragment bands in a "manual" gel? It make no sense in a gel using a "finishing post" type detector. Tony In bionet.molbio.methds-reagnts HARDIES@THORIN.UTHSCSA.EDU said: >Path: >pipeline!newsjunkie.ans.net!howland.reston.ans.net!spool.mu.edu!olivea!biosci!TH >ORIN.UTHSCSA.EDU!HARDIES >From: HARDIES@THORIN.UTHSCSA.EDU >Newsgroups: bionet.molbio.methds-reagnts >Subject: Gradients with automated DNA sequencers? >Date: 2 Aug 1995 08:32:06 -0700 >Organization: BIOSCI International Newsgroups for Molecular Biology >Lines: 14 >Sender: daemon@net.bio.net >Distribution: world >Message-ID: <950802103414.21c437@thorin.uthscsa.edu> >NNTP-Posting-Host: net.bio.net > >Hi Netters: > >Does anyone use (or know of) wedge gels (or other gradient configurations) used >on an ABI sequencer (or other DNA sequencer with a fixed sensor position)? I'm >writing a grant proposal about gradient systems in DNA sequencing, and am under >the impression that they aren't applied much to automated sequencers. Am I >misinformed? > >Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio >Hardies@uthscsa.edu > >For information on our graduate program in Biochemistry: >http://bioc02.uthscsa.edu/biochem.html -- Tony Murray Research & Business Development Pharmacia Biotech Inc. 800 Centennial Ave Piscataway NJ 08855-1327 USA (908)-457-8392 (tel) (908)-457-8246 (fax) From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!News.Uni-Marburg.DE!papin.HRZ.Uni-Marburg.DE!klieber From: klieber@papin.HRZ.Uni-Marburg.DE (Hans-Georg Klieber) Newsgroups: bionet.molbio.methds-reagnts Subject: need some help starting molecular biology Date: 2 Aug 1995 12:37:37 GMT Organization: Hochschulrechenzentrum der Universitaet Marburg Lines: 30 Message-ID: <3vnrih$65i@surz03.HRZ.Uni-Marburg.DE> NNTP-Posting-Host: papin.hrz.uni-marburg.de X-Newsreader: TIN [version 1.2 PL2] Our group is about to enter the field of molecular biology of ion transport systems and we need some help to get started. More specifically: - we need to retrieve published sequence data from certain ion channels. Where and how can I do this? - we need some software (DOS/Windows) to look at the sequence information, identify the features, introns, exons, cutting sites, etc... - we are planning to do PCR to demonstrate the expression of certain ion channels in our cells, therefore we need to locate existing primers and/or need a software to design them. Where can I find them? - any other hints to software, lab protocolls, suppliers etc that might be interesting for such work are most welcome Thank you very much for your help! Hans-Georg --------------------------------------------------- Dr. Hans-Georg Klieber Institute for Physiology, University of Marburg Deutschhausstrasse 2, 35033 Marburg, Germany Office: +49-6421-282290, Home: +49-6421-25680 From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!uunet!in1.uu.net!cis.ohio-state.edu!nntp.sei.cmu.edu!news.psc.edu!hudson.lm.com!news.math.psu.edu!news.cac.psu.edu!newsserver.jvnc.net!newsserver2.jvnc.net!netnews.upenn.edu!NewsWatcher!user From: maris@email.chop.edu (John Maris, MD) Newsgroups: bionet.molbio.methds-reagnts Subject: Protein Truncation Assay Date: Wed, 02 Aug 1995 13:23:39 -0400 Organization: Oncology, The Children's Hospital of Phila. Lines: 6 Message-ID: NNTP-Posting-Host: 159.14.43.9 Does anyone have experience with in vitro transcription/translation for mutation detection? I would like to use this technique to screen a newly described gene to see if it MAY be involved in a certain disease. I would prefer not to go the SSCP route. Is a commercially produced kit available? From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!COMPBIO.MED.WAYNE.EDU!jeff From: jeff@COMPBIO.MED.WAYNE.EDU (Jeff Kramer) Newsgroups: bionet.molbio.methds-reagnts Subject: RE: pET vectors, good or bad? Date: 2 Aug 1995 10:30:43 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 67 Sender: daemon@net.bio.net Distribution: world Message-ID: <9508021728.AA01214@compbio.med.wayne.edu> NNTP-Posting-Host: net.bio.net Dr. Browning writes: ----- Begin Included Message ----- >>Mike Holloway wrote: -------------------------------------------------------------------------------- >>I'm giving up on the pET system and wondered if anyone else has had >>similar experiences. >> ...stuff deleted... >>Anyone had experience with pET vectors? -------------------------------------------------------------------------------- >Jeff Kramer wrote: >I have spentf years of my life trying to get lipoxygenase expression using >pET 3a while a grad student working on my PhD. Even after I got expression, >I >rarely got anywhere near reproducible amounts of protein. The specific >activity was, thankfully, pretty reproducible. ... stuff deleted...So in >short, my advice is, give up on the pET vectors. I believe NEB now sells >them, and I love NEB (no affiliation, etc). I have never gotten anything >from them that wasn't excellant (I got the pET 3a before NEB liscenced the >system), but I wouldn't encourage anyone to use pET vectors, if another >system is readily available... ---------------------------------------------------------------------------- We have used pET vectors to very successfully express several plant proteins (mg amounts with activities equal to the native protein), so I do not think pET is to blame for problems. Some proteins just do not express well, fold properly, get modified, etc. in E. coli systems. If there are problems with expression, try another form of the pET vector. We had one protein that did not express well in pET3d, but did in another pET vector! We also have to test different host strains to optimize expression. However, sometimes it is necessary to use fusion expression to get some proteins expressed in E. coli or in the worst cases bacculovirus or yeast expression is the only choice. Also, some people use co-expression of groEL and groES chaparone proteins to get good expression/activity. If pET does not work for your protein, try something else. But don't knock a system just because it did not work for your particular protein. Karen S. Browning, Ph.D. Department of Chemistry and Biochemistry University of Texas Austin, TX 78712 512-471-4562 512-471-8696 FAX kbrowning@mail.utexas.edu ----- End Included Message ----- Perhaps my opinion here is a bit colored by the years of frustration while working with the pET system, by I have to stick with what I wrote above. I am not saying anyone should stay away from pET vectors based solely on my exper- ience. I have been collecting complaints from this very net, however, to send to my old boss. I have collecyed about ten complaints so far. I'd be willing to bet a few others will have more to say on the subject in response to Mike Holloway's original query yesterday... -Jeff Jeffrey A. Kramer, Ph.D. Wayne State University Medical Center C.S. Mott Center for Human Growth and Development 275 E. Hancock Detroit, MI 48201 jeff@compbio.med.wayne.edu http://compbio.med.wayne.edu/~jeff/ From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!daresbury!not-for-mail From: RMCMAH93@IRLEARN.UCD.IE Newsgroups: bionet.molbio.methds-reagnts Subject: quantity of cDNA in PCR Date: 2 Aug 1995 18:21:09 +0100 Lines: 6 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3voc65$t23@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk I wish to do PCR on a number of cDNA sources isolated from different libraries. How much cDNA would be required in a PCR to isolate a single copy gene? Thanks, Ruth From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!daresbury!is.bbsrc.ac.uk!bcc.ac.uk!t-chappell.mcbl.ucl.ac.uk!user From: t.chappell@ucl.ac.uk (tom chappell) Subject: Re: Gradients with automated DNA sequencers? Sender: news@ucl.ac.uk (Usenet News System) Message-ID: Date: Wed, 2 Aug 1995 16:54:44 GMT References: <950802103414.21c437@thorin.uthscsa.edu> Organization: MRC laboratory for molecular cell biology X-Newsreader: Yet Another NewsWatcher 2.0b30 Lines: 26 In article <950802103414.21c437@thorin.uthscsa.edu>, HARDIES@THORIN.UTHSCSA.EDU wrote: > Hi Netters: > > Does anyone use (or know of) wedge gels (or other gradient configurations) > used on an ABI sequencer (or other DNA sequencer with a fixed sensor > position)? I'm writing a grant proposal about gradient systems in > DNA sequencing, and am under the impression that they aren't applied > much to automated sequencers. Am I misinformed? > > Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio > Hardies@uthscsa.edu > > For information on our graduate program in Biochemistry: > http://bioc02.uthscsa.edu/biochem.html > Why would you want to use a wedge gel in an ABI sequencer? The only point of a wedge is to slow down the lower MW fragments in the wedge, so they don't run off the bottom of the gel while you're resolving the higher MW fragments. On an ABI sequencer you want the fragments to fall off the bottom of the gel after they pass the sensor. Tom Chappell MRC Laboratory for Molecular Cell Biology From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!spool.mu.edu!torn!news.unb.ca!coranto.ucs.mun.ca!leif!ynakao From: ynakao@kean.ucs.mun.ca Newsgroups: bionet.molbio.methds-reagnts Subject: NMU and tobacco Date: 2 Aug 95 14:28:06 -0230 Organization: Memorial University. St.John's Nfld, Canada Lines: 13 Message-ID: <1995Aug2.142806.1@leif> NNTP-Posting-Host: leif.ucs.mun.ca Dear Netters Would you please tell me the relationship between tobacco smoke and N- nitrosomethylurea (NMU)? Though tobacco smoke contains many kinds of nitrosoamines, NMU is not in the list of them. I know NMU is used anti cancer drug and not amines but ureas. How about the possibility that a nitrosomamine becomes NMU after metabolism? If you know any refernces of the origin of NMU and NMU as a metabolite of nitrosoamine containied tobacco smoke, please send me a Email. (I suppose NMU does not relate with tobacco specific nitrosoamines... .. However, I am not sure.) Thanks in advance Yoshifumi Nakao ynakao@kean.ucs.mun.ca From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!swrinde!hookup!noc.tor.hookup.net!japhale!japhale From: japhale@cangene.com (Jayant S. Aphale) Newsgroups: bionet.molbio.methds-reagnts Subject: Genomic DNA from Campylobacter Date: Wed, 2 Aug 1995 16:44:23 GMT Organization: Cangene Corp. Lines: 19 Distribution: na Message-ID: NNTP-Posting-Host: japhale.tor.hookup.net Summary: Genomic DNA digestion problems Keywords: Qiagen, restriction digestion X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] I am having problems digesting chromosomal DNA isolated from three Campylobacter species. The genomic DNA was isolated using the Qiagen genomic DNA kit Cat# 13343 according to the manufacterers instructions. The O.D. 260:280 ratio is 1.80 plus/minus 0.05 a nd looks intact on an EtBr agarose gel but will not cut with either BamH1 or EcoRI even after extended digestion with excess enzyme. The problem is probably not trace EtOH because I precipitataed the genomic DNA a second time, spooled, washed 70% EtOH and dried it as long as I dared. Still didn't cut. The problem may be DNA modification or something I am missing. According to the NEB catalog neither EcoRI or BamHI are blockered by methylation. Does anyone have experience cutting Campylobacter genomic DNA? Glenn From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.uoregon.edu!vixen.cso.uiuc.edu!uchinews!ellis!grandall From: grandall@ellis.uchicago.edu (Glenn Randall) Subject: need a bidirectional promotor X-Nntp-Posting-Host: midway.uchicago.edu Message-ID: Sender: news@midway.uchicago.edu (News Administrator) Reply-To: grandall@midway.uchicago.edu Cc: I am trying to locate a mammalian expression vector with a Organization: The University of Chicago Date: Wed, 2 Aug 1995 16:30:32 GMT Lines: 1 From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!news.sprintlink.net!news.bluesky.net!solaris.cc.vt.edu!news.duke.edu!soprano.cellbio.duke.edu!user From: ChunLin_Lu@cellbio.duke.edu (ChunLin_Lu) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: pET vector cloning of hydrophobic protein gene Date: 2 Aug 1995 16:29:51 GMT Organization: Duke University Lines: 35 Message-ID: References: <3vltmi$qn5@warp.cris.com> NNTP-Posting-Host: soprano.cellbio.duke.edu Yes, I do have similar experiments like you. I want to express Sul A ( a bacteria division inhibitor) in BL21 using PET11b vector. I amplified sulA gene from DH5a, and cloned into PET vector, transformed into DH5a. I got recombinant vector (hope it not be rearranged), but can not see a transformant from BL21 transformation. I repeated this seveal times, no sucess at all. Many peoples here are using pET system to express prteins, do not have any problems. I think some SulA may be made even without any inducer, because excess SulA is toxic to bacteria, which inhibites cell division. I am thinking about how to solve my problem, could give some suggestions. Thanks. Chunlin In article <3vltmi$qn5@warp.cris.com>, mike.holloway@stjude.org (Mike Holloway) wrote: > I'm giving up on the pET system and wondered if anyone else has had > similar experiences. Even with the thioredoxin fusion protein vector, > I can not isolate a transformant with my sequence and an intact vector. > I do get many rearrangements - things that are not whole pET vector or > PCR template from the preparation of my insert. The transformants are of > various sizes, with various restriction sites missing. Other cloning > projects using the same bugs and reagents are working just fine, without > any evidence of contamination. > > I have to conclude that some protein is being made, despite what anyone > says about the T7 promoter not being used without the polymerase being > pumped out, and that its toxic. This has been the previous experience > in our lab with this protein using other bacterial expression vectors. > Apparently this isn't an isolated experience, since several companies > have recently come out with this thioredoxin fusion protein scheme that's > supposed to solve everything. > > Anyone had experience with pET vectors? > > ========================================================================= >============= From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!news.mid.net!sbctri.tri.sbc.com!newspump.wustl.edu!cerberus-138.wustl.edu!NewsWatcher!user From: anderson@pharmdec.wustl.edu (Eric C. Anderson) Newsgroups: bionet.molbio.methds-reagnts Subject: apology/expalanation re: TIBS post Date: Wed, 02 Aug 1995 11:10:45 -0500 Organization: Dept. of Molecular Biol. & Pharmacology Lines: 40 Message-ID: NNTP-Posting-Host: 128.252.181.14 To all: I am writing to explain and apologize for a statement that I made regarding Dr. Paul Hengen's July TIBS article on His-Tag protein purification. In a reply intended to go to a single individual whom I knew (but mistakenly sent to the entire group), I made the following comment: > i am the person mentioned in the TIBS article (i.e., the one who did all > the work and got none of the credit, but never mind that). This was written in jest and intended for one individual. I never meant to state or imply that Dr. Hengen did not give me credit for the work I did (and that he subsequently wrote about), nor give the impression to the BIOSCI/bionet community that he would "steal" protocols, ideas, procedures, etc. and publish them under his own name. I do not believe that he would do anything of this sort and I apologize for implying that he might. I have personally benefitted from a number of suggestions which Dr. Hengen has offered both in this group and in his column and find them to be beneficial, interesting, well written and insightful. I would like to take this opportunity to encourage people posting to this group to continue to contribute ideas and protocols to both this group and to Dr. Hengen if he deems them of particular interest to the TIBS readership and decides to include them in his column. If asked in the future to contribute a protocol for the column, I would gladly do so with no reservations. Eric Anderson -- "i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in." eric c. anderson anderson@pharmdec.wustl.edu dept. of molecular bio. and pharm. (314)362-3963 (lab) washington univ. school of medicine (314)362-7058 (FAX) 660 s. euclid box 8103 (314)962-2407 (home) st. louis, mo 63110 From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!dns.crocker.com!wizard.pn.com!news.xensei.com!news From: chi@healthtech.com (Cambridge Healthtech Institute) Newsgroups: bionet.molbio.methds-reagnts Subject: Microfabrication Technology meeting announcement Date: Wed, 02 Aug 1995 16:08:25 GMT Organization: The Xensei Corporation Lines: 44 Message-ID: <3vo7q6$omq@xensei3.xensei.com> NNTP-Posting-Host: chi.xensei.com X-Newsreader: Forte Free Agent 1.0.82 Microfabrication Technology for Research and Diagnostics: September 28-29, 1995, Miyako Hotel, San Francisco, CA Many of the assays, screen and analytical test used in biomedical research and diagnostics are suitable for miniaturization. The development and production of devices based on emerging microfabrication technology offers advantages such as unprecedented speed, while minimizing sample requirements and the operations in a massively parallel fasion on chips produced at such a low cost as to be considered disposable. Engineers are borrowing from technology already developed for microelectronics, as well as coming up with new advances in micromachining, materials and other areas needed to meet the challenges presented by these devices. Academics and commercial scientists with microfabrication experience will duscuss the enormous opportunies and critical challenges to be met. If you are involved in the development or use of analytical instruments or diagnostic assays, this timely conference offers you a series of perspectives on an extremely powerful trend that will help shape how these activities will be performed. Take advantage of this unique forum to get a glimpse of that future. Call for Additional Exhibitors and Posters Organizations interested in exhibiting technology or services should contact Jim MacNeil at (617) 630-1341 for more information. Researchers are encouraged to present results of their work in poster form. Poster boards are 8' x 4 ' landscape. A one page summary of the poster should be submitted by September 1 for inclusion in the conference binder. For more information on attending this meeting, contact: Cambridge Healthtech Institute chi@healthtech.com World Wide Web: http://www.healthtech.com/conferences --------------------------------------------------------------------------------------- Cambridge Healthtech Institute 1037 Chestnut Street Newton Upper Falls, MA 02164 tel: 617.630.1300 fax. 617.630.1325 From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!spool.mu.edu!howland.reston.ans.net!swrinde!sdd.hp.com!night.primate.wisc.edu!nntp.msstate.edu!fiona.umsmed.edu!fiona.umsmed.edu!hutchins From: hutchins@fiona.umsmed.edu (Jim Hutchins) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RT-PCR and gels Date: 2 Aug 1995 15:55:27 GMT Organization: University of Mississippi Medical Center Lines: 38 Distribution: world Message-ID: <3vo75f$42r@fiona.umsmed.edu> References: <9507281634.aa00482@etsuodt.etsu.edu> NNTP-Posting-Host: fiona.umsmed.edu X-Newsreader: TIN [version 1.2 PL1] GORDON BETTS (betts@ORION.ETSU.EDU) wrote: : I believe it is possible to run RT then take 3 ul or so and run : on a 1% agarose gel to verify you have a product. Correct? I doubt whether this will work; you would need >1 ug of cDNA to see a product, and if you are using specific primers this is unlikely. : If not, then how can you check to see if your RT worked before : wasting reagents on PCR? Yes. The `standard' method is to `spike' the RT reaction, or a duplicate reaction consisting of 1/10 of the RT reaction, with a small amount of 32P-labeled nucleotide (32Palpha-dCTP, e.g.). Then, from the known molar amounts of each reactant, and making assumptions about the size of your cDNA, you can calculate a percent incorporation after separating the reaction from the cDNA on a spun column (or better, two spun columns). Most people use Cerenkov counts to get a guesstimate of the percent incorporated. In my hands, this number is very, very low. For example, in a kinase reaction, we expect 20-50% incorporation, but less that 1% is not uncommon with an RT reaction. Anyone else have any comments on this? What do other labs get as a percent incorp in a `typical' RT reaction? Oh, I just remembered, some people use TCA-precipitable counts, which is cheaper and easier than the spun column unless you already have them around. Hope this helps. -- Jim Hutchins Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr http://fiona.umsmed.edu/~hutchins/ *** E-mail: hutchins@umsmed.edu ``It became necessary to destroy the town in order to save it.''--American infantry officer firing on Ben Tre, Vietnam, 2/8/68 From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!lyra.csx.cam.ac.uk!macop207-1.mrc-lmb.cam.ac.uk!user From: ik103@hermes.cam.ac.uk () Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Address of HT Biotechnology? Followup-To: bionet.molbio.methds-reagnts Date: 2 Aug 1995 15:30:25 GMT Organization: University of Cambridge, England Lines: 18 Distribution: bionet Message-ID: References: <806753087snz@pzoptic.demon.co.uk> <1995Jul26.151432@ania.path.ox.ac.uk> <3v5jobINNro2@s-crim1.dl.ac.uk> NNTP-Posting-Host: macop207-1.mrc-lmb.cam.ac.uk The address is: HT Biotechnology Ltd, Unit 4, 61 Ditton Walk, Cambridge CB5 8QD Ingrid In article <3v5jobINNro2@s-crim1.dl.ac.uk>, mbbtn@s-crim1.dl.ac.uk (D. Brittain) wrote: > > Does anyone know the address of HT Biotechnology, Cambridge? > > Thanks in advance. From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!THORIN.UTHSCSA.EDU!HARDIES From: HARDIES@THORIN.UTHSCSA.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: Gradients with automated DNA sequencers? Date: 2 Aug 1995 08:32:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <950802103414.21c437@thorin.uthscsa.edu> NNTP-Posting-Host: net.bio.net Hi Netters: Does anyone use (or know of) wedge gels (or other gradient configurations) used on an ABI sequencer (or other DNA sequencer with a fixed sensor position)? I'm writing a grant proposal about gradient systems in DNA sequencing, and am under the impression that they aren't applied much to automated sequencers. Am I misinformed? Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio Hardies@uthscsa.edu For information on our graduate program in Biochemistry: http://bioc02.uthscsa.edu/biochem.html From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!MAIL.UTEXAS.EDU!kbrowning From: kbrowning@MAIL.UTEXAS.EDU (Karen Browning) Newsgroups: bionet.molbio.methds-reagnts Subject: RE: pET vectors, good or bad? Date: 2 Aug 1995 08:05:30 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: <199508021441.JAA17718@mail.utexas.edu> NNTP-Posting-Host: net.bio.net >>Mike Holloway wrote: -------------------------------------------------------------------------------- >>I'm giving up on the pET system and wondered if anyone else has had >>similar experiences. >> ...stuff deleted... >>Anyone had experience with pET vectors? -------------------------------------------------------------------------------- >Jeff Kramer wrote: >I have spentf years of my life trying to get lipoxygenase expression using >pET 3a while a grad student working on my PhD. Even after I got expression, >I >rarely got anywhere near reproducible amounts of protein. The specific >activity was, thankfully, pretty reproducible. ... stuff deleted...So in >short, my advice is, give up on the pET vectors. I believe NEB now sells >them, and I love NEB (no affiliation, etc). I have never gotten anything >from them that wasn't excellant (I got the pET 3a before NEB liscenced the >system), but I wouldn't encourage anyone to use pET vectors, if another >system is readily available... ---------------------------------------------------------------------------- We have used pET vectors to very successfully express several plant proteins (mg amounts with activities equal to the native protein), so I do not think pET is to blame for problems. Some proteins just do not express well, fold properly, get modified, etc. in E. coli systems. If there are problems with expression, try another form of the pET vector. We had one protein that did not express well in pET3d, but did in another pET vector! We also have to test different host strains to optimize expression. However, sometimes it is necessary to use fusion expression to get some proteins expressed in E. coli or in the worst cases bacculovirus or yeast expression is the only choice. Also, some people use co-expression of groEL and groES chaparone proteins to get good expression/activity. If pET does not work for your protein, try something else. But don't knock a system just because it did not work for your particular protein. Karen S. Browning, Ph.D. Department of Chemistry and Biochemistry University of Texas Austin, TX 78712 512-471-4562 512-471-8696 FAX kbrowning@mail.utexas.edu From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!NewsWatcher!user From: MAlexeyev@biost1.thi.tmc.edu (Mikhail Alexeyev) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Endonuclease activity: Crude extraction from E.coli Date: 2 Aug 1995 14:53:53 GMT Organization: Texas Heart Institute Lines: 50 Message-ID: References: NNTP-Posting-Host: 128.249.210.32 Hi, Patrice: In a past I found useful the following technique: 1. Spin down 1.5 ml of stationary-phase E. coli culture in Eppendorf tube 2. Resuspend pellet in 0.5 ml ice-cold 20 mM Tris pH 7.4-7.6, 50 mM NaCL, 5-10 mM 2-mercaptoethanol 3. sonicate on ice (time and intensity depends on culture and model of sonicator, generally 6 x 10 sec with 10 sec. intervals, 50% pulse duration and 50-90 power is OK in most cases) 4. spin down in microcentrifuge for 2 min @ max speed at 4 C (room t works as well) 5. incubate 10 ul of lambda DNA (0.1 ug/ul in appropriate restriction buffer) with 3 ul of resulting lysate for 10-30 min @ 37 C 6. run the gel This procedure worked in my hands with about dosen of naturally occuring producers of restriction enzymes as well as with genes cloned in E. coli. In a latter case contaminating nucleases never presented a problem. However, if this happens, some old remedy suggests to add t-RNA to titer out nonspecific nucleases. Some adjustments might be necessary with above procedure (increase amount of pellet, change buffer pH, salt, etc Hope, this will help. M. Alexeyev In article , woodj@acs.ucalgary.ca (Julian Wood) wrote: > I'm working on a restriction/methylation system in Rhizobium. A construct > where the methylase has been knocked down and the endonuclease is intact, > has been introduced into DH10B cells. The culture is growing slowly and > many cell wall debris are present in the culture. This is in favour of an > active endonuclease. > > I'm looking for a quick method to make a crude extraction containing the > endonuclease (and possibly get rid of non-specific endonuclease activity). > > The extract will be used to test for the activity of the endonuclease on a > target DNA. > > Thanks in advance for any help > > Patrice Rochepeau, PhD e-mail: prochepe@acs2.acs.ucalgary.ca > University of Calgary > 2500 University Dr.N.W. > Calgary, AB, T2N 1N4 > Canada From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!tank.news.pipex.net!pipex!warwick!griffin.nott.ac.uk!ppdgtm.nottingham.ac.uk!user From: pdzah@pdn1.gene.nottingham.ac.uk (Alan Hair) Newsgroups: bionet.molbio.methds-reagnts Subject: pSPT64T Followup-To: bionet.molbio.methds-reagnts Date: 2 Aug 1995 14:37:35 GMT Organization: Nottingham University Lines: 4 Message-ID: NNTP-Posting-Host: ppdgtm.nottingham.ac.uk Does anyone have the sequence for pSPT64T or the location where I can find its sequence? Cheers Al From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!newsfeed.internetmci.com!uunet!in1.uu.net!newsflash.concordia.ca!canopus.cc.umanitoba.ca!kunz2 From: ramacha@ccu.umanitoba.ca (Karthi Rama) Newsgroups: bionet.molbio.methds-reagnts Subject: Supressible URA3 marker Date: 2 Aug 1995 14:28:07 GMT Organization: U of Manitoba Lines: 9 Message-ID: <3vo21n$df9@canopus.cc.umanitoba.ca> NNTP-Posting-Host: kunz2.microbio.umanitoba.ca Keywords: URA3, Ochre, Supression X-Newsreader: News Xpress Version 1.0 Beta #4 Dear Bio-netters, I need a Ochre supressible URA3 marker for the budding yeast. If anyone has such a gene could you please let me know if you can give it to me. Please help. Thank you verymuch Karthi. R From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!news.sprintlink.net!EU.net!Germany.EU.net!nntp.gmd.de!news.rwth-aachen.de!news.rhrz.uni-bonn.de!news.uni-stuttgart.de!news.belwue.de!fu-berlin.de!klipha3.ukbf.fu-berlin.DE!not-for-mail From: Bohnemeier@ukbf.fu-berlin.de (Holger Bohnemeier) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RT-PCR and gels Date: 2 Aug 1995 11:32:28 GMT Organization: Clinical Pharmacology FU-Berlin Lines: 14 Message-ID: <3vnnoc$m78@fu-berlin.de> References: <9507281634.aa00482@etsuodt.etsu.edu> <3vln2j$die@fu-berlin.de> <3vlv47$h9c@hermes.oc.com> Reply-To: bohnemeier@ukbf.fu-berlin.de NNTP-Posting-Host: klipha3.ukbf.fu-berlin.de (160.45.191.31) X-Access: 16 17 19 X-Newsreader: WinVN 0.92.6 In article <3vlv47$h9c@hermes.oc.com>, Gordon Betts says: -shortened- > >OK, but in RT I am using either oligo-dT or random primers. Won't >this result in ALL mRNA being RT'ed and getting lots of cDNA regardless >of whether the transcript I'm looking for is there? >Gordon You could use the primers you suggested as a controll for your RT-reaction in total labeling them. Another possibility would be if you use a specific oligo complementary to the cDNA you are interested in as an RT-primer. Holger From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!tank.news.pipex.net!pipex!warwick!bham!usenet From: altabios@bham.ac.uk (John E. Fox) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Trouble with a Millipore "Expedite" Oligonucleotide synthesiser Date: 2 Aug 1995 13:18:16 GMT Organization: Alta Bioscience Lines: 5 Message-ID: <3vntuo$jf2@sun4.bham.ac.uk> References: NNTP-Posting-Host: bcs118.bham.ac.uk X-Newsreader: WinVN 0.90.6 In article , reynard@accord.caltech.edu (Greg Reynard) says: Contact me for a fix for this. John Fox From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!news.moneng.mei.com!news.ecn.bgu.edu!vixen.cso.uiuc.edu!newsrelay.iastate.edu!gw1.phibred.com!news From: Subject: Re: PCR of lambda gt10 Message-ID: Sender: news@phibred.com (USENET News System) Organization: Pioneer Hi-Bred International, Inc. References: <3vhr2d$5kl@nntp3.u.washington.edu> Date: Wed, 2 Aug 1995 13:00:15 GMT Lines: 25 stardog@u.washington.edu (Kevin Lease) wrote: > > Could someone please send me a good protocol for PCR with a lambda gt10 > clone as my template DNA? I am particulary interested in hearing how > people begin their PCR to denature the capsid. > > Thanks, > Kevin Lease > Kevin, I simply add 1 ul of phage in SM. I denature normally, ie, 3-4 minutes at denaturing temperature. The minimal amount of Mg in SM hasn't mattered to me so far (I use 1.5 mM Mg in a commercial buffer) and the gelatin was used in early buffers anyway. I then follow cycling conditions appropriate for the oligo set in use and the size of the amplimer. My rxn volumes are either 50 or 100 ul. I've used this technique to amplify clones from library stocks and to score primary picks (it cuts down on the number of secondary lifts that you need to do). When I'm stuck using a library that doesn't have an easy plasmid pop-out method, I'll also clone inserts this way, it's easier than a lambda DNA prep! One caveat is avoid the chloroform until after you're done with PCR, or you'll have to wait a few weeks! John McElver mcelverja@phibred.com From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!news.sprintlink.net!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: dprince225@aol.com (DPrince225) Newsgroups: bionet.microbiology,bionet.molbio.methds-reagnts Subject: Re: ATCC book on freeze drying methods Date: 2 Aug 1995 08:36:38 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 6 Sender: root@newsbf02.news.aol.com Message-ID: <3vnrgm$9hf@newsbf02.news.aol.com> References: <813424462wnr@genesys.demon.co.uk> Reply-To: dprince225@aol.com (DPrince225) NNTP-Posting-Host: newsbf02.mail.aol.com Xref: biosci bionet.microbiology:2851 bionet.molbio.methds-reagnts:31652 Duncan- You may be thinking of a teaching workshop offered by the ATCC dealing with cryopreservation. It's possible that at this workhop or some other that they cover the subject of interest. Suggest that you request from ATCC a master list of their workshops. dprince225@aol.com From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!tank.news.pipex.net!pipex!sunsite.doc.ic.ac.uk!daresbury!not-for-mail From: pennings.knoware.nl@knoware.nl (Henk Pennings) Newsgroups: bionet.molbio.methds-reagnts Subject: mtDNA specific PCR primers Date: 2 Aug 1995 10:11:45 +0100 Lines: 10 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3vnfgh$3d4@mserv1.dl.ac.uk> X-Sender: pennings@pop.knoware.nl Original-To: methods@dl.ac.uk In protoplast fusion experiments between onion (Allium cepa) and leek (Allium ampeloprasum) we are looking for PCR based methods to distinguish the mitochondrial DNA from both fusion partners. Can someone help me out with DNA sequences to develop specific PCR primers or with literature references ? Thanks in advance, Henk Pennings. From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!swrinde!tank.news.pipex.net!pipex!newsfeed.internetmci.com!usenet.eel.ufl.edu!news.gmi.edu!msunews!harbinger.cc.monash.edu.au!news.cs.su.oz.au!metro!socs.uts.edu.au!uts.edu.au!usenet From: Peter Hains Newsgroups: bionet.molbio.methds-reagnts Subject: (no subject) Date: 2 Aug 1995 04:20:15 GMT Organization: UTS Lines: 12 Message-ID: <3vmudv$jce@cassia.itd.uts.edu.au> NNTP-Posting-Host: 138.25.241.85 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.methds-reagnts I am looking for a supplier of a fluorescent phospholipid for use in an assay system. The specific phospholipid I need is 10-pyrene PA monomethylester (abbrev). Full description: 1-palmitoyl(2-pyrenyldecanoyl)-sn-glycero-3-monomethyl phosphatidic acid. I have tried Sigma but they do not have it, nor do Molecular Probes. I would prefer a supplier in Australia but overseas is okay. Thanks in advance, Peter. From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!newsfeed.internetmci.com!usenet.eel.ufl.edu!news.gmi.edu!msunews!harbinger.cc.monash.edu.au!news.cs.su.oz.au!metro!socs.uts.edu.au!uts.edu.au!usenet From: Peter Hains Newsgroups: bionet.molbio.methds-reagnts Subject: Fluorescent phospholipid Date: 2 Aug 1995 04:31:32 GMT Organization: UTS Lines: 11 Message-ID: <3vmv34$jce@cassia.itd.uts.edu.au> NNTP-Posting-Host: 138.25.241.85 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.methds-reagnts I am looking for the supplier of a fluorescent phopholipid for use in an assay system. The phospholipid I require is 10-pyrene PA monomethylester (abbrev). Full description: 1-palmitoyl-2-(10-pyrenyldecanoyl)-sn-glycero-3-monomethyl phophatidic acid. I have tried Sigma and Molecular Probes but neither sell the compound. I would prefer an Australian supplier if possible. Thanks in Advance, Peter. From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!dispatch.news.demon.net!demon!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!News.Uni-Marburg.DE!news.th-darmstadt.de!news.uni-mainz.de!usenet From: kochj000@mzdmza.zdv.uni-mainz.de (Jan Oliver Koch) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: infor. on PCR machine Date: Thu, 03 Aug 1995 04:27:49 GMT Organization: Institute for Virology, University of Mainz, Germany Lines: 12 Message-ID: <3vojcm$vfh@kralle.zdv.Uni-Mainz.DE> References: NNTP-Posting-Host: dialin07.zdv.uni-mainz.de X-Newsreader: Forte Free Agent v0.55 hfang@darwin.mbb.sfu.ca (Hung Fang) wrote: >Hello there: >Our lab. is interested in buying a PCR machine. We'd like to buy one like Biometra's Trio Thermoblock which one can run three different program at the same time. The problem is Biometra's machine are quite expensive, we are wondering whether any other companies making tri-block or twin-block PCR machine. Any information or personal experience on this will be very helpful for us and will be very much appreciated. We bought a Biometra Trio Thermoblock about a year ago. It is running all day (and night) and we never had a problem. It is said that Pelletier-cooling elements do not live very long, but so far everything is ok. Oliver From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!gatech!news.uoregon.edu!newsfeed.internetmci.com!tank.news.pipex.net!pipex!dish.news.pipex.net!pipex!sunic!sunic.sunet.se!umdac!news From: Eric Hanson Newsgroups: bionet.molbio.methds-reagnts Subject: DIG Gelshifts??? Date: 2 Aug 1995 15:19:39 GMT Organization: University of Umea, Sweden Lines: 9 Message-ID: <3vo52b$fsg@studium.student.umu.se> NNTP-Posting-Host: lth.chem.umu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) Is anyone out there familiar with non-radioactive gelshifts? More specifically I am looking for any comments on the new DIG gelshift system offered by Boehringer. Any assistance/comments would be most appreciated. Please respond to my E-mail. Thanks, Eric Hanson From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!warp.cris.com!usenet From: mike.holloway@stjude.org (Mike Holloway) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: FLAG epitope - sources and advice Date: 3 Aug 1995 02:16:27 GMT Organization: none Lines: 27 Message-ID: <3vpbhr$ut@warp.cris.com> References: <3vooil$e67@vixen.cso.uiuc.edu> NNTP-Posting-Host: crc4-fddi.cris.com X-Newsreader: WinVN 0.92.2 In article <3vooil$e67@vixen.cso.uiuc.edu>, David Clayton says: >1) is there a commercial source for FLAG-epitope related reagents? IBI has a kit with pFLAG vectors. >2) Anybody want to offer opinions as to the utility of FLAG, vs. other >candidates such as His, myc, HA, etc? Flag works well in my hands for both Westerns and immunofluorescence. ========================================================================= Mike Holloway |* On average, 8 people a day on the waiting list mike.holloway@stjude.org | for a transplant die for lack of a donor. ____________________________|* The end-stage diseases treated by transplant do not recognize age, race, nationality or pocketbook. * There is no justifiable reason to deny organ donation, but many myths and misunderstandings. * Next of kin must allow donation. Your family must know your wishes. * Only a small fraction of the next of kin of potential donors allow donation to take place. * In general, medical professionals do not recognize their obligation to support and promote donation. Questions? See FAQ: http://www.lib.ox.ac.uk/internet/news/faq/bit.listserv.transplant.html or ftp://rtfm.mit.edu/pub/usenet/bit.listserv.transplant/ ========================================================================= From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.sprintlink.net!warp.cris.com!usenet From: mike.holloway@stjude.org (Mike Holloway) Newsgroups: bionet.molbio.methds-reagnts Subject: RE: pET vectors, good or bad? Date: 3 Aug 1995 02:12:41 GMT Organization: none Lines: 36 Message-ID: <3vpbap$ut@warp.cris.com> References: <199508021441.JAA17718@mail.utexas.edu> NNTP-Posting-Host: crc4-fddi.cris.com X-Newsreader: WinVN 0.92.2 In article <199508021441.JAA17718@mail.utexas.edu>, kbrowning@MAIL.UTEXAS.EDU (Karen Browning) says: >Also, some people use co-expression of groEL and groES chaparone >proteins to get good expression/activity. Thanks. That's interesting, though in my case it probably wouldn't make much difference since I can't even get the plasmid to replicate in JM109. >If pET does not work for your protein, try something else. But don't knock >a system just because it did not work for your particular protein. Won't hear a condemnation from me. It was my first choice, after all. When it works, it seems to work beautifully. But Jeff Kramer's advice fits nicely with my own thoughts. Time to try Pichia yeast. Nice to know I'm not the only one. If only I could figure out what the bacteria's problem is. It might be useful information about my protein's function. ========================================================================= Mike Holloway |* On average, 8 people a day on the waiting list mike.holloway@stjude.org | for a transplant die for lack of a donor. ____________________________|* The end-stage diseases treated by transplant do not recognize age, race, nationality or pocketbook. * There is no justifiable reason to deny organ donation, but many myths and misunderstandings. * Next of kin must allow donation. Your family must know your wishes. * Only a small fraction of the next of kin of potential donors allow donation to take place. * In general, medical professionals do not recognize their obligation to support and promote donation. Questions? See FAQ: http://www.lib.ox.ac.uk/internet/news/faq/bit.listserv.transplant.html or ftp://rtfm.mit.edu/pub/usenet/bit.listserv.transplant/ ========================================================================= From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!uunet!in1.uu.net!gwu.edu!sullivan From: sullivan@gwis2.circ.gwu.edu (Steven Sullivan) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR of lambda gt10 Date: 3 Aug 1995 00:14:28 GMT Organization: The George Washington University, Washington DC Lines: 32 Message-ID: <3vp4d4$775@cronkite.seas.gwu.edu> References: <3vhr2d$5kl@nntp3.u.washington.edu> NNTP-Posting-Host: 128.164.127.252 X-Newsreader: TIN [version 1.2 PL2] mcelverja@phibred.com wrote: : stardog@u.washington.edu (Kevin Lease) wrote: : > : > Could someone please send me a good protocol for PCR with a lambda gt10 : > clone as my template DNA? I am particulary interested in hearing how : > people begin their PCR to denature the capsid. : > : > Thanks, : > Kevin Lease : > : Kevin, : I simply add 1 ul of phage in SM. I denature normally, ie, 3-4 : minutes at denaturing temperature. The minimal amount of Mg in SM : hasn't mattered to me so far (I use 1.5 mM Mg in a commercial buffer) : and the gelatin was used in early buffers anyway. I then follow : cycling conditions appropriate for the oligo set in use and the : size of the amplimer. My rxn volumes are either 50 or 100 ul. : I've used this technique to amplify clones from library stocks and : to score primary picks (it cuts down on the number of secondary : lifts that you need to do). When I'm stuck using a library that : doesn't have an easy plasmid pop-out method, I'll also clone inserts : this way, it's easier than a lambda DNA prep! : One caveat is avoid the chloroform until after you're done with PCR, : or you'll have to wait a few weeks! : John McElver : mcelverja@phibred.com How do you know you're not missing some large inserts, due to the size limits of normal PCR? From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!uunet!in1.uu.net!gwu.edu!sullivan From: sullivan@gwis2.circ.gwu.edu (Steven Sullivan) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Library screening alternatives? Date: 3 Aug 1995 00:11:12 GMT Organization: The George Washington University, Washington DC Lines: 29 Message-ID: <3vp470$775@cronkite.seas.gwu.edu> References: <3vomhu$65j@cronkite.seas.gwu.edu> <3vp325$6tp@cwis-20.wayne.edu> NNTP-Posting-Host: 128.164.127.252 X-Newsreader: TIN [version 1.2 PL2] Anton Scott Goustin (asg@cmb.biosci.wayne.edu) wrote: : sullivan@gwis2.circ.gwu.edu (Steven Sullivan) wrote: : > : > : >It's been some years since I screened a lambda gt11/cDNA library, using : >the tried-and-tru (but labor intensive and slow) plaque hybridization : >method -- I'm wondering if in the interim any cool new (e.g. PCR based?) : >methods have arisen to make library screening quicker, easier, etc. : > : I don't find screening to be a drag. It's rather fast if you have a good hot probe, 2 days from plating to picking the plaques. Th= : e problem for me is lambda purification. Well, I meant the *whole* process, including the dreaded plaque purification step. That takes time, especially if you like crowded plates (50,000 plaques per 150 mm plate), = : which requires secondary and tertiary purifications, and then--DREAD!--growing up a good-sized high-titer phage stock for DNA purifi= : cation. Remember, to get 1 µg of 2 kb insert you need 20 µg of pure recombinant DNA. I find it very easy and convenient to pop out= : the inserts using PCR. New England Biolabs sells forward and reverse primers for lambda gt11 flanking the Eco RI site (24-mers). = : They work easily, especially if the insert is not cut by Eco RI, allowing a very quick shuttle from phage->PCR product->EcoRI-cut PC= : R product->plasmid. do you worry at all about fidelity of the PCR product? From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!newsfeed.internetmci.com!usenet.eel.ufl.edu!news.gmi.edu!isclient.merit.edu!cwis-20.wayne.edu!usenet From: Anton Scott Goustin Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Library screening alternatives? Date: 2 Aug 1995 23:51:33 GMT Organization: Wayne State University Lines: 16 Message-ID: <3vp325$6tp@cwis-20.wayne.edu> References: <3vomhu$65j@cronkite.seas.gwu.edu> NNTP-Posting-Host: dyn51-49.biosci.wayne.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) X-URL: news:3vomhu$65j@cronkite.seas.gwu.edu sullivan@gwis2.circ.gwu.edu (Steven Sullivan) wrote: > > >It's been some years since I screened a lambda gt11/cDNA library, using >the tried-and-tru (but labor intensive and slow) plaque hybridization >method -- I'm wondering if in the interim any cool new (e.g. PCR based?) >methods have arisen to make library screening quicker, easier, etc. > I don't find screening to be a drag. It's rather fast if you have a good hot probe, 2 days from plating to picking the plaques. Th= e problem for me is lambda purification. That takes time, especially if you like crowded plates (50,000 plaques per 150 mm plate), = which requires secondary and tertiary purifications, and then--DREAD!--growing up a good-sized high-titer phage stock for DNA purifi= cation. Remember, to get 1 µg of 2 kb insert you need 20 µg of pure recombinant DNA. I find it very easy and convenient to pop out= the inserts using PCR. New England Biolabs sells forward and reverse primers for lambda gt11 flanking the Eco RI site (24-mers). = They work easily, especially if the insert is not cut by Eco RI, allowing a very quick shuttle from phage->PCR product->EcoRI-cut PC= R product->plasmid. From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!newsfeed.internetmci.com!news.sprintlink.net!uunet!in2.uu.net!news.connectnet.com!pm-1-1.connectnet.com!imnr From: imnr@connectnet.com (Immune Response) Newsgroups: bionet.molbio.methds-reagnts Subject: DNA methylation - effect on transfectoma expression Date: Wed, 2 Aug 1995 16:40:26 Organization: The Immune Response Corporation Lines: 17 Message-ID: NNTP-Posting-Host: pm-1-1.connectnet.com Keywords: DNA, methylation, transfectoma, expression X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Hello! I am wondering what is known about the effect of the amount of methylation in a transfected plasmid on the resultant expression of its genes in mammalian cells. Also, does anyone know about the amount of methylation in plasmid preps from bacteria harvested in log phase versus stationary (saturated) phase versus stationary (amplified: eg chloramphenicol)? Thanks! Jose E. N. Gonzales The Immune Response Corporation Note: if you wish to reply to me directly, please E-mail me at: JOSEGONZ@IMNR.COM From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!darwin.sura.net!fconvx.ncifcrf.gov!fcsparc6!pnh From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) Subject: Re: amp resistance gene Message-ID: Summary: Source of many cassettes for plasmid creation Sender: Paul N. Hengen Nntp-Posting-Host: fcsparc6.ncifcrf.gov Organization: Frederick Cancer Research and Development Center References: <3vjjph$38q@newsbf02.news.aol.com> Distribution: bionet Date: Wed, 2 Aug 1995 23:33:40 GMT Lines: 66 In article <3vjjph$38q@newsbf02.news.aol.com> rdaines@aol.com (R Daines) writes: | I am looking for a clonable DNA fragment containing the amp structural | gene (yes the same one found in many E. coli vectors) hopefully with some | common restriction sites at the ends. If any one has such a item, I would | greatly appreciate hearing from you. | | Thanks in advance | | Richard Daines | Pfizer, Inc | rdaines@aol.com You're in luck! In the latest issue of Gene there is a paper on a number of DNA cassettes and omega fragments that can be used for constructing your own plasmids.... @article{Alexeyev1995c, author = "M. F. Alexeyev and I. N. Shokolenko and T. P. Croughan", title = "Improved antibiotic-resistance gene cassettes and omega elements for {{\em Escherichia coli}} vector construction and {{\em in vitro}} deletion/insertion mutagenesis", journal = "Gene", volume = "160", pages = "63-67", year = "1995"} The ampicillin resistance gene in the omega fragment is actually from pBR322, but for the construction of it, the gene was cleaved out of another cassette described in the paper below.... @article{Elhai.Wolk1988, author = "J. Elhai and C. P. Wolk", title = "A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers", journal = "Gene", volume = "68", pages = "119-138", comment = "amp resistance cassette", year = "1988"} The authors are making a set of plasmids available to those who want them. To request, contact: Dr. T. P. Croughan Rice Research Station P.O. Box 1429 Crowley, LA 70527-1429 USA e-mail: agexp86@lsuvm.sncc.lsu.edu ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - - - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/ - - - - - - * * - - - Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - * ******************************************************************************* From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!agate!howland.reston.ans.net!cs.utexas.edu!uunet!in1.uu.net!news.maz.net!news.omnilink.net!news.gni.net!thorin!rz.uni-karlsruhe.de!news.uni-stuttgart.de!news.rhrz.uni-bonn.de!news.rwth-aachen.de!news.mu-luebeck.de!molbio From: sievert@molbio.mu-luebeck.de Newsgroups: bionet.molbio.methds-reagnts Subject: Re: test, please ignore Date: Thu, 03 Aug 1995 00:08:34 GMT Organization: Inst. f. Med. Informatik, Med. Univ. Luebeck Lines: 2 Message-ID: <3votal$ok8@gwsun.medinf.mu-luebeck.de> References: Reply-To: sievert@molbio.mu-luebeck.de NNTP-Posting-Host: biopc2.biologie.mu-luebeck.de this is the old one, far more beautyful, but posts only *sometimes* From owner-methds-reagnts@net.bio.net Tue Aug 01 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!news.ksu.ksu.edu!pstpm.pp.ksu.edu!user From: PST@KSU.KSU.Edu (Paul St. Amand) Newsgroups: bionet.molbio.methds-reagnts Subject: /\\/\\ Oligo melting point calculation question /\\/\\ Date: Wed, 02 Aug 1995 18:30:55 -0500 Organization: USDA/ARS and KSU Agronomy Dept. Lines: 17 Message-ID: NNTP-Posting-Host: pstpm.pp.ksu.edu X-Newsreader: Value-Added NewsWatcher 2.0b27+ In Maniatis the formula for calculating the Tm of an oligo is: Tm = 81.5 + 16.6 (log10 [Na+]) + 0.41 (%G+C) - (600/length) Why does this formula only consider Na+ ions? Wouldn't other monovalent cations also have the same or similar effects? Should the concentration of ALL monovalent cations (or multi-valent cations for that matter) be included? For example, our PCRs include both Na and K salts. I guess that the original work on this only examined Na ions (Bolton and McCarthy 1962. PNAS 48:1390). Paul C. St. Amand (PST@KSU.KSU.Edu) USDA Research Geneticist // \\ // \\ // \\ // \\ // 2004 Throckmorton Hall, KSU // | :,\\': | \\ // | :,\\': | \\ // | Manhattan KS 66506-5501\\ // | | // \\ | |\\ // | | // \\ | | \\ // | | (913) 532-6168 \\ | :,\\': | // \\ |:,\\': | // \\ | :,\\': | // Fax (913) 532-5692 \\ // \\ // \\// \\ // \\ // \\ // From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!EU.net!i2unix!galactica.galactica.it!usenet From: Giorgio Spagnol Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Consultant company stalks this board Date: 3 Aug 1995 10:23:25 GMT Organization: University of Milan Lines: 17 Distribution: world Message-ID: <3vq82t$mvg@galactica.galactica.it> References: NNTP-Posting-Host: 151.99.164.2 X-UserAgent: Version 1.1.3 X-XXDate: Thu, 3 Aug 95 12:39:15 GMT In article Paul N Hengen, pnh@fcsparc6.ncifcrf.gov writes: > I think this is a common ploy. When we have commercial shows at NIH, the > salespeople (?) line the halls and trap you into answering by asking if you run > gels. Hmmmm...novel thought, eh? One time a salesperson stood between me and > the door and asked "Do you do lab work?" I replied, "No, I'm here to buy a new > car." Her jaw dropped and I left through the door while she tried to think of > something to say next. Why beeing so contemptous against salespeople? I don't see anything bad in doing oneself's job. Giorgio. > From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!EU.net!i2unix!galactica.galactica.it!usenet From: Giorgio Spagnol Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Consultant company stalks this board Date: 3 Aug 1995 10:22:12 GMT Organization: University of Milan Lines: 17 Distribution: world Message-ID: <3vq80k$mk5@galactica.galactica.it> References: NNTP-Posting-Host: 151.99.164.2 X-UserAgent: Version 1.1.3 X-XXDate: Thu, 3 Aug 95 12:38:02 GMT In article Paul N Hengen, pnh@fcsparc6.ncifcrf.gov writes: > I think this is a common ploy. When we have commercial shows at NIH, the > salespeople (?) line the halls and trap you into answering by asking if you run > gels. Hmmmm...novel thought, eh? One time a salesperson stood between me and > the door and asked "Do you do lab work?" I replied, "No, I'm here to buy a new > car." Her jaw dropped and I left through the door while she tried to think of > something to say next. Why be so contemptous against salespeople? After all we all depend on them. I do not think there is anything bad in doing oneself's job. Giorgio. > From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!EU.net!i2unix!galactica.galactica.it!usenet From: Giorgio Spagnol Newsgroups: bionet.molbio.methds-reagnts Subject: Re: HELP: inconsistent genomic PCR troubleshooting Date: 3 Aug 1995 10:18:03 GMT Organization: University of Milan Lines: 36 Distribution: world Message-ID: <3vq7or$mk5@galactica.galactica.it> References: <1995Jul28.184151.1540@newsserver.rrzn.uni-hannover.de> NNTP-Posting-Host: 151.99.164.2 X-UserAgent: Version 1.1.3 X-XXDate: Thu, 3 Aug 95 12:33:52 GMT In article Ted M., tedm@darkwing.uoregon.edu writes: In article Ted M., tedm@darkwing.uoregon.edu writes: > > I'm trying to amplify 2 parts of a single copy gene from human DNA > prepared from buffy > > coat with Qiagens QIAamp Blood Kit. I'm using 2 pairs of primers already > used in published > > literature (published annealing temp. 65!/68!) designed to yield > fragments of ~190 and > > ~250 bp. 100!l reaction volumes contain 200 !M dNTPs each, 1.5 mM MgCL2, > primers 50 > > pmoles each, 1 !g DNA and 2 u cloned Taq (USB) in buffer supplied. > Cycles consist of 1 min > > denaturation 95!, 1 min anealing 58! and 45s extension 72! with 5 min > initial denaturation > > and final extension in a Crocodile II cycler from Appligene. > > MY PROBLEM ARE INCONSISTENT AMPLIFICATION RESULTS: EITHER I GET NOTHING > (LOOKS LIKE A > > PERFECT NEGATIVE CONTROL) OR I GET SINGLE BANDS BUT I CAN'T REPRODUCE THEM THE > > OTHER DAY. SOME DNAS YIELD SINGLE BANDS WITH ONE PRIMER PAIR AND NOTHING > WITH THE > > OTHER AND VICE VERSA. I'VE ALSO FAILED TO REAMPLIFY A SINGLE BAND I ONCE GOT! > > Any hints, suggestions or comments wellcome !!! If you know the sequence (or part of it) of the gene you are trying to amplify, try using a nested, internal primer. Since I amplify with a nested primer I solved problems like the ones you have. best wishes. Giorgio. From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!EU.net!i2unix!galactica.galactica.it!usenet From: Giorgio Spagnol Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PCR of lambda gt10 Date: 3 Aug 1995 10:33:06 GMT Organization: University of Milan Lines: 15 Distribution: world Message-ID: <3vq8l2$mvg@galactica.galactica.it> References: <3vhr2d$5kl@nntp3.u.washington.edu> NNTP-Posting-Host: 151.99.164.2 X-UserAgent: Version 1.1.3 X-XXDate: Thu, 3 Aug 95 12:48:55 GMT In article <3vhr2d$5kl@nntp3.u.washington.edu> Kevin Lease, stardog@u.washington.edu writes: > Could someone please send me a good protocol for PCR with a lambda gt10 > clone as my template DNA? I am particulary interested in hearing how > people begin their PCR to denature the capsid. I just destroy it by heating at 95!C for 5'. It always worked. I'm amplifying from a lambda library since two months and results are good. I would not recommend a special protocol, but I solved every problem I had with PCR by using the PCR Optimizer kit from INVITROGEN and by always using a nested primer. If you experience any problem feel free to E-Mail me. No affiliation whatsoever. Best wishes. Giorgio. From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!dispatch.news.demon.net!demon!uknet!inmos-root!ftel.co.uk!bham!warwick!griffin.nott.ac.uk!usenet From: Nigel Kenward Newsgroups: bionet.molbio.methds-reagnts Subject: Re: RFLP of birds Date: 3 Aug 1995 15:37:27 GMT Organization: Dept. Biochemistry, University of Nottingham, U.K. Lines: 17 Message-ID: <3vqqfn$9qq@griffin.ccc.nottingham.ac.uk> References: <3vokaf$2r3@hermes.oc.com> NNTP-Posting-Host: macjm2.biochem.nottingham.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: betts@orion.etsu.edu X-URL: news:3vokaf$2r3@hermes.oc.com Hi there, I don't know the name of the guy, but there's a guy who'd be ideal to help you in the genetics dept (address below). I guess a letter to the departmental secretary would get passed on. All the best, Nigel Kenward (in Biochemistry at the same address below) Dept. of Genetics Queens Medical Centre Nottingham U.K. From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!uknet!inmos-root!ftel.co.uk!bham!warwick!griffin.nott.ac.uk!usenet From: Nigel Kenward Newsgroups: bionet.molbio.methds-reagnts Subject: Re: northerns Date: 3 Aug 1995 15:30:43 GMT Organization: Dept. Biochemistry, University of Nottingham, U.K. Lines: 23 Message-ID: <3vqq33$9qq@griffin.ccc.nottingham.ac.uk> References: <-3107951204100001@mac67114.salk.edu> <3vkga0$415@ari.net> NNTP-Posting-Host: macjm2.biochem.nottingham.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:3vkga0$415@ari.net Soon Paik wrote: >Aardraa Potnis wrote, >>How do you get rid of background bands of 18s, 28s RNA on the >northerns?? >>I have used all kinds of stringent conditions and still have them ! >>Please help! > >How about treating the blot with RNase during the wash step? > >Soon Paik >Lombardi Cancer Center > DON'T ! Unless you're sure your probe is a 100% match with your required RNA sequence ! This is an idea used in RNAse protection assays and = works well only under the conditions required for these assays. If you're not set up to do RNAse protection assays, keep RNAse well = out of the way of your Northerns ! Nigel Kenward > From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!news.sprintlink.net!dispatch.news.demon.net!demon!uknet!daresbury!not-for-mail From: MIcha Ron Newsgroups: bionet.molbio.methds-reagnts Subject: lambda gtWES Date: 3 Aug 1995 16:48:45 +0100 Lines: 14 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3vqr4t$oo@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk Who can give some advice on lambda gtWES? I need a priming site for PCR from the left and right arms outside the ecoRI site of gtWES. Searching for the sequence in the standard places(genbank or BRL, whom markets the vector, came up with nothing). Its a very old vector and not many researchers around today have experience with it. The restriction maps for gtWES and wildtype lambda are exactly the same according to Maniatis (1982). Can I assume homology for the sequences flanking the ecoRI site and base my primers on the sequence of wildtype lambda? Mark Band ARO The Volcani Center Israel From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!not-for-mail From: "lidaniel" Newsgroups: bionet.molbio.methds-reagnts Subject: Summary of strataclean prep protocol Date: 3 Aug 1995 17:22:56 +0100 Lines: 11 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3vqt50$2jk@mserv1.dl.ac.uk> Original-To: methods@dl.ac.uk The core silica gel particles (Fractosil 500, 230-400 mesh) were washed sequentially with dilute nitric acid, distilled water, and high purity methanol and then dried in a vaccuum oven. These cleansed particles were heated with aminophenyltrimethoxysilane in a mixture of isopropyl alcohol, water, and acetic acid. This polymerizes a thick layer of aminophenyl silane on the surface of the particles. These particles are treated with sodium nitrite in sulfuric acid at 0-5 degrees to make the diazonium salt and this is heated in sulfuric acid to decompose to the phenol. Now you have phenol bonded particles.- Anal. biochem. 181,66-74 (1989) Let me know if you try it. Dan Schindler LIDANIEL@WEIZMANN.WEIZMANN.AC.IL From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!DOLPHIN.UPENN.EDU!stein From: stein@DOLPHIN.UPENN.EDU Newsgroups: bionet.molbio.methds-reagnts Subject: postdoc position-wanted Date: 3 Aug 1995 17:33:43 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <199508040031.UAA25578@dolphin.upenn.edu> NNTP-Posting-Host: net.bio.net *** Please, don't response by command "reply" *** SUBJECT: Seeking for postdoctoral position in US FIELD: Molecular Biology & Genetics of Oncogenes or Antioncogenes SKILLS: Cloning, sequencing, mutagenesis, PCR (RT-PCR), Southern & Northern blotting, immunoprecipitation, CAT, tissue culture, tumor induction, PC, publications registered in CC, etc. WHEN: available immediately, further information upon request ADDRESS: stein@dolphin.upenn.edu From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!solaris.cc.vt.edu!news.duke.edu!mmorales From: mmorales@acpub.duke.edu (Michael Morales) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Summary of strataclean prep protocol Date: 4 Aug 1995 00:21:12 GMT Organization: Duke University, Durham, NC, USA Lines: 18 Distribution: bionet Message-ID: <3vrp5o$o70@news.duke.edu> References: <3vqt50$2jk@mserv1.dl.ac.uk> NNTP-Posting-Host: godzilla.acpub.duke.edu X-Newsreader: TIN [version 1.2 PL2] lidaniel (lidaniel@wiccmail.weizmann.ac.il) wrote: : The core silica gel particles (Fractosil 500, 230-400 mesh) were washed : sequentially with dilute nitric acid, distilled water, and high purity : methanol and then dried in a vaccuum oven. These cleansed particles were : heated with aminophenyltrimethoxysilane in a mixture of isopropyl alcohol, : water, and acetic acid. This polymerizes a thick layer of aminophenyl silane : on the surface of the particles. These particles are treated with sodium : nitrite in sulfuric acid at 0-5 degrees to make the diazonium salt and this : is heated in sulfuric acid to decompose to the phenol. Now you have phenol : bonded particles.- Anal. biochem. 181,66-74 (1989) Let me know if you try it. : Dan Schindler LIDANIEL@WEIZMANN.WEIZMANN.AC.IL How about just using phenyl-sepharose from pharmacia? One this scale I bet it would be cheap. Mike Morales From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: celinn@aol.com (Celinn) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: UV Sterilization Date: 3 Aug 1995 20:17:09 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 46 Sender: root@newsbf02.news.aol.com Message-ID: <3vrou5$f3u@newsbf02.news.aol.com> References: <2077C117C@axe.acadiau.ca> NNTP-Posting-Host: newsbf02.mail.aol.com X-Newsreader: AOL Offline Reader In article <2077C117C@axe.acadiau.ca>, 002233W@AXE.ACADIAU.CA ("ANTHONY WILSON") writes: > I am interested in knowing possible protocols for UV >sterilization. Our lab has a UV cross-linker which we wish to use >for sterilization of PCR tubes. Any information regarding the >intensity of UV light and the optimal exposure time for UV >sterilization would be appreciated. > > Stratagene will fax you a protocol for using their crosslinker if you call them. I've routinely used this method to "decontaminate" pcr tubes and equipment. Notice that I said "decontaminate" not "sterilize". Sterilization (killing of organisms) is best done by gas, autoclave or 80 C heat for a period of time. Decontamination is the process of destroying or inactivating polynucleotides that might be present even after sterilization. I've used the following process to assure PCR decontamination: Cleaning (reusable supplies and equipment, glass): 1) Remove any visible contaminant. 2) Wash in a _mild_ solution of glassware detergent (don't overdo the detergent) 3) Rinse well with tap water. 4) Rinse well with deionized water. 5) Rinse with 70% EtOH. 6) Soak in a 10% Bleach solution for 10 min. 7) Rinse well with deionized or distilled water. Sterilize: 1) Autoclave or 2) Place in an 80 C oven for 3 hours to overnight. Decontaminate: 1) 10 minutes at highest setting in the Stratagene. Please note that most plastics break down if repeatedly exposed to UV and will eventually interfere with PCR, electroporation, etc. I'd only do this 2-3 times if I'm reusing. Most of the time I go with disposables anyway. Charles CELinn@aol.com From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!rutgers!gatech!newsfeed.internetmci.com!news.sprintlink.net!howland.reston.ans.net!torn!news.bc.net!rover.ucs.ualberta.ca!mac04.biochem.ualberta.ca!user From: colin_rasmussen@darwin.biochem.ualberta.ca (Dr. Colin Rasmussen) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Help!!!! how to clone a PCR fragment? Date: Thu, 03 Aug 1995 17:54:14 -0700 Organization: University of Alberta Lines: 17 Distribution: world Message-ID: References: NNTP-Posting-Host: mac04.biochem.ualberta.ca In article , hmmoss@MAIL.MED.CORNELL.EDU (Heidi Moss) wrote: > I have a cloning project that involves inserting a PCR fragment with one > cohesive (Bam HI) and one blunt end into pBS. We usually do the following. After PCR we EtOH precipitate the DNA. We then resuspend in Klenow reaction buffer along with dNTPs, 1 mM ATP and add Klenow and T4 PNK. We then run out on an LMP agarose gel, excise the band and clone into pGEM cut with SmaI and CIPd. So far it has never failed. One other you could try to obviate the need for the Klenow is to use Pfu which leaves a blunt-end. Colin From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!news.sprintlink.net!rockyd!cmcl2!mcrcr6!huange01 From: huange01@mcrcr6.med.nyu.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Ecoli Strain for protein expression Date: 3 Aug 95 12:49:15 EDT Organization: NYU Medical Center, New York, NY 10016, USA Lines: 6 Message-ID: <1995Aug3.124915@mcrcr6> NNTP-Posting-Host: mcrcr6.med.nyu.edu I am working on Prokaryotic Expression, but i am having problems of breakingdown. The gene is in pGEX and cell line is Ecoli XO-1 blue. Is there a line of Ecoli that is lacking of proteases?? Thanks From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!CS.Arizona.EDU!news.Arizona.EDU!hamblin.math.byu.edu!park.uvsc.edu!news.caldera.com!news.cc.utah.edu!5c210mac4.genetics.utah.edu!hmcbride From: Helen McBride Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Non-radioactive Southerns and Northerns Date: 3 Aug 1995 22:54:44 GMT Organization: Univ of Utah Lines: 15 Distribution: world Message-ID: <3vrk3k$pmu@news.cc.utah.edu> NNTP-Posting-Host: 5c210mac4.genetics.utah.edu X-UserAgent: X-XXMessage-ID: X-XXDate: Thu, 3 Aug 95 16:54:41 GMT We have used the Boehrenger Genius sytem for non-radioactive Southerns with great success. The only real difference is that you need to use their membrane (positively charged) for it to work best. All the steps are the same except for different prehyb/hyb solutions. The detection is just like any other ECL system and exposure times are usually short (recommended time of 1 hr is usually just fine). I tried to use this system for Northerns and had a major problem-NO SIGNAL. I followed all their directions and used the recommended prehyb/hyb which is different from that used with Southerns and it still didn't work. I probed the same blot with a radioactive probe made from the same batch of cut DNA with no problem, so I don't know what went wrong. Others I have spoken with at our institution have also had no success using it for Northerns. I hope this helps. hmcbride@hlthsci.med.utah.edu From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!uknet!strath-cs!st-and!Aberdeen!gen126 From: gen126@abdn.ac.uk (f.r.byrne) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA purification from agarose gels Date: 3 Aug 1995 13:53:57 GMT Organization: University of Aberdeen, Scotland Lines: 30 Message-ID: <3vqkdl$600@fs2.abdn.ac.uk> References: <3sp9rk$1tp@lyra.csx.cam.ac.uk> <3u3viq$9gl@threed.uchc.edu> NNTP-Posting-Host: sysb.abdn.ac.uk X-Newsreader: TIN [version 1.2 PL2] Tim (tkuwada@neuron.uchc.edu) wrote: : In article : bio.tamu.edu writes: : >I am interested in what other people think about Qiagens new Qiaquick : >columns compared to Promega's wizard PCR preps. Has anybody done a direct : >comparison of these two kits for purification of DNA from gels? : We have not used the Qiagen kit but have had poor yields with the : Promega kit. : When we have attempted to purify plasmids (>3kb) with the Promega kit : we have consistently : recovered a 1.5kb. All of our reagents have been filtered, thus it is : unlikely : that this a contaminating band. To date we have 0% yield of the : desired DNA. : and : I would be interested in hearing from anyone else who has had similar : problems. : t.kuwada@neuron.uchc.edu I've always liked the bio rad prep a gene kit - never had any problems isolating from gelas although I prefer promegas for cleaning up solutions. We've messed aruhnd with this a lot in our lab - basic message is prepaa gene gfor gels, magic clean for all others Fergus Byrne Aberdeen University From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!not-for-mail From: Andrei Popov Newsgroups: bionet.molbio.methds-reagnts Subject: RE: QUICK & DIRTY RT-PCR Date: 3 Aug 1995 23:47:28 +0100 Lines: 4 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3vrjm0$kg7@mserv1.dl.ac.uk> Importance: High Sensitivity: Company-Confidential Original-To: methods@dl.ac.uk (Receipt Notification Requested) (Non Receipt Notification Requested) (IPM Return Requested) Have a look at Meth. in Enzym. vol 225 pp611-623 regards Andrei From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!MAIL.MED.CORNELL.EDU!hmmoss From: hmmoss@MAIL.MED.CORNELL.EDU (Heidi Moss) Newsgroups: bionet.molbio.methds-reagnts Subject: Help!!!! how to clone a PCR fragment? Date: 3 Aug 1995 15:29:13 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I have a cloning project that involves inserting a PCR fragment with one cohesive (Bam HI) and one blunt end into pBS. I can foresee two problems with this, namely: 1) the presence of the nontemplate specific A residue that is added at the end of PCR fragments, which gives me a non-blunt end, and 2) the absence of the 5' phosphate of the primer on the "blunt" end. I know the theory about what should be done to eliminate these problems, but what I need is someone with a little experience doing this. I'll start with the nontemplated A issue: I've heard that incubation with DNA pol I will "polish" the end by the activity of the exonuclease. Does anyone have practical experience with this, and is this even the best way to go about this? If so, can one accomplish this in the PCR buffer including dNTPs? Second, I've imagined that the T4 PNK reaction would ocuur more efficiently if I phenol extract the DNA before the PNK reaction. True? Finally, if phenol extractions are necessary, I'm going to be concerned about loss of sample, so the method that accomplishes the goals with the fewest extractions is the most valuable to me. From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!news.itd.umich.edu!frogger.rs.itd.umich.edu!jpsmith From: jpsmith@umich.edu (James Smith) Newsgroups: bionet.molbio.methds-reagnts Subject: pET15b ligation probs. Date: 3 Aug 1995 15:43:55 GMT Organization: University of Michigan Lines: 36 Message-ID: <3vqqrr$f1b@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: frogger.rs.itd.umich.edu X-Newsreader: TIN [version 1.2 PL2] I have posted here before and have corresponded with some of you via email, but I'm still having ligation problems. I have recently switched restriction sites in hopes that I may have better luck. I have been successfully amplifying a 1.5kb DNA fragment via RT-PCR with a Bam HI restriction site included in the primers, 3 bases from the end of the fragment (according to NEB's catalog, Bam HI appears to be a good end-cutter). I've been digesting the plasmid with Bam for 3h (checked on gel for complete linearization) and then treating with CIAP. I've been using Bio-Rad's Prep-a-Gene kit to purify the plasmid. The DNA insert was digested for 3 h with Bam and also purified with Prep-a-Gene. My ligation mix has included DNA & plasmid in a 3:1 (approx.) ratio. I've been using 2 U of T4 DNA ligase (BM) and the included 10x ligase buffer. My ligase appears to be good as it ligated a HindIII lambda digest ladder in an hour at RT. Following an hour ligation, I ran 10 uL of the 30 uL ligation mix on a gel and noted a fairly intense smear from my 1.5kb fragment up to a point corresponding approximately to the location of circular plasmid with no insert (although no *distinct* bands were observed). The smear continued up beyond the location of linear plasmid, but was less intense in this region. I attempted transformation with this ligation rxn, but no colonies were observed (transformation was successful on the pUC18 control plates). I'm at a loss. I've been trying to get this to work for about a month now with no success. I'm new at these procedures, but it doesn't seem like it should be this problematic! The smear on the gel described above concerns me - it seems that if my insert was dimerizing, etc., I would see distinct bands corresponding to 1.5kb, 3kb, 4.5kb, etc. instead of a smear. However I've never seen a successful ligation rxn run on a gel. I'd appreciate any comments or advice anyone could offer. Thank you very much! Jim Smith University of Michigan From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!agate!trib.apple.com!olivea!venus.sun.com!nntp-hub2.barrnet.net!news.Stanford.EDU!usenet From: Matt Springer Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Non-radioactive Southerns and Northerns Date: 4 Aug 1995 06:17:00 GMT Organization: Stanford University Lines: 27 Message-ID: <3vse0s$2h6@nntp.Stanford.EDU> References: <3vrk3k$pmu@news.cc.utah.edu> NNTP-Posting-Host: r312-pb540c.stanford.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:3vrk3k$pmu@news.cc.utah.edu Helen McBride wrote: > We have used the Boehrenger Genius sytem for non-radioactive >Southerns with great success. The only real difference is that you need >to use their membrane (positively charged) for it to work best. All the >steps are the same except for different prehyb/hyb solutions. The >detection is just like any other ECL system and exposure times are >usually short (recommended time of 1 hr is usually just fine). > I tried to use this system for Northerns and had a major problem-NO >SIGNAL. I followed all their directions and used the recommended >prehyb/hyb which is different from that used with Southerns and it still >didn't work. I probed the same blot with a radioactive probe made from >the same batch of cut DNA with no problem, so I don't know what went >wrong. Others I have spoken with at our institution have also had no >success using it for Northerns. I hope this helps. > >hmcbride@hlthsci.med.utah.edu I and several other people I know have used the Genius system for northerns according to the manufacturer's conditions and have gotten wonderful results. Not that this really helps, but they really can work. Only problem from my viewpoint is major deterioration of results with each round of stripping. -Matt Springer From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!uunet!in2.uu.net!news.ios.com!ppp-nyc-1-4.ios.com!user From: Efrain@soho.ios.com (Efrain Gonzalez) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Needed: Sigma Gel clone app. for Mac Date: 4 Aug 1995 04:36:25 GMT Organization: SUNY/Maimonides Medical Center Lines: 13 Message-ID: NNTP-Posting-Host: ppp-nyc-1-4.ios.com Does anyone know if a program that does the same thing as SigmaGel by Jandel exists for the Mac? The program can replace the functions of an electorphoretic gel analysis system using a computer and a scanner. If anyone knows of anything, please E-mail me. Thank you. -- -- Efrain Gonzalez Efrain@soho.ios.com From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!xlink.net!rz.uni-karlsruhe.de!news.uni-stuttgart.de!uni-regensburg.de!lrz-muenchen.de!fauern!gs.dfn.de!news.mdc-berlin.de!ppc3002.mdh3.mdc-berlin.de!user From: mstrehle@mdc-berlin.de (Michael Strehle) Newsgroups: bionet.molbio.methds-reagnts Subject: Test Date: 1 Aug 1995 11:39:58 GMT Organization: Max-Delbrueck-Centrum Berlin-Buch Lines: 1 Message-ID: NNTP-Posting-Host: ppc3002.mdh3.mdc-berlin.de From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!rutgers!gatech!swrinde!howland.reston.ans.net!ix.netcom.com!netnews From: babco@ix.netcom.com (Tom Anderson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: peptide synthesis Date: 4 Aug 1995 05:11:07 GMT Organization: Netcom Lines: 20 Distribution: world Message-ID: <3vsa5b$6nd@ixnews5.ix.netcom.com> References: <3uoem3$rra@threed.uchc.edu> NNTP-Posting-Host: ix-wc1-05.ix.netcom.com In <3uoem3$rra@threed.uchc.edu> tkuwada@neuron.uchc.edu (Tim) writes: > >Can anyone recommend a company for reasonably priced peptide synthesis? > >Thanks in advance, > >Tim Kuwada >Univ. of CT Health Center > >E-Mail:tkuwada@neuron.uchc.edu >203-679-2906 Try Quality Control Biochemicals in Hopkinton MA. We have had them wynthesize over 100 peptides, and every one has been a gem. There are helpful, accurate, and on time. --Tom Anderson Berkeley Antibody Company (BAbCO) From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!math.ohio-state.edu!ohstpy.mps.ohio-state.edu!miavx1!news.muohio.edu!miavx1!gliang Newsgroups: bionet.molbio.methds-reagnts Subject: Spirulina platensis strain Message-ID: <1995Aug3.213638@miavx1> From: gliang@miavx1.acs.muohio.edu Date: 3 Aug 95 21:36:38 -0500 Organization: Miami University NNTP-Posting-Host: miavx1.acs.muohio.edu Lines: 6 Hi, is anyone using spirulina platensis or maxima? I would like to know more about it. Could you contact me please if you know where to find this strain? Any kind of strains. Thanks My e-mail address is: Gliang@miavx1.muohio.edu Guiqing From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!rutgers!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!cisco-ts5-line41.uoregon.edu!user From: tedm@darkwing.uoregon.edu (Ted M.) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: pET15b ligation probs. Date: 4 Aug 1995 02:53:47 GMT Organization: U of O Lines: 37 Message-ID: References: <3vqqrr$f1b@lastactionhero.rs.itd.umich.edu> NNTP-Posting-Host: cisco-ts5-line41.uoregon.edu In article <3vqqrr$f1b@lastactionhero.rs.itd.umich.edu>, jpsmith@umich.edu (James Smith) wrote: > I have posted here before and have corresponded with some of you via > email, but I'm still having ligation problems. I have recently switched > restriction sites in hopes that I may have better luck. I have been > successfully amplifying a 1.5kb DNA fragment via RT-PCR with a Bam HI > restriction site included in the primers, 3 bases from the end of the > fragment (according to NEB's catalog, Bam HI appears to be a good end-cutter). > I've been digesting the plasmid with Bam for 3h (checked on gel for > complete linearization) and then treating with CIAP. I've been using > Bio-Rad's Prep-a-Gene kit to purify the plasmid. The DNA insert was > digested for 3 h with Bam and also purified with Prep-a-Gene. > > Jim Smith > University of Michigan Jim, I'm concerned about the "purification" of the BamH I digested insert, it is essential that you get rid of the cut ends, primers and dNTP's to get a good ligation. Good ways to purify this insert include isolation from an agarose gel or a size exclusion method. Typically I'll use a Microcon 30 (from Amicon) spin unit to remove all the cut ends and dNTP's/primers from a PCR/digestion. This leaves you with a little template DNA and your primary PCR product with digested ends. If the amplification isn't very clean, a gel is the way to go. With all the ends and/or primers present it is possible to get big ugly TA ligated smears and the like. If the Prep-a-Gene kit is used in conjunction with gel purification, ignore this point, as I am unfamiliar with this procedure. Concatamers are a problem with an insert with compatible ends. If this is the case try a lower ratio of insert to vector and possibly PEG to help your chances. Good luck. Ted Michelini Institute of Molecular Biology University of Oregon tedm@darkwing.uoregon.edu From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news-e1a.megaweb.com!newstf01.news.aol.com!uunet!in2.uu.net!nwfocus.wa.com!immunex.wa!hollingswor From: hollingswor@immunex.wa.com Newsgroups: bionet.molbio.methds-reagnts Subject: LongRanger on Automated Sequencer? Message-ID: <1995Aug3.175425.328@immunex.wa.com> Date: 3 Aug 95 17:54:25 PST Organization: Immunex Corporation, Seattle, WA Lines: 6 The recent question about gradient gels on an ABI373 prompted this question. Has anyone tried Long Ranger gels? What percentages, running parameters, matrix/mobility concerns? How were the results and lenth of read? Thanks Tamy Hollingsworth From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.starnet.net!wupost!waikato!celebrian.otago.ac.nz!sanger.otago.ac.nz!bmcm From: bmcm@sanger.otago.ac.nz (Brendan McMorran) Newsgroups: bionet.molbio.methds-reagnts Subject: DIG gel shift. Good or bad? Date: 4 Aug 1995 01:24:15 GMT Organization: University of Otago, Dunedin, NZ Lines: 1 Message-ID: <3vrsrv$tnd@celebrian.otago.ac.nz> NNTP-Posting-Host: sanger.otago.ac.nz X-Newsreader: TIN [version 1.2 PL2] From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!rutgers!gatech!newsfeed.internetmci.com!news.sprintlink.net!cs.utexas.edu!howland.reston.ans.net!news.starnet.net!wupost!news.missouri.edu!NewsWatcher!user From: c601591@mizzou1.missouri.edu (Don Haut) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA purification from agarose gels Date: 4 Aug 1995 00:20:29 GMT Organization: Dept of Molecular Micro and Immunology Lines: 13 Message-ID: References: <3sp9rk$1tp@lyra.csx.cam.ac.uk> <3u3viq$9gl@threed.uchc.edu> <3vqkdl$600@fs2.abdn.ac.uk> NNTP-Posting-Host: 128.206.12.143 We use both electro-elution and Qia-Quick. The Qia-Quick is great for anything under about 4 kb. After that, our yeild drops big time!!! So we use electo-elution. The QiaQuick is MUCH faster and for smaller things, the yeild is good. Don Don Haut Molecular Microbiology and Immunology University of Missouri-Columbia C601591@showme.missouri.edu 314-882-3171 From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!news.sprintlink.net!uunet!in2.uu.net!newsflash.concordia.ca!canopus.cc.umanitoba.ca!gietz.hgen.umanitoba.ca!user From: rennie@bldghsc.lan1.umanitoba.ca (S. L. Rennie) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Non-radioactive Southerns and northerns-help Date: Thu, 03 Aug 1995 11:23:07 -0600 Organization: U of Manitoba Lines: 19 Message-ID: References: NNTP-Posting-Host: gietz.hgen.umanitoba.ca In article , "Mary P. Remington" wrote: > I need some input on non-radioactive Southerns and northerns. What is > available? Are the protocols easier or more difficult than hot > protocols? What type of membrane is used with the non-radioactive > protocols? Please respond via email as I'm in a hurry. Thanks, Mary -------------------------------- I know Boehinger-Mannheim has kits for this (its their cause of the decade??) but I've never had a chance to try it as your probe needs to be made with their tag chemical on the end......apperently it works very well though and all the reagents are supposed to be able to be stored for 2 years or so.... worth a try to contact them, they are VERY eager to help. I don't think the protocols are any harder just different. Good luck Sharon From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!plug.news.pipex.net!pipex!tank.news.pipex.net!pipex!warwick!bham!bham.ac.uk!D.J.Glover From: D.J.Glover@bham.ac.uk (David Glover) Newsgroups: bionet.molbio.methds-reagnts Subject: RNA Extraction (RVCs) Date: Thu, 3 Aug 1995 11:49:58 Organization: The University of Birmingham Lines: 10 Message-ID: NNTP-Posting-Host: bcs177.bham.ac.uk X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] The literature states that when using ribonucleoside vanadyl complexes (RVC) as an RNase inhibitor during RNA extraction, high concentrations of EDTA and SDS should be avoided as they may cause dissociation of the complexes and thus their inactivation. Does anyone know the lowest molar concentration of EDTA that causes dissociation of the compexes? There is certainly a difference in the colour of the solution when RVCs are dissolved in buffer containing 10mM and 2mM EDTA. Thanks in advance. David Glover. From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!ns1.faseb.org!darwin.sura.net!mother.usf.edu!news From: tabibzadeh@rics.moffitt.usf.edu (SIAMAK TABIBZADEH) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: FLAG epitope - sources and advice Date: 3 Aug 1995 11:12:35 GMT Organization: Moffitt Cancer Center at USF Lines: 16 Message-ID: <3vqav3$3sf@mother.usf.edu> References: <3vooil$e67@vixen.cso.uiuc.edu> NNTP-Posting-Host: pc3160.moffitt.usf.edu Mime-Version: 1.0 X-Newsreader: WinVN 0.93.11 In article <3vooil$e67@vixen.cso.uiuc.edu>, dclayton@uiuc.edu says... > >I need to put a small epitope onto a protein to allow detection of it in >cultured cells and perhaps tissues. I have the impression that the FLAG >epitope might be a good choice for this. Questions: >1) is there a commercial source for FLAG-epitope related reagents? >2) Anybody want to offer opinions as to the utility of FLAG, vs. other >candidates such as His, myc, HA, etc? >Any advice appreciated! >>>David dclayton@uiuc.edu 217-244-4525 > > For the Flag system contact the IBI/Kodak people. Mike gruidl From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!howland.reston.ans.net!newsserver.jvnc.net!news.caren.net!news.join.ad.jp!wnoc-tyo-news!news.iij.ad.jp!wincgw1!creamy!icspub!odins-suita!odins-sci!usenet From: shiraga4@bio.sci.osaka-u.ac.jp (Shin-ichiro Hiraga) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Is there a shareware plasmid drawing program? Message-ID: <3vq7fl$b7u@ns2.sci.osaka-u.ac.jp> Date: 3 Aug 95 10:13:09 GMT References: <3v9i9i$ej8@netserv.unmc.edu> Organization: Osaka University Lines: 12 NNTP-Posting-Host: grape.bio.sci.osaka-u.ac.jp Mime-Version: 1.0 Content-Type: text/plain; charset=iso-2022-jp Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: dchakrav@netserv.unmc.edu X-URL: news:3v9i9i$ej8@netserv.unmc.edu For Macintosh, there is a program called "MacPlasMap". I think it can be obtainded from "ftp://fly.bio.indiana.edu". I am using it and I think it is worth to get. I don't know of other platforms such as PC or UNIX. Sorry. Good luck! Shin-ichiro Hiraga Graduate student Osaka University shiraga4@bio.sci.osaka-u.ac.jp From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!cs.utexas.edu!swrinde!hookup!nstn.ns.ca!newsflash.concordia.ca!CC.UMontreal.CA!news.uqam.ca!UQuebec.CA!Pierre_Talbot From: Pierre_Talbot@Infopuq.UQUEBEC.CA () Subject: Video Photodocumentation of gels Sender: news@UQuebec.CA (news) Message-ID: Date: Thu, 3 Aug 1995 01:41:02 GMT Nntp-Posting-Host: panoramix.uqss.uquebec.ca Organization: Universite du Quebec X-Newsreader: TIN [version 1.2 PL2] Lines: 11 What is the best video apparatus for getting digital images of gels (Nucleic acids, proteins, etc.) in place of Polaroid prints: Bio-Rad, UVP, Applied Innotech, or others I do not know about? Pierre Talbot, Ph.D. Armand-Frappier Institute Laval, Quebec, Canada H7N 4Z3 E-mail: Pierre.Talbot@iaf.uquebec.ca Fax: 514-686-5531 From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!newsfeed.internetmci.com!EU.net!Austria.EU.net!newsfeed.ACO.net!mail.boku.ac.at!news From: Iain Wilson Newsgroups: bionet.molbio.methds-reagnts Subject: Re: pET vectors, good or bad? Date: 3 Aug 1995 08:28:25 GMT Organization: BOKU Wien Lines: 21 Message-ID: <3vq1b9$au0@mail.boku.ac.at> References: <9508021728.AA01214@compbio.med.wayne.edu> NNTP-Posting-Host: 141.244.58.101 > Perhaps my opinion here is a bit colored by the years of frustration while > working with the pET system, by I have to stick with what I wrote above. I am > not saying anyone should stay away from pET vectors based solely on my exper- > ience. I have been collecting complaints from this very net, however, to send > to my old boss. I have collecyed about ten complaints so far. I'd be willing > to bet a few others will have more to say on the subject in response to Mike > Holloway's original query yesterday... > > -Jeff I would just add that in my previous lab, we got good results using pET-16b with a transmembrane-domain deleted mannosyltransferase. With the His10 tag were able to immobilise the enzyme in an active form on His-Bind resin with no refolding. Perhaps we were just lucky ... Iain wilson@edv1.boku.ac.at From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!dispatch.news.demon.net!demon!mail2news.demon.co.uk!genesys.demon.co.uk!Duncan From: Duncan Clark Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Endonuclease activity: Crude extraction from E.coli Date: Wed, 02 Aug 1995 15:12:39 GMT Organization: GeneSys Ltd. Lines: 22 Message-ID: <808462965wnr@genesys.demon.co.uk> References: Reply-To: Duncan@genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk X-Broken-Date: Wednesday, Aug 02, 1995 15.12.39 X-Newsreader: Newswin Alpha 0.7 In article: woodj@acs.ucalgary.ca (Julian Wood) writes: > I'm looking for a quick method to make a crude extraction containing the > endonuclease (and possibly get rid of non-specific endonuclease activity). > > The extract will be used to test for the activity of the endonuclease on a > target DNA. Simple, harvest cells and resuspend in say 10mM KPi (pH 7.4), 10mM B-EtSH, 0.1mM EDTA,10% glycerol. Sonicate, centrifuge and assay supernatant (possibly using dilutions) against your DNA substrate. It ios not possible to get rid of non-specific endonuclease unless you have a thermophilic enzyme ('cos then you can heat treat the lysate). If DH10 is endA then the E.coli is as non-specific nuclease minus as you will get. If you need better then you will have to purify your enzyme. Duncan -- ----------------------------------------------------------------------------- My mind's made up. Don't confuse me with the facts! From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!dispatch.news.demon.net!demon!mail2news.demon.co.uk!genesys.demon.co.uk!Duncan From: Duncan Clark Newsgroups: bionet.molbio.methds-reagnts Subject: Re: strataclean--homemade version? Date: Wed, 02 Aug 1995 15:07:37 GMT Organization: GeneSys Ltd. Lines: 16 Message-ID: <700910729wnr@genesys.demon.co.uk> References: Reply-To: Duncan@genesys.demon.co.uk X-NNTP-Posting-Host: genesys.demon.co.uk X-Broken-Date: Wednesday, Aug 02, 1995 15.07.37 X-Newsreader: Newswin Alpha 0.7 In article: sbv@pop.cwru.edu (Scott Vande Pol) writes: > > We have become addicted to Strataclean: the more we use it, the more we > want to use it. But it is very pricey. Soon I will be knocking off liquor > stores to support our growing habit. What IS this stuff--does anyone know > how to make it (maybe a 12 step protocol)?? Thanks in advance. It is covered in US patent 4923978. From what I remember it looked pretty horrendous to make. Duncan -- ----------------------------------------------------------------------------- My mind's made up. Don't confuse me with the facts! From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!uunet!in1.uu.net!news.maz.net!news.omnilink.net!news.gni.net!thorin!rz.uni-karlsruhe.de!news.uni-stuttgart.de!news.rhrz.uni-bonn.de!news.rwth-aachen.de!news.mu-luebeck.de!biopc2.biologie.mu-luebeck.de!SIEVERT From: SIEVERT@molbio.mu-luebeck.de (Volker Sievert) Newsgroups: bionet.molbio.methds-reagnts Subject: test, please ignore Date: Wed, 2 Aug 1995 23:57:57 UNDEFINED Organization: Inst. f. Med. Informatik, Med. Univ. Luebeck Lines: 1 Message-ID: NNTP-Posting-Host: biopc2.biologie.mu-luebeck.de X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #1] This is a test using a new newsreader. hopefully this one is better... From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!swrinde!ringer.cs.utsa.edu!lonestar.jpl.utsa.edu!blong From: "Billy B. Long" Newsgroups: bionet.molbio.methds-reagnts Subject: paired domains? Date: Thu, 3 Aug 1995 16:06:36 -0500 Organization: The University of Texas at San Antonio Lines: 12 Message-ID: NNTP-Posting-Host: lonestar.jpl.utsa.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Hi, netters! I have recently come across the term "paired domains" while reading a paper on a transcriptional activator. I am very familiar with homeodomains, but my efforts to find a description on paired domains in literature/texts have been futile. If anyone can describe/define them, or provide for me a review article or text chapter, or any info for that matter, it would be greatly appreciated. Thanks in advance! C-ya! Bob Long From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!swrinde!gatech!newsfeed.internetmci.com!news.sprintlink.net!sunic!sunic.sunet.se!news.uni-c.dk!news.daimi.aau.dk!biobase!ali From: ali@biobase.dk (Ali Karami) Newsgroups: bionet.molbio.methds-reagnts Subject: looking fo E.coli RY13 Date: 3 Aug 1995 14:22:46 GMT Organization: The Danish BioBase Lines: 12 Message-ID: <3vqm3m$f8v@biovax.biobase.dk> NNTP-Posting-Host: biobase.dk X-Newsreader: TIN [version 1.2 PL2] Dear netters I am looking for E.coli RY13 strain containing plasmid encoding EcoRI methylase. If you have this strain or you know how can I find it please send me an Email to ali@biobase.DK Thank you Ali Copenhagen University From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!bcm!news.msfc.nasa.gov!newsfeed.internetmci.com!gatech!howland.reston.ans.net!vixen.cso.uiuc.edu!uchinews!morla540c.bsd.uchicago.edu!user From: amorla@midway.uchicago.edu (Alex Morla) Subject: Plasmid Sequence Database? X-Nntp-Posting-Host: morla540c.bsd.uchicago.edu Message-ID: Sender: news@midway.uchicago.edu (News Administrator) Organization: The Univ. of Chicago Date: Thu, 3 Aug 1995 09:13:48 GMT Lines: 11 I am looking for a source of vector plasmid sequences (I am actually looking for the sequence of the PECE vector). I have not had much luck with GeneBank and I was wondering if there was some database somewhere (either on the www, or gopher, ftp, etc.) that would have such sequences. Any info will be appreciated. -- Dr. Alex Morla Dept. of Pathology The Univ. of Chicago From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!NewsWatcher!user From: MAlexeyev@biost1.thi.tmc.edu (Mikhail Alexeyev) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: What is SELEX? Date: 3 Aug 1995 15:03:08 GMT Organization: Texas Heart Institute Lines: 11 Distribution: world Message-ID: References: NNTP-Posting-Host: 128.249.210.32 > there is a method, called SELEX, for determing the target sequences of > RNA-binding proteins (?) > How does it work? > References? > > Stephan SELEX stands for Systematic Evolution of Ligands by EXponential enrichment. Try the original article by C. Tuerk and L. Gold in Science v.249 1990 pp. 505-510 From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!convex!news.duke.edu!solaris.cc.vt.edu!newsfeed.internetmci.com!gatech!swrinde!howland.reston.ans.net!ee.und.ac.za!nntp.und.ac.za!pc048.ag.unp.ac.za!Scholfield From: Scholfield@bchm.unp.ac.za (Niccy.Scholfield) Newsgroups: bionet.molbio.methds-reagnts Subject: RNA denaturant: methylmercuric hydroxide Date: Thu, 3 Aug 1995 13:37:23 GMT Organization: University of Natal , Pietermaritzburg, South Africa Lines: 12 Message-ID: NNTP-Posting-Host: pc048.ag.unp.ac.za I need to denature my double-stranded viral RNA before I do RT-PCR and it seems that methylmercuric hydroxide is the best way to do this, being reversible by reaction with thiol compounds. The ubiquitous Molecular Cloning (2nd Ed) by Sambrook, Fritsch and Maniatis refers to said compund and its use in denaturing gels for RNA size fractionation, but I have not been able to find the compound for sale in *any* of a number of catalogues (Merck, Sigma, Promega, Fluka, Boerhinger Mannheim etc), in any version of its molecular formula (CH3HgOH or CH4HgO) or as mercury, hydroxymethyl or hydroxymethyl mercury or as methylated mercuric hydroxide! If someone can please tell me where I can get hold of some of this mysteriously unavailable chemical, I would be delighted! Nic Scholfield (SCHOLFIELD@BIOCHEM.UNP.AC.ZA) From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!newsfeed.internetmci.com!news.mid.net!netserv.unmc.edu!netserv.unmc.edu!not-for-mail From: dchakrav@netserv.unmc.edu (Dhruba Chakravarti) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Is there a shareware plasmid drawing program? Date: 3 Aug 1995 09:47:16 -0500 Organization: University of Nebraska Medical Center, Omaha, NE, USA Lines: 6 Message-ID: <3vqnhk$aic@netserv.unmc.edu> References: <3v9i9i$ej8@netserv.unmc.edu> <3vq7fl$b7u@ns2.sci.osaka-u.ac.jp> NNTP-Posting-Host: netserv.unmc.edu X-Newsreader: TIN [version 1.2 PL2] Thank you all for the advice. Regards, Dhruba. From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!uwvax!newssinet!news.u-tokyo.ac.jp!news.nc.u-tokyo.ac.jp!t-server!mech.t.u-tokyo.ac.jp!130.69.160.103!yokada From: yokada@kinesin.kaibo1.m.u-tokyo.ac.jp (Yasushi OKADA) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: pET vector cloning of hydrophobic protein gene Date: 3 Aug 1995 14:36:07 GMT Organization: Dept. Anatomy & Cell Biol. Facul. Med. Univ. Tokyo. Lines: 26 Message-ID: References: <3vltmi$qn5@warp.cris.com> NNTP-Posting-Host: kinesin.kaibo1.m.u-tokyo.ac.jp In-reply-to: mike.holloway@stjude.org's message of 1 Aug 1995 19:01:38 GMT In article <3vltmi$qn5@warp.cris.com> mike.holloway@stjude.org (Mike Holloway) writes: > I have to conclude that some protein is being made, despite what > anyone says about the T7 promoter not being used without the > polymerase being pumped out, and that its toxic. This has been the > previous experience in our lab with this protein using other > bacterial expression vectors. Apparently this isn't an isolated > experience, since several companies have recently come out with this > thioredoxin fusion protein scheme that's supposed to solve > everything. Do you consider that the protein is made even in the bugs that do not have T7 pol gene? If you can subclone your PCR product in bugs without T7 pol, you can induce the expression of the protein by the infection with phage that have T7 pol gene. Such phage can be obtained from Novagen or Invitrogen. Hope this helps. -- ****************************************************************************** * Yasushi Okada, MD. | Email:yokada@kinesin.kaibo1.m.u-tokyo.ac.jp * * Dept. Anatomy & Cell Biology | Tel:81-3-3812-2111 ext.3336 * * Univ. Tokyo JAPAN | Fax:81-3-5689-4856 * ****************************************************************************** From owner-methds-reagnts@net.bio.net Wed Aug 02 23:00:00 1995 Path: biosci!daresbury!not-for-mail From: "lidaniel" Newsgroups: bionet.molbio.methds-reagnts Subject: Re:strataclean-homemade version Date: 3 Aug 1995 15:38:33 +0100 Lines: 17 Sender