From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!daresbury!not-for-mail From: "David C. Logan" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: PLEASE VISIT: http://www.muetec.com/sgk/index.htm Date: 2 Jun 1996 14:01:00 +0100 Organization: Oxford University - Plant Science Lines: 19 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <4os3ac$hch@mserv1.dl.ac.uk> Reply-To: david.logan@Plant-Sciences.oxford.ac.uk X-mailer: Pegasus Mail/Mac (v2.1.2) Original-To: gk1702@ix.netcom.com >Subject: PLEASE VISIT: http://www.muetec.com/sgk/index.htm Why? *************************** david.logan@plants.ox.ac.uk David C. Logan Department of Plant Science University of Oxford South Parks Road Oxford OX1 3RB *************************** From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!surfnet.nl!ruu.nl!stofwis3.biol.ruu.nl!N.P.Dantuma From: N.P.Dantuma@biol.ruu.nl Newsgroups: bionet.molbio.methds-reagnts Subject: DEAE-dextran transfection/divalent cations Date: Sun, 2 Jun 1996 15:26:13 Organization: Rijksuniversiteit Utrecht Lines: 21 Message-ID: NNTP-Posting-Host: stofwis3.biol.ruu.nl Hi, COS cells can be transfected according to the protocol of Cullen (Meth. Enzym. 152: 684-704) by incubating associated cells with DNA-DEAE- dextran complexes in PBS without Ca and Mg. This DEAE-dextran transfection works nicely and the efficiency is high. However, I noticed that many cells dissociate during the 30 min incubation with the DNA transfection mix when the Ca and Mg are omitted (as expected since COS cells require divalent cations for attachment). Am I right that Ca and Mg have to be omitted according to this protocol (it is not clearly indicated)? What is the reason? Does it disturb transfection? Is it worth trying to transfect in the presence of Ca and Mg? Suggestions/answers preferable by e-mail. Thanks. Nico Dantuma From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!IMPSAT1.COM.AR!ppadula From: ppadula@IMPSAT1.COM.AR Newsgroups: bionet.molbio.methds-reagnts Subject: Re: H E L P ! ! Date: 2 Jun 1996 09:54:34 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <31B1FE92.673A@impsat1.com.ar> References: <31AE17AC.4C27@impsat1.com.ar> NNTP-Posting-Host: net.bio.net Please, could you tell me how I get off this mailing-list? What must to say the e-mail? Thanks! From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!news.starnet.net!newsreader.wustl.edu!biodec.wustl.edu!graham From: graham@biodec.wustl.edu (James Graham) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: mRNA extraction from Gram positive bacteria Date: 2 Jun 1996 12:45:51 -0500 Organization: Washington University Biology, St. Louis, MO Lines: 19 Message-ID: References: NNTP-Posting-Host: @biodec.wustl.edu X-Newsreader: NN version 6.5.0 #1 (NOV) I think you will find a oscillating or high-speed reciprocating shaker almost essential for efficient isolation from many gram positives. These devices shake the pelleted suspention at high speeds (6K rpm) with zirconimum fragments (beads) and will increase the total RNA yield from say Mycobacterium almost 10X compared to other grinding and lysis procedures, in my experience. Two such devices are "Fast-prep" from Gibco and "Mini Bead Beater". As for Trizol, its just colored buffered phenol of low PH, with a buffer system that supposedly allows recovery of the DNA from the organic phase. Published reports show that up to 20% of the nucleic acids recovered from the aqueous phase may be DNA. See Fischetti's recent Analytical Biochemisty article for details. Jim J. Graham Biology Department From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!newsfeed.internetmci.com!in2.uu.net!newsflash.concordia.ca!news.mcgill.ca!athena.ulaval.ca!news From: aaa186@agora.ulaval.ca (gwen jenkins) Newsgroups: bionet.molbio.methds-reagnts Subject: pcr problem with gc rich sequences Date: Mon, 03 Jun 1996 00:18:27 GMT Organization: Universite Laval Lines: 10 Message-ID: <4ot046$7d5@athena.ulaval.ca> NNTP-Posting-Host: ppp-a9.ulaval.ca X-Newsreader: Forte Agent .99c/32.126 We are trying to amplify a gene with many repeats in GC rich sequences. After PCR, no amplification seems to appear. I've heard that the Polymerase is blocked in some way by secondary structures. We have tried SSB proteins to stabilize it but no improvement was observed. Is somobody know a way to do it ? If so please contact us by e-mail at BHXM@musicb.mcgill.ca Stephane Angers From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!chi-news.cic.net!news.compuserve.com!news.production.compuserve.com!news From: Survey Administration <74750.1341@CompuServe.COM> Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.molbio.recombination,bionet.molbio.yeast Subject: WE NEED YOUR INPUT Date: 3 Jun 1996 03:20:05 GMT Organization: Kitty Knight Corporation Lines: 25 Message-ID: <4otll5$3ni$6@mhade.production.compuserve.com> Xref: biosci bionet.molbio.methds-reagnts:45225 bionet.molbio.proteins:8022 bionet.molbio.recombination:193 bionet.molbio.yeast:5356 Please excuse this brief intrusion, but we are trying to locate any and all holders of academic degrees conferred within the last ten (10) years. The Kitty Knight Corporation, Boston, MA, is currently searching for qualified individuals to participate in a ***PAID*** study focusing on post-secondary, graduate, and professional education in the United States. Holders of ALL types of degrees in ALL fields of study are needed. We would like to hear from you as long as your degree was earned at an accredited institution in the United States. After an initial screening, qualified participant will be asked to complete a questionaire of approx 150 questions. Everyone who completes the survey will receive a $100 stipend. Naturally, *ALL* information will be held in the strictest confidence. To express interest, please send your name, mailing address, and photocopies of **ALL** degrees you have earned to: The Kitty Knight Corporation, Attn: Study 96-3H, Back Bay Annex, P O Box 546, Boston, MA 02117. (If not obvious from the degree, please indicate the field of study.) Thank You Very Much! From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!howland.reston.ans.net!news-e2a.gnn.com!newstf01.news.aol.com!newsbf02.news.aol.com!not-for-mail From: johnnyhugh@aol.com (JohnnyHugh) Newsgroups: bionet.molbio.methds-reagnts Subject: ProK Digestion Date: 2 Jun 1996 22:55:16 -0400 Organization: America Online, Inc. (1-800-827-6364) Lines: 4 Sender: root@newsbf02.news.aol.com Message-ID: <4otk6k$ci7@newsbf02.news.aol.com> Reply-To: johnnyhugh@aol.com (JohnnyHugh) NNTP-Posting-Host: newsbf02.mail.aol.com I would like to treat small tissue sections with Pro K. Could somebody kindly suggest the optimal buffer and pH conditions for its digestion? Thanks. From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!agate!howland.reston.ans.net!newsfeed.internetmci.com!in2.uu.net!nih-csl!postman From: bernard@elsie.nci.nih.gov (Bernard Murray) Subject: Re: Anyone using "the hook"? Message-ID: <1996Jun3.005655.8030@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: ermintrude.nci.nih.gov Organization: National Institutes of Health, Bethesda, MD X-Newsreader: WinVN 0.99.7 References: <31MAY96.19034119@shrsys.hslc.org> Date: Mon, 3 Jun 1996 00:56:55 GMT Lines: 76 In article <31MAY96.19034119@shrsys.hslc.org>, jovermeyer@shrsys.hslc.org says... >I am considering trying Invitrogen's new transfection kit using a phook >vector that entails magnetic beads on the cell surface to help select for a >trsnsfected population of cells. Does anyone have any experience with this? > Yes indeedy-do, I am currently in the process of playing with this. Our current "gold standard" for post transfection selection is an expression vector for the human IL2 receptor and we can then capture the cells with anti-IL2R-mab coated Dynal beads. This system works extremely well but we want to sort cells that already have IL2R so I am trying this Capture-Tec kit. Whilst the overall theory is sound the kit seems to have been put together by Homer Simpson. It does work but needs some fiddling. I've used the pHook-1 system (does not contain a MCS in the vector). Here are some thoughts and tips; 1) Dump the pCR3LacZ test vector that comes with the kit. It gives the lowest LacZ expression of any vector I have seen. It also uses the same promoter (CMV) as the pHook plasmid (doh!) so they would probably interfere with each other. I've used the MacGregor pRSVZ or one of the SV derivatives (pSVbeta or pCH110) and seen nice in situ staining of sorted cells. 2) They put a neo/kan cassette in the vector which already has an amp cassette (doh!). This is entirely pointless as if you are going for a stable line you'll use pSV2neo at the start and not bother with the sorting and if you are sorting cells for transient transfection you don't need another selectable marker. That's 0.8 kb of DNA wasted guys! 3) Invitrogen obviously hope to make most of their cash from the PhOX beads which are obscenely over-priced, even by their own standards. Look at the price of Dynal beads and do the maths to check the mark-up. Alternatives to selling major organs to pay for this are to use standard MACS with an antibody against either the HA or myc epitope (I haven't tried this) or to make your own derivatised beads. Since the hapten for their sFv is cheap and spontaneously amine-reactive it should be possible to either react it with protein coated beads (eg. an irrelavant antibody) or to react it with a diamino spacer reagent (eg. diaminooctane) and thence with tosylated dynabeads. I shall let you know how this works out. 4) The kit does not include a magnet (doh!) so get a good one. 5) If you've ever done any kind of MACS before using the kit is a breeze. The one major practical problem is that Invitrogen warn you against using trypsin to harvest cell prior to sorting and suggest dispersion in PBS/EDTA. Since many adherent cell lines just shrug off such treatment this results in unsortable clumps. The IL2R system I use has the advantage that the surface antigen is trypsin resistant so I can make nice single-cell suspensions for sorting. Thankfully preliminary results with the Capture-Tec kit suggest that Invitrogen are being over-cautious and that light exposure of transfected cells to trypsin does not stop them from being sorted. I'll try and confirm this next time. 6) I have no idea just how high the affinity of the antibody is for the beads. Since the system is designed for positive selection, rather than depletion, this only affects the yield. I aim to perform a selection and then check the remaining cells for the presence of the sFv by immunofluorescent staining for the myc or HA epitopes. Okay, that's all I have for now. I shall pass on any other tips as they occur to me. Bernard [No affiliation with Invitrogen, Dynal or The Simpsons] Bernard Murray, Ph.D. bernard@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA) From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!news.ac.net!news.cais.net!van-bc!van.istar!news-w.ans.net!newsfeeds.ans.net!lantana.singnet.com.sg!nuscc.nus.sg!mcbtayhn From: mcbtayhn@leonis.nus.sg (Tay Hou Ngee Agnes) Newsgroups: bionet.molbio.methds-reagnts Subject: MJ thermal cycler problems Date: 3 Jun 1996 01:54:24 GMT Organization: National University of Singapore Lines: 9 Message-ID: <4otgkg$b5t@nuscc.nus.sg> NNTP-Posting-Host: mcbtayhn@leonis.nus.sg X-Newsreader: TIN [version 1.2 PL2] I have owned an MJPTC200 thermal cycler for 6 months and have gone through 4 alpha blocks. Each replacement block lasts a month or so before drifting as much as 3C or having sensor failure. The latest replacement did not even make it to me as our local service engineer tried testing it before sending it on to me and it failed the tests. I know of at least 2 others from 2 different countries with the same problems with MJ machines. Not sure what's happening because the PTC-100's used to work well. The PTC-200 is a great disappointment and MJ has been very slow to respond to our complaints. From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!munnari.OZ.AU!news.mel.connect.com.au!news.uwa.edu.au!newsman.murdoch.edu.au!usenet From: oxberry@numbat.murdoch.edu.au (Megan) Newsgroups: bionet.molbio.methds-reagnts Subject: Autoradiography of PAGE gels - please help Date: 3 Jun 1996 01:45:20 GMT Lines: 17 Sender: -Not-Authenticated-[3595] Message-ID: <4otg3g$bfv@newsman.murdoch.edu.au> NNTP-Posting-Host: vetmac26.murdoch.edu.au X-Posted-From: InterNews 1.0.1@vetmac26.murdoch.edu.au Xdisclaimer: No attempt was made to authenticate the sender's name. I am trying to identify the protein to which a drug is binding in a unicellular organism using autoradiography and native PAGE gels. I have been adding tritium labelled drug to sonicated cells (with protease inhibitor), incubating the lysate at 4¡C for 24h then running it on a gel. I then fix the gel in 2-propanol/water/acetic acid (25:65:10) for 30min and soak it in Amlify (Amersham) for 30min in the dark. I then dry the gel at 80¡C for 2h on to filter paper and place it against Hyperfilm MP (preflashed) in a cassette for 1-2 weeks at -80¡C. I have had no success at all with this technique even with the positive control I have been using. I would be most grateful for any suggestions regarding this. Thankyou, Megan From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!COUGAR.VUT.EDU.AU!s9033036 From: s9033036@COUGAR.VUT.EDU.AU (Dianne Emslie) Newsgroups: bionet.molbio.methds-reagnts Subject: (none) Date: 2 Jun 1996 18:04:56 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net subscribe methods From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!agate!usenet.kornet.nm.kr!snunews.snu.ac.kr!usenet From: swine@plaza.snu.ac.kr (Dae-Young Kim) Newsgroups: bionet.molbio.methds-reagnts Subject: Help : Detection of nucleotide on TLC Date: Mon, 03 Jun 1996 04:39:05 GMT Organization: Vet Pathology at Seoul National University Lines: 8 Message-ID: <4otu6s$ptv@snunews.snu.ac.kr> Reply-To: swine@plaza.snu.ac.kr NNTP-Posting-Host: 147.46.155.71 X-Newsreader: Forte Free Agent v0.55 My message was bounced, so I try again. I am trying to detect mononucleotide, which was purified with HPLC, on TLC plate. I wanna see, by naked eyes, the position of it on TLC. If anyone has a good, please reply me. E-mail : swine@plaza.snu.ac.kr From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!swrinde!sgigate.sgi.com!uunet!in2.uu.net!world!oravaxcm From: oravaxcm@world.std.com (Charles A Miller) Subject: How do you determine % homology vs. stringency in a Southern Blot? Message-ID: Organization: The World Public Access UNIX, Brookline, MA X-Newsreader: TIN [version 1.2 PL2] Date: Mon, 3 Jun 1996 04:02:32 GMT Lines: 33 I have been trying to determine how to calculate the Stringency of hybridization in a Southern Blot versus the % homology of the probe with its various target sequences. For instance, from what I have been able to determine, if you drop the stringency of a hybridization by 1 to 1.5 'C from the Tm of the probe, you also decrease the necessary homology for hybridization by 1%. This is from Maniatis by the way. What I cannot determine, is exactly at what point this applies, and how exactly to determine the Tm using the equation (can't remember it exactly but it looks something like this: Tm=81.5+16(log salt)- molar content of G's+C's plus % formamide etc...). What I have to wonder is this: When determining the Tm for the given conditions, do you then have to determine the conditions separately for the hybridization as well as the more stringent washes? The protocol I am using uses formamide in the hybridization to lower the Tm, but the later washes do not use formamide. So, do I have to determine the Tm of the hybrid for this final wash, subtract the ACTUAL temperature used in under the conditions of salt and formamide plugged into the equation and then use THAT to determine the % change in homology? ALSO, if I have digested the target DNA before I bind the probe, do I only use the part of the probe that is actually binding to the fragment of most interest when I am trying to determine the proper temperature? Do I also only use the number of basepairs that are binding to this particular fragment in the Tm equation (ignoring the rest of the probe that will bind to another fragment that was cut away? Is there a decent shareware/freeware mac or pc program for doing all of this calculation? Thanks! Chuck Miller oravaxcm@world.std.com From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!news.uoregon.edu!platform.uoregon.edu!netnews.nwnet.net!news.nodak.edu!plains!comstock From: comstock@plains.nodak.edu (Clay Comstock) Newsgroups: bionet.molbio.methds-reagnts Subject: DNA/RNA Stain Date: 3 Jun 1996 04:20:50 GMT Organization: North Dakota Higher Education Computing Network (NDHECN) Lines: 18 Message-ID: <4otp72$7sm@daily-planet.nodak.edu> NNTP-Posting-Host: plains.nodak.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit X-Newsreader: TIN [version 1.2 PL2] I have a question.. does any one know of a common lab chemical that is either DNA or RNA specific. I'm looking for something that is cheap and easy that will tell me what I have extracted, for I am on a limited budget. Thanks. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Clay Comstock e-mail comstock@plains.nodak.edu homepage http://murphy220.phys.dsu.nodak.edu/students/clay/comstock.htm PGP Public Key available through most key servers. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!MOLECULE.BIO.UTS.EDU.AU!michelle From: michelle@MOLECULE.BIO.UTS.EDU.AU (Michelle Gleeson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Acrylamide Gel setting too quickly -- IDEAS?? Date: 2 Jun 1996 15:48:14 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <4oqlfi$8iu@dfw-ixnews9.ix.netcom.com> NNTP-Posting-Host: net.bio.net On Sat, 1 Jun 1996, Brian MacNevin wrote: > Well, when I mix up my acrylamide gel solution and begin pipetting it > between the glass plates for electrophoresis, I have found that it > starts to set before I can get it evenly dispersed in the plates. > My first thought is that this might be due to the temp of the > solution. It seems to me that I don't have this problem if the > solution is allowed to cool a little before adding the final solution. > I was wonderin if anyone else has had these problems and if it really > is due to the temp of the solution? > Thanks! > Brian > macnevin@ix.netcom.com > > > Dear Brian, Yes, it is definitely the temperature. Acrylamide gels always take longer to set on cold days. Try chilling the acrylamide mix before adding the TEMED and APS until it feels cool to touch. Also, don't use high heat to dissolve the urea. It will take a little longer but not nearly as long as preparing a new set of plates! Good luck, Michelle michelle.gleeson@uts.edu.au From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!swrinde!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!lyra.csx.cam.ac.uk!macr1-1.welc.cam.ac.uk!user From: jpcd0@mole.bio.cam.ac.uk (John Dixon) Newsgroups: bionet.molbio.methds-reagnts Subject: PCR from lambda - primer help Date: 2 Jun 1996 23:09:45 GMT Organization: Wellcome CRC Institute Lines: 20 Message-ID: NNTP-Posting-Host: macr1-1.welc.cam.ac.uk X-Newsreader: Yet Another NewsWatcher 2.0b27.5 Hi there, now that long range PCR is working like a treat, I would like to PCR out some inserts cloned into lambda FIX. But I cant find any published sequence for primers that flank the cloning site. The clones I have were made by cloning into the XbaI sites and destroying them in the process so that I cannot cut the insert out. Has anyone got anything like this? P.S. on the map the lambda regions where the primers must bind are git and lom. Cheers John -- John Dixon Lab 44 (1223) 334131 Wellcome/CRC Institute Fax 44 (1223) 334134 Department of Genetics Cambridge University United Kingdom e-m: jpcd0@mole.bio.cam.ac.uk From owner-methds-reagnts@net.bio.net Sat Jun 01 23:00:00 1996 Path: biosci!LSUVM.SNCC.LSU.EDU!JCUROLE From: JCUROLE@LSUVM.SNCC.LSU.EDU (Jason Curole) Newsgroups: bionet.molbio.methds-reagnts Subject: Cloning Vectors Date: 2 Jun 1996 17:53:41 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 25 Sender: daemon@net.bio.net Distribution: world Message-ID: <960602.195005.CDT.JCUROLE@LSUVM.SNCC.LSU.EDU> NNTP-Posting-Host: net.bio.net To all: We are interested in setting up TA Cloning in our laboratory. I have seen the kit from Invitrogen (and others), but would prefer to buy the components separately and make my own T-vector (sans. Marchuk et. al. Nucleic Acids Research 19:1154). My first question is: Does anyone out there have any experience with making T-vectors this way? Marchuk et. al. make the procedure sound rather simple and claim to have great sucess with it (9 of 9 PCR products were successfully cloned). Is it really this easy? Secondly, Marchuck et. al. used Stratagene's Bluescript vector, but claim any vector with a unique blunt end restriction site will work. Is there any reason to use Stratagene's vectors over, say, Promega or Gibco's vectors? If there is an FAQ or any previous discussion of this topic on the net please let me know. Feel free to e-mail me directly: jcurole@lsuvm.sncc.lsu.edu Thank you JASON P. CUROLE DEPARTMENT OF ZOOLOGY AND PHYSIOLOGY LOUISIANA STATE UNIVERSITY BATON ROUGE LA 70803-1725 From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!news.cse.psu.edu!news.cc.swarthmore.edu!netnews.upenn.edu!cronkite.ocis.temple.edu!astro.ocis.temple.edu!waring From: waring@astro.ocis.temple.edu (Richard Waring) Newsgroups: bionet.molbio.methds-reagnts Subject: expressing truncated proteins Date: 3 Jun 1996 16:33:22 GMT Organization: Temple University, Academic Computer Services Lines: 5 Message-ID: <4ov44i$6eu@cronkite.ocis.temple.edu> NNTP-Posting-Host: astro.ocis.temple.edu X-Newsreader: TIN [version 1.2 PL2] -- Best wishes Richard Waring From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!netnews.upenn.edu!cronkite.ocis.temple.edu!astro.ocis.temple.edu!waring From: waring@astro.ocis.temple.edu (Richard Waring) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: expressing truncated proteins Date: 3 Jun 1996 16:49:27 GMT Organization: Temple University, Academic Computer Services Lines: 9 Message-ID: <4ov52n$6eu@cronkite.ocis.temple.edu> References: <4ov44i$6eu@cronkite.ocis.temple.edu> NNTP-Posting-Host: astro.ocis.temple.edu X-Newsreader: TIN [version 1.2 PL2] We need to express truncated versions of an already small protein (137aa) In E. coli and have read that it is better to fuse them to another protein like DHFR. Does anyone know of any references or have any experience in trying this? -- Best wishes Richard Waring From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!swrinde!tank.news.pipex.net!pipex!lade.news.pipex.net!pipex!tube.news.pipex.net!pipex!dish.news.pipex.net!pipex!pipex-sa.net!iafrica.com!und.ac.za!wabe.csir.co.za!csir.co.za!jburger From: jburger@csir.co.za (Johan Burger) Newsgroups: bionet.molbio.methds-reagnts Subject: Vancomycin resistance Date: Mon, 3 Jun 1996 16:29:57 LOCAL Organization: CSIR Foodtek Lines: 8 Message-ID: NNTP-Posting-Host: 146.64.120.62 X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #4] Can someone please help: The neomycin-phosphotransferase (NPT-II) gene confers resistance to a few antibiotics (e.g. neomycin, kanamycin, geneticin). Is vancomycin included in this list? If not, what gene is responsible for vancomycin resistance? Thanks very much Johan Burger From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!news.cse.psu.edu!news.math.psu.edu!chi-news.cic.net!newsfeed.internetmci.com!in2.uu.net!newsfeed.pitt.edu!newsflash.concordia.ca!news.mcgill.ca!news From: Eric Campeau Newsgroups: bionet.molbio.methds-reagnts Subject: inverse PCR on cDNA: any protocol available ? Date: 3 Jun 1996 15:09:53 GMT Organization: McGill University Computing Centre Lines: 9 Message-ID: <4ouv81$4ba@sifon.cc.mcgill.ca> NNTP-Posting-Host: g-01.das.mcgill.ca X-Newsreader: SPRY News 3.03 (SPRY, Inc.) Hi, I am currently trying to setup a protocol to do inverse PCRs at the cDNA level. We have already a protocol working for genomic DNA. I would like to know if anyone have tried inverse PCRs on cDNA, especially if it worked ! Thanks Eric Campeau graduate student McGill Universty e-mail: popa0272@po-box.mcgill.ca From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!caen!reeve.research.aa.wl.com!usenet From: Greg Roland Newsgroups: bionet.molbio.methds-reagnts Subject: ABI 377 sequencer Date: Mon, 03 Jun 1996 11:24:41 -0400 Organization: Parke-Davis Lines: 2 Message-ID: <31B303B9.AB6@wl.aa.com> NNTP-Posting-Host: etpc12.research.aa.wl.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Win16; I) Have a new machine want all short cuts and tips. Especially info regarding premade acryl gels. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!chi-news.cic.net!nntp.coast.net!news2.acs.oakland.edu!condor.ic.net!gate1.mika.com!news.cencom.net!NewsWatcher!user From: ajpchem@cencom.net (Protein Chemistry Facility) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: SEAP assay Date: Sun, 02 Jun 1996 18:00:55 -0400 Organization: WAJCSC Lines: 23 Message-ID: References: NNTP-Posting-Host: saranac_2nd_ppp_34.cencom.net In article , thompsop@burnet.mbcmr.unimelb.edu.au (Phillip R Thompson) wrote: > I am using Clontech's Great Escape SEAP Genetic reporter system & am > having a major problem with reproducibility. Some of my duplicates differ > by as much as 300%! The assays are being carried out essentially as > described in the manual & read in 96-well black plates in a Wallach plate > reader. I'm being very careful with pipetting & I don't believe pipetting > error could result in such large errors anyway. Any help would be > appreciated! > I don't know the details of the Clontech protocol, I have used one from Tropix Inc. . Killing the endogenous alkaline phosphatase by heating at 65 C is essential though. I do this directly in the 96 well plate in a hyb. oven. Hope this helps, Breandan Kennedy, Protein Chemistry Facility, WAJCSC, L. Placid, NY 12946 Ph 518-523-1268 fax 518-523-4970/1849 From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!ODYSSEE.NET!dellaire From: dellaire@ODYSSEE.NET (Graham Dellaire) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DEAE-dextran transfection/divalent cations Date: 3 Jun 1996 19:03:35 -0700 Organization: McGill Div. of Experimental Medicine Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <31B3967B.5A55@odyssee.net> NNTP-Posting-Host: net.bio.net Hello Nico, I transfect cells (murine fibroblasts) with DEAE-dextran and I don't worry about Ca or Mg ion content. It works fine... I found the mol weight of the DEAE-dextran you use has more of an effect. Try to get 100,000 or higher. G. Dellaire dellaire@odyssee.net From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!news.cse.psu.edu!news.cc.swarthmore.edu!netnews.upenn.edu!taurus.fccc.edu!thalis.rm.fccc.edu!user From: tsichlis@thalis.rm.fccc.edu (Tsichlis Lab) Newsgroups: bionet.molbio.methds-reagnts Subject: RDA (?) Followup-To: bionet.molbio.methds-reagnts Date: Mon, 03 Jun 1996 19:02:45 -0400 Organization: FCCC Lines: 14 Message-ID: NNTP-Posting-Host: thalis.rm.fccc.edu I would like some feedback from someone using RDA-representational display analysis to look for differences in RNA between two samples. Are there any new tricks to incorporate since the MCB Fli paper? -- Tsichlis Lab Fox Chase Chase Center 7701 Burholme Ave Phila, PA 19111 Phone: 215-728-3636 FAX: 215-728-2741 Tsichlis@thalis.rm.fccc.edu From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!uwm.edu!news.cse.psu.edu!news.math.psu.edu!news.cac.psu.edu!psuvm!txs25 Organization: Penn State University Date: Mon, 3 Jun 1996 14:24:11 EDT From: Toshio SAKAMOTO, Dr. Message-ID: <96155.142411TXS25@psuvm.psu.edu> Newsgroups: bionet.molbio.methds-reagnts Subject: HELP/glucose assay kit of Sigma #635 Lines: 14 Does anybody use glucose assay kit of Sigma #635 ? This is the kit for colorimetric assay of glucose using o-Toluidine. I have o-Toluidine Reagent, catalog #635-6, and I can make 3% TCA and glucose solutions. Unfortunately, I have no indications for the kit !! If you know the instruction of the kit, please tell me. I went to the library to see the original references, but I could not get it, because the references are too old or in Spanish. Thanks a lot in advance. Toshi Sakamoto FAX 814-863-7024 From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!tank.news.pipex.net!pipex!oleane!jussieu.fr!math.ohio-state.edu!uwm.edu!news.cse.psu.edu!news.cc.swarthmore.edu!netnews.upenn.edu!taurus.fccc.edu!thalis.rm.fccc.edu!user From: tsichlis@thalis.rm.fccc.edu (Tsichlis Lab) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: beta-galactosidase assays using microtitre plates Followup-To: bionet.molbio.methds-reagnts Date: Mon, 03 Jun 1996 18:53:40 -0400 Organization: FCCC Lines: 22 Message-ID: References: <4ofb5s$ci3@sun4.bham.ac.uk> NNTP-Posting-Host: thalis.rm.fccc.edu In article <4ofb5s$ci3@sun4.bham.ac.uk>, K.A.Barne@bham.ac.uk (Kerry Barne) wrote: > Does anybody have a protocol for assaying beta-galactosidase activity > in E. coli using microtitre plates rather than test-tubes. > We have used this protocol extensively and are very happy with it. Bignon, C., D. Daniel, and J. Djiane. 1993. b-Galactosidase and chloramphenicol acetyltransferase assays in 96-well plates. Biotechniques 15: 243-245. -- lee grimes Fox Chase Chase Center 7701 Burholme Ave Phila, PA 19111 Phone: 215-728-3636 FAX: 215-728-2741 L_grimes@fccc.edu From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!oleane!in2p3.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!News.Uni-Marburg.DE!nnex01.ppp.Uni-Marburg.DE!wagenkne From: wagenkne@mailer.Uni-Marburg.DE (Chr. Wagenknecht) Newsgroups: bionet.molbio.methds-reagnts Subject: HELP! T4-RNA Ligase Date: Tue, 4 Jun 1996 00:29:30 LOCAL Organization: Uni Marburg, Inst. f. Physiologische Chemie Lines: 7 Message-ID: NNTP-Posting-Host: nnex01.ppp.uni-marburg.de X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #4] Hi! Has anybody worked with T4-RNA Ligase before? I want to ligate an RNA-Linker to mRNA. Can You give me a protocol about it? Thanks in advance, Christian From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!STUDENTS.UIUC.EDU!iyu From: iyu@STUDENTS.UIUC.EDU (I-ching Yu) Newsgroups: bionet.molbio.methds-reagnts Subject: Quantify DNA Date: 3 Jun 1996 15:38:20 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <199606032234.RAA83474@ux7.cso.uiuc.edu> NNTP-Posting-Host: net.bio.net Hello, I am posting a question for a friend. My project is to look at the apoptosis or programmed cell death in the chicken ovary. One of the biochemical hallmark for apoptosis is the fragmentation of genomic DNA. Once the cells are destined to undergo apoptosis, a specific DNase which makes cut in between nucleosomes will be activated. If you run the isolated DNA from the apoptotic cells on an EB agrose gel, you will get a laddering pattern which the space between the bands is about the size of a nucleosome (160-200bp). In order to compare the degree of apoptosis among different treatment groups, I need to use the densitometry to quantify the level of DNA laddering. Unfortunately, there is no densitometry available in our building. Another problem is that I am not sure if densitometry can read black-white picture or a ethidium bromide gel. It will be highly appreciated if you can give me some info about it. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!in2.uu.net!taligent!ames!news.tulane.edu!news.starnet.net!newsreader.wustl.edu!biodec.wustl.edu!graham From: graham@biodec.wustl.edu (James Graham) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.molbio.recombination,bionet.molbio.yeast Subject: Re: WE NEED YOUR INPUT Date: 3 Jun 1996 16:51:09 -0500 Organization: Washington University Biology, St. Louis, MO Lines: 12 Message-ID: References: <4otll5$3ni$6@mhade.production.compuserve.com> NNTP-Posting-Host: @biodec.wustl.edu X-Newsreader: NN version 6.5.0 #1 (NOV) Xref: biosci bionet.molbio.methds-reagnts:45267 bionet.molbio.proteins:8029 bionet.molbio.recombination:195 bionet.molbio.yeast:5360 > To express interest, please send your name, mailing address, and > photocopies of **ALL** degrees you have earned to: Careful folks. Why would it be necessary to send actual copies of anything in order to merely "express interest" in a survey? (They're probalby building a directed marketing database from the information you send "expressing interest".) Jim J. Graham PhD Biology Department From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!chi-news.cic.net!nntp.coast.net!howland.reston.ans.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!in1.uu.net!world!news From: rrohan@world.std.com (Richard Rohan) Subject: Re: RNAse protection assays Message-ID: <31b2b8d5.1783799@news.std.com> Sender: news@world.std.com (Mr Usenet Himself) Nntp-Posting-Host: world.std.com Organization: The World @ Software Tool & Die X-Newsreader: Forte Agent .99d/16.182 References: <4on34p$d1g@mercury.hgmp.mrc.ac.uk> Date: Mon, 3 Jun 1996 12:23:52 GMT Lines: 17 We use the RPA protocol from Current Methods in Molecular Biology and have been able to detect 0.07 pg of a 400 nt target in 20 micrograms of total RNA. Probes are synthesized with 800 Ci/mmole UTP. Less than 20 micrograms of sample RNA can be used but we always add yeast tRNA as carrier to bring the total to 20. Contact me if you want more details. Rich Rohan tking@hgmp.mrc.ac.uk (Dr. T.F. King) wrote: >I need a good RNAse protection assay kit or protocol which is optimised >for minimal amounts of RNA. What is the minimal amount of total RNA anyone >has used to detect low messages in eukayotic cells >Thanks >Tim King From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!glaxo.com!lansing~tj From: lansing~tj@glaxo.com (Tim Lansing) Newsgroups: bionet.molbio.methds-reagnts Subject: (no subject) Date: 3 Jun 1996 05:38:18 -0700 Organization: Glaxo Wellcome Research and Development Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <9606031232.AA15493@ussunk.glaxo.com> Reply-To: lansing~tj@glaxo.com NNTP-Posting-Host: net.bio.net Does anyone have the sequence of the vector pRK-5 that they could send me? My E-mail address is lansing~tl@glaxo.com. Many thanks in advance! Tim Lansing Glaxo Wellcome R & D From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!swrinde!tank.news.pipex.net!pipex!lade.news.pipex.net!pipex!newshost.zeneca.co.uk!usenet From: John Brennand Newsgroups: bionet.molbio.methds-reagnts Subject: Re: supplier for human serum Date: 3 Jun 1996 12:05:02 GMT Organization: zeneca.co.uk Lines: 11 Message-ID: <4oukde$k0l@mailhost.zeneca.co.uk> References: <4ouk1r$dq4@rigel.rz.uni-ulm.de> NNTP-Posting-Host: 156.71.144.158 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: sabine.hanelt@medizin.uni-ulm.de X-URL: news:4ouk1r$dq4@rigel.rz.uni-ulm.de Sabine There are literally dozens of suppliers of human serum in all its formulation (even Sigma). Check Linscott's directory page 189 for full listing (contact them in Sweden on Fax: 46-23-39232) John From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!lhc.nlm.nih.gov!nih-csl!postman From: bernard@elsie.nci.nih.gov (Bernard Murray) Subject: T7 RNA polymerase termination signals? Message-ID: <1996Jun4.050619.20308@alw.nih.gov> Sender: postman@alw.nih.gov (AMDS Postmaster) Nntp-Posting-Host: ermintrude.nci.nih.gov Organization: National Institutes of Health, Bethesda, MD X-Newsreader: WinVN 0.99.7 References: <4oqlfi$8iu@dfw-ixnews9.ix.netcom.com> Date: Tue, 4 Jun 1996 05:06:19 GMT Lines: 33 Bottom line: What are the sequences of phage polymerase termination signals? Yes, I am still trying to get good in vitro transcription of a particular sequence for use in RNase protection analysis. Basically I can only really use one region and this is next to a T7 promoter so I am fiddling with the conditions. The transcript I get is mainly full length but the yield is awful. I tried 15degC for 3 h. It was worse then 37degC for 1 h. I tried the Maslak & Martin buffer and this may have improved things but the salt/detergent in the buffer screw up the gel purification of the probe (I am working on this). I tried beefing up the amount of enzyme or the amount of limiting nucleotide (32P-CTP) but this didn't really help. I have yet to try T4g32 protein or its equivalent. In the back of the Ambion MAXIscript guide their is a vague reference to accidental incorporation of phage RNA polymerase termination signals in a template being a reason for poor yield. No information is given as to what these sequences actually are. I am wondering if I have blundered into one of these at the T7 end of my transcript. Can some virally-enhanced person help a poor toxicologist and give me a pointer to what a termination signal for T7 polymerase (and SP6 and T3 if possible) look like? Much obliged for any pointers. Bernard Bernard Murray, Ph.D. bernard@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA) From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!aol.com!WSchick From: WSchick@aol.com Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Thermal cyclers for less than $3,000? Date: 3 Jun 1996 21:23:48 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <960604001944_548789970@emout16.mail.aol.com> NNTP-Posting-Host: net.bio.net In a message dated 96-06-03 14:29:52 EDT, mremingt@umabnet.ab.umd.edu (Mary P. Remington) writes: > >Are there any thermal cyclers on the market for less than $3,000? >Thanks, Mary Both Biometra and MJ Research have personal cyclers under $3000 in the U.S. They are probably more in other countries. Several other companies offer units very near this price. Walt Schick From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!math.ohio-state.edu!uwm.edu!post.its.mcw.edu!usenet From: Luanne Newsgroups: bionet.molbio.methds-reagnts Subject: Re: HELP! T4-RNA Ligase Date: Mon, 03 Jun 1996 22:44:30 -0500 Organization: Medical College of Wisconsin; Milwaukee Wisconsin Lines: 4 Message-ID: <31B3B11E.2F1C@post.its.mcw.edu> References: NNTP-Posting-Host: post.its.mcw.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0b3 (Win95; I) Sounds funky, can't you just make a plasmid w/ RNA pol prom and transcribe it? Luanne From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!howland.reston.ans.net!newsjunkie.ans.net!newsfeeds.ans.net!rcogate.rco.qc.ca!altitude!usenet From: Achim Recktenwald Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Acrylamide Gel setting too quickly -- IDEAS?? Date: Mon, 03 Jun 1996 10:00:51 -0500 Organization: IBEX Technologies, Inc. Lines: 17 Message-ID: <31B2FE23.6FEF@ibex.ca> References: <4oqlfi$8iu@dfw-ixnews9.ix.netcom.com> NNTP-Posting-Host: ibex.hip.cam.org Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; PPC) Brian MacNevin wrote: > > Well, when I mix up my acrylamide gel solution and begin pipetting it > between the glass plates for electrophoresis, I have found that it > starts to set before I can get it evenly dispersed in the plates. > My first thought is that this might be due to the temp of the > solution. It seems to me that I don't have this problem if the > solution is allowed to cool a little before adding the final solution. > I was wonderin if anyone else has had these problems and if it really > is due to the temp of the solution? > Thanks! > Brian > macnevin@ix.netcom.com How much TEMED are you using ? Try with half the amount. Achim From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!newsfeed.internetmci.com!info.ucla.edu!nnrp.info.ucla.edu!usenet From: Ed Castro Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA/RNA Stain Date: Mon, 03 Jun 1996 17:27:05 -0700 Organization: UCLA Lines: 1 Message-ID: <31B382D9.7A82@physci.ucla.edu> References: <4otp72$7sm@daily-planet.nodak.edu> Reply-To: ecastro@physci.ucla.edu NNTP-Posting-Host: henderson1.physci.ucla.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) To: Clay Comstock How about methylene blue staining? Super cheap From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!info.ucla.edu!nnrp.info.ucla.edu!usenet From: Ed Castro Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA/RNA Stain Date: Mon, 03 Jun 1996 17:26:57 -0700 Organization: UCLA Lines: 1 Message-ID: <31B382D1.3D3D@physci.ucla.edu> References: <4otp72$7sm@daily-planet.nodak.edu> Reply-To: ecastro@physci.ucla.edu NNTP-Posting-Host: henderson1.physci.ucla.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) To: Clay Comstock How about methylene blue staining? Super cheap From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!info.ucla.edu!nnrp.info.ucla.edu!usenet From: Ed Castro Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Acrylamide Gel setting too quickly -- IDEAS?? Date: Mon, 03 Jun 1996 17:22:36 -0700 Organization: UCLA Lines: 2 Message-ID: <31B381CC.7FB@physci.ucla.edu> References: <4oqlfi$8iu@dfw-ixnews9.ix.netcom.com> Reply-To: ecastro@physci.ucla.edu NNTP-Posting-Host: henderson1.physci.ucla.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) Temperature matters, try cooling mix prior to addition of APS and TEMED or reduce the amount of TEMED you are using. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!KAIS.KYOTO-U.AC.JP!kinoshit From: kinoshit@KAIS.KYOTO-U.AC.JP (masato Kinoshita) Newsgroups: bionet.molbio.methds-reagnts Subject: How many ug of RNA for differential display Date: 3 Jun 1996 19:46:54 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <9606040241.AA25025@kais.kais.kyoto-u.ac.jp> NNTP-Posting-Host: net.bio.net Hi, netter Please tell me how much RNA ( total RNA or mRNA) is required for differential display analysis. Any advice and information is available for me. Thank you. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!ODYSSEE.NET!dellaire From: dellaire@ODYSSEE.NET (Graham Dellaire) Newsgroups: bionet.molbio.methds-reagnts Subject: Microinjection of Murine ES cells... any experiences? What is involved? Date: 3 Jun 1996 19:13:28 -0700 Organization: McGill Div. of Experimental Medicine Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <31B398C4.6E5@odyssee.net> NNTP-Posting-Host: net.bio.net Hello All, I am thinking of trying microinjection of mouse ES cells and I was wondering what is involved as far as equipement (including rough pricing??) and time. I have seen papers where they inject several thousand cells (many a paper by Capecchi) and I was wondering how difficult this is to do. We routinely inject pronuclei in mouse oocytes. Is it much more involved than this? Any and all hints, suggestions are appreciated G. Dellaire McGill dept of Medicine Div. of Experimental Medicine. dellaire@odyssee.net From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!fconvx.ncifcrf.gov!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.ums.edu!umabnet.ab.umd.edu!umabnet.ab.umd.edu!mremingt From: "Mary P. Remington" Newsgroups: bionet.molbio.methds-reagnts Subject: Thermal cyclers for less than $3,000? Date: Mon, 3 Jun 1996 12:03:48 -0400 Organization: University of Maryland at Baltimore Lines: 3 Message-ID: NNTP-Posting-Host: umabnet.ab.umd.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Are there any thermal cyclers on the market for less than $3,000? Thanks, Mary From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.ageing,bionet.molbio.bio-matrix,bionet.molbio.evolution,bionet.molbio.gene-linkage,bionet.molbio.gene-org,bionet.molbio.methds-reagnts Path: biosci!daresbury!yama.mcc.ac.uk!bofh.dot!thor.cf.ac.uk!news From: Nick Jacobsen Subject: Re: DNA ligation help Sender: news@cf.ac.uk (USENET News System) Message-ID: Date: Mon, 3 Jun 1996 09:20:40 GMT To: xcl@med.unc.edu X-Nntp-Posting-Host: d053.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii References: <4opneu$nke@newz.oit.unc.edu> Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics Cardiff Lines: 14 Xref: biosci bionet.molbio.ageing:2742 bionet.molbio.bio-matrix:730 bionet.molbio.evolution:4574 bionet.molbio.gene-linkage:1078 bionet.molbio.methds-reagnts:45234 Hi, I find that the majority of problems with DNA ligation inefficiency are due to ATP degradation in the reaction buffer. If in doubt order new buffer and freeze in small aliquots. You can also order Pharmacia ligase - you have to order separate 10mM ATP with it to add to the reaction as required. You have to be fairly accurate with the amount of ATP added as too much is as bad as too little. I hope this is of help to you cheers Nick Jacobsen. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.ageing,bionet.molbio.bio-matrix,bionet.molbio.evolution,bionet.molbio.gene-linkage,bionet.molbio.gene-org,bionet.molbio.methds-reagnts Path: biosci!daresbury!yama.mcc.ac.uk!bofh.dot!thor.cf.ac.uk!news From: Nick Jacobsen Subject: Re: DNA ligation help Sender: news@cf.ac.uk (USENET News System) Message-ID: Date: Mon, 3 Jun 1996 09:20:12 GMT To: xcl@med.unc.edu X-Nntp-Posting-Host: d053.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii References: <4opneu$nke@newz.oit.unc.edu> Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics Cardiff Lines: 14 Xref: biosci bionet.molbio.ageing:2741 bionet.molbio.bio-matrix:729 bionet.molbio.evolution:4573 bionet.molbio.gene-linkage:1077 bionet.molbio.methds-reagnts:45233 Hi, I find that the majority of problems with DNA ligation inefficiency are due to ATP degradation in the reaction buffer. If in doubt order new buffer and freeze in small aliquots. You can also order Pharmacia ligase - you have to order separate 10mM ATP with it to add to the reaction as required. You have to be fairly accurate with the amount of ATP added as too much is as bad as too little. I hope this is of help to you cheers Nick Jacobsen. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.ageing,bionet.molbio.bio-matrix,bionet.molbio.evolution,bionet.molbio.gene-linkage,bionet.molbio.gene-org,bionet.molbio.methds-reagnts Path: biosci!daresbury!yama.mcc.ac.uk!bofh.dot!thor.cf.ac.uk!news From: Nick Jacobsen Subject: Re: DNA ligation help Sender: news@cf.ac.uk (USENET News System) Message-ID: Date: Mon, 3 Jun 1996 09:19:49 GMT To: xcl@med.unc.edu X-Nntp-Posting-Host: d053.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii References: <4opneu$nke@newz.oit.unc.edu> Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics Cardiff Lines: 14 Xref: biosci bionet.molbio.ageing:2740 bionet.molbio.bio-matrix:728 bionet.molbio.evolution:4572 bionet.molbio.gene-linkage:1076 bionet.molbio.methds-reagnts:45232 Hi, I find that the majority of problems with DNA ligation inefficiency are due to ATP degradation in the reaction buffer. If in doubt order new buffer and freeze in small aliquots. You can also order Pharmacia ligase - you have to order separate 10mM ATP with it to add to the reaction as required. You have to be fairly accurate with the amount of ATP added as too much is as bad as too little. I hope this is of help to you cheers Nick Jacobsen. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!in2.uu.net!tank.news.pipex.net!pipex!oleane!in2p3.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.uni-kl.de!rz.uni-karlsruhe.de!news.urz.uni-heidelberg.de!usenet From: Rico Laage Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Acrylamide Gel setting too quickly -- IDEAS?? Date: Mon, 03 Jun 1996 10:31:30 +0200 Organization: Neurobiology, Uni. Heidelberg Lines: 28 Message-ID: <31B2A2E2.23F0@urz.uni-heidelberg.de> References: <4oqlfi$8iu@dfw-ixnews9.ix.netcom.com> Reply-To: rico.laage@urz.uni-heidelberg.de NNTP-Posting-Host: mac222.nbio.uni-heidelberg.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; PPC) solution. > > I was wonderin if anyone else has had these problems and if it really > > is due to the temp of the solution? > > Thanks! > > Brian > > Dear Brian, > Yes, it is definitely the temperature. Acrylamide gels always take > longer to set on cold days. Try chilling the acrylamide mix before adding the > TEMED and APS until it feels cool to touch. Also, don't use high heat to > Michelle > michelle.gleeson@uts.edu.au yes temp is important, but I would suggest to reduce the Temed concentration, because this catalizes the reaction and as far as I know determines the speed of polymerization. try it ! Rico -- ********************************************************** * Neurobiology Tel. +49 (6221) 54-4863 * * Uni Heidelberg FAX 54-4496 * * Im Neuenheimer Feld 364 * * Germany * * World-Wide-Web: * * http://server.nbio.uni-heidelberg.de/Neurobiology.html * ********************************************************** From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!math.ohio-state.edu!jussieu.fr!oleane!hole.news.pipex.net!pipex!weld.news.pipex.net!pipex!plug.news.pipex.net!pipex!tank.news.pipex.net!pipex!blackbush.xlink.net!news.fhg.de!usenet From: seegert@ita.fhg.de (Dirk Seegert) Newsgroups: bionet.molbio.methds-reagnts Subject: Murine T-cell line responsive to IL-12? Date: Mon, 03 Jun 1996 08:38:31 GMT Organization: Fraunhofer Gesellschaft Lines: 17 Message-ID: <4ou896$a7g@news.fhg.de> NNTP-Posting-Host: 153.96.224.59 X-Newsreader: Forte Free Agent 1.0.82 Hi there, does anybody know (or have) a murine T-cell line which is highly responsive to IL-12 signals, especially by producing IFNg in a high range? Additionally preparation of these cell line from Balb/c or/and C3H mice would be great. Thanks in advance Dirk Seegert, Ph.D. Fraunhofer Institute Hannover Nikolai-Fuchs-Straße 1 30625 Hannover e-mail: seegert@ita.fhg.de Tel.: +49 511 5350 255 From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!DOGSURI.HOSEO.AC.KR!hbkim From: hbkim@DOGSURI.HOSEO.AC.KR (Han Bok Kim) Newsgroups: bionet.molbio.methds-reagnts Subject: [Q] mechanism of formation of pancreatic tail cancer? Date: 3 Jun 1996 01:04:22 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <199606030739.QAA00998@biochem.kaist.ac.kr> NNTP-Posting-Host: net.bio.net I have a cancer patient who is suspected and diagnosed to have hepatic and splenic metastasis from pancreatic tail cancer. They say most of pancreatic cancer is from its head not tail. For the patient of 70, operation is impossible, and even chemo- therapy is also not recommended. Is there any possible treatment in this case? Does any molecular biologists of pancreatic cancer have information about this and effective treatment for this case? I need your help, thank you in advance! From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!sgigate.sgi.com!sdd.hp.com!night.primate.wisc.edu!ames!news.tulane.edu!news.starnet.net!newsreader.wustl.edu!biodec.wustl.edu!graham From: graham@biodec.wustl.edu (James Graham) Newsgroups: bionet.molbio.methds-reagnts,bionet.microbiology Subject: Persistant phenotypic differences (serial subculture)? Date: 3 Jun 1996 16:35:43 -0500 Organization: Washington University Biology, St. Louis, MO Lines: 44 Message-ID: NNTP-Posting-Host: @biodec.wustl.edu X-Newsreader: NN version 6.5.0 #1 (NOV) Xref: biosci bionet.molbio.methds-reagnts:45266 bionet.microbiology:6256 Hello folks. Here's an interesting problem which I've yet to hear any good ideas on, but one which has become increasingly evident in my work, yet is apprarently largely unrecognized in the area of my research. When one grows several broth batch cultures of a single bacterial strain inoculated from say an saturated overnight culture, a single colony, or from a frozen glycerol stock, one will notice a variety of differences between the two cultures, at least in terms of iducability of promoters, or perhaps the transformation efficiency of subsequently prepared calcium competent cells. Rumor has it that a two-dimensional PAGE analysis of identically cultured cellls prepared from two cultures originating from slightly different inocula will show considerable differences, far beyond that which could be attributed to differences among any nondividing cells originally introduced. Personally, I have noticed some time ago that Staphylococcus aureus cultures grown in a defined medium with 0.5 M NaCl frequently (but not always) grow in visible clumps to a greater extent than when grown at higher or lower salt concentrations. More importantly for this discussion, those cultures which show the clumped or "smooth" growth characterisitc will continue to show such growth in multiple subsequent serial batch subcultures, as if some kind of "founder effect" could be propagated under otherwise identical conditions. Is there any evidence for such a "heritable" change in gene expression in bacterial cells growing in batch cultures? If so, how many such serial subcultures would it take to obtain two identical patterns of gene expression among two cultures of the same strain taken from two different inocula? What is the nature of the propagation of the phenotypic differences in these cases? Do the intial inocula perhaps conditionthe culture media differently? Do regualtory cascades perpetuate different types of gene expression among the progeny of differently cultured cells growing under identical conditions? If so, through how many batch subculturings can such effects persist? Any refereneces to such discussion or experiments most appreciated. Thanks. Please send me a copy of any responses directly. Jim J. Graham PhD Biology Department Washington University of St. Louis From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!aol.com!BADIDION From: BADIDION@aol.com Newsgroups: bionet.molbio.methds-reagnts Subject: culture society disc group Date: 3 Jun 1996 14:43:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <960603173858_209628838@emout14.mail.aol.com> NNTP-Posting-Host: net.bio.net Does anyone know the email address for the soc.culture.nordic? thanks From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!ns1.faseb.org!lamarck.sura.net!fconvx.ncifcrf.gov!cockleberry!pnh From: pnh@cockleberry.ncifcrf.gov (Paul N Hengen) Subject: Re: TKO miniflourometer (long) Message-ID: Sender: usenet@ncifcrf.gov (C News) Nntp-Posting-Host: cockleberry.ncifcrf.gov Organization: Frederick Biomedical Supercomputer Center X-Newsreader: TIN [version 1.2 PL2] References: <4nvdne$o4c@newz.oit.unc.edu> <4opn1m$g5l@elna.ethz.ch> Date: Mon, 3 Jun 1996 21:17:14 GMT Lines: 19 Alexander Kraev (kraev@bc.biol.ethz.ch) wrote: : I absolutely subscribe to what Philip said! And this article really : belongs to the FAQ list! (Paul, are you listening?) Yeah, I'm here! :-) I'll put that in the FAQ list. Is there anyone here who has used their TKO mini to detect strand separation using 2-aminopurine? -- ******************************************************************************* * 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 -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - - * * - TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - - * * - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/ * ******************************************************************************* From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!chi-news.cic.net!newsfeed.internetmci.com!tank.news.pipex.net!pipex!lade.news.pipex.net!pipex!newshost.zeneca.co.uk!usenet From: John Brennand Newsgroups: bionet.molbio.methds-reagnts Subject: Re: how many bases does an RE cover? Date: 3 Jun 1996 11:56:49 GMT Organization: zeneca.co.uk Lines: 11 Message-ID: <4ouju1$k0l@mailhost.zeneca.co.uk> References: NNTP-Posting-Host: 156.71.144.158 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: G X-URL: news:Ds6BBx.KAx@info.swan.ac.uk Gareth It varies. The New England Biolabs catalogue (p 238-9) is a good place to start They are at http://www.neb.com John From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!chi-news.cic.net!nntp.coast.net!howland.reston.ans.net!vixen.cso.uiuc.edu!uchinews!news From: Richard Moldwin Subject: Re: Vancomycin resistance X-Nntp-Posting-Host: rmoldwin.bsd.uchicago.edu Content-Type: text/plain; charset=us-ascii Message-ID: <31B2D593.572E@midway.uchicago.edu> Sender: news@midway.uchicago.edu (News Administrator) Content-Transfer-Encoding: 7bit Organization: U. of Chicago Hospitals References: Mime-Version: 1.0 Date: Mon, 3 Jun 1996 12:07:47 GMT X-Mailer: Mozilla 2.0 (WinNT; I) Lines: 25 Johan Burger wrote: > > Can someone please help: > > The neomycin-phospho-transferase (NPT II) gene confers resistance to Kanamycin > and Neomycin. Does it also confers resistance to Vancomycin?? If not, > which gene does? > > Thanks > Johan Burger Neomycin, Kanamycin, and G418 are all aminoglycoside antibiotics. The Neo(R) gene confers resistance to the whole class. Vancomycin is a complex glycopeptide antibiotic unrelated to the aminoglycosides. Since those of us who worry about antibiotic resistance view Vanco as the last line we have against a growing list of pathogenic organisms, I would suggest that you avoid using any kind of Vanco-resistance selection system. A discussion with a clinical microbiologist or Infectious disease specialist might be useful. Why do you want to use Vanco anyway? --Rich From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.reston.ans.net!math.ohio-state.edu!jussieu.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!news.uni-ulm.de!sponti From: sabine.hanelt@medizin.uni-ulm.de (Sabine Hanelt) Newsgroups: bionet.molbio.methds-reagnts Subject: supplier for human serum Date: Mon, 03 Jun 96 11:58:59 GMT Organization: University of Ulm, Germany Lines: 6 Message-ID: <4ouk1r$dq4@rigel.rz.uni-ulm.de> NNTP-Posting-Host: sponti.medizin.uni-ulm.de X-Newsreader: News Xpress Version 1.0 Beta #4 Hey netters, does anybody know a supplier for human serum. We have heared about a supplier named biobee, but we don't know the address. Who knows either a source for human serum or the address of biobee??? Thank's for your help Sabine From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!chi-news.cic.net!nntp.coast.net!oleane!in2p3.fr!swidir.switch.ch!scsing.switch.ch!news.belwue.de!fu-berlin.de!cs.tu-berlin.de!uni-erlangen.de!news.th-darmstadt.de!news From: joerg hauf Newsgroups: bionet.molbio.methds-reagnts Subject: repetitive dna Date: 3 Jun 1996 11:17:45 GMT Organization: Technische Hochschule Darmstadt Lines: 10 Message-ID: <4ouhkp$iku@rs18.hrz.th-darmstadt.de> NNTP-Posting-Host: fkz1.bio.th-darmstadt.de Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.2N (Windows; I; 16bit) Hello everybody, is there anyone experienced in cloning repetitive DNA? I've tried to clone tandemly repeated DNA in E. coli strains Sure and DH5alpha several times, but neither of them contained the desired plasmid. The Sure strain produced no clones at all, DH5alpha produced only plasmids with wrong insertions. My question is: Does anybody know of any other strain which may be used for that purpose? From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!in2p3.fr!swidir.switch.ch!swsbe6.switch.ch!news.belnet.be!news.rediris.es!power.ci.uv.es!usenet From: Inmaculada Garcia Robles Newsgroups: bionet.molbio.methds-reagnts Subject: NaBr gradients versus KBr Date: 3 Jun 1996 11:31:37 GMT Organization: Universitat de Valencia Lines: 7 Message-ID: <4ouiep$1h8k@power.ci.uv.es> NNTP-Posting-Host: biogen.geneti.uv.es Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Does anybody know if there is a great difference between perform a NaBr gradient (to separate certain proteins) or a KBr gradient? In fact I always read in all papers about NaBr ones but I wonder if it is possible to do it with KBr. Thanks From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!daresbury!yama.mcc.ac.uk!bofh.dot!news.salford.ac.uk!aber!not-for-mail From: jms93@aber.ac.uk (jms93) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Subcloing PCR Products Date: 3 Jun 1996 10:44:32 GMT Organization: University of Wales, Aberystwyth Lines: 42 Message-ID: <4oufmg$991@infoserv.aber.ac.uk> References: <4of7pm$m1c@abel.cc.sunysb.edu> NNTP-Posting-Host: pccbif.ccu.aber.ac.uk Mime-Version: 1.0 X-Disclaimer: Warning - Sender not verified X-Newsreader: WinVN 0.93.11 In article , bijgh@zeus.bris.ac.uk says... > >Minsoo Yoon (ms.yoon@auckland.ac.nz) wrote: > >: Why don't you try T-A cloning? >: You can either buy (from Promega or Invitro gene) or make your own t vector. >: I've been using t vector to clone my PCR products. It's much easier and >: more efficient than traditional blunt-end ligation. > >I'd have to agree with this and add that once you have your PCR safely in >the TA plasmid you can amplify up as much as you want, and when it comes >to restriction, you *know* that you have cut at both sites. > >Jared > >-- >Jared Head at the Department of Biochemistry, University of Bristol > > "If anybody wants to clap," said Eeyore when he had read this, > "now is the time to do it." I would agree with this too! PCR with non-proofreading enzymes such as Taq, Tfl etc., produce A overhangs which facilitate it's sub-cloning into a vector with T overhangs. We use the pGEM-T vector from Promega. First we band prep/purify our DNA products before sub-cloning our procedures which is a very wise thing to do. This way you are sure it is the band(s) of interest that you are sub-cloning. Routinely, we band purify 40 microlitres of a standard PCR reaction and get good yield back. We estimate the concentration of the DNA simply by band intensity on an ethidium bromide gel and then use the appropriate amount in the ligation reaction. Haapy cloning! Jonathan Shillingford jms93@aber.ac.uk From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!chi-news.cic.net!news.cais.net!usenet.seri.re.kr!bofh.dot!news.kaist.ac.kr!biochem.kaist.ac.kr!hbkim From: hbkim@biochem.kaist.ac.kr (Hanbok Kim) Newsgroups: bionet.molbio.methds-reagnts Subject: mechanism of formation of pancreatic tail cancer? Date: 3 Jun 1996 10:15:54 GMT Organization: Biochemistry Lab, KAIST Lines: 10 Message-ID: <4oue0q$sar@worak.kaist.ac.kr> NNTP-Posting-Host: biochem.kaist.ac.kr X-Newsreader: TIN [version 1.2 PL2] I have a patient who was diagnosed to liver and spleen cancer from pancreatic tail cancer. It is said that cancer in tail is rare, since most of cancer in pancreas occurs in head. Is there any molecular biologists of oncology who can explain this and give some information about effective treatments? Operation is impossible, and chemotherapy is also hard. Thank you in advance! hbkim@biochem.kaist.ac.kr From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!daresbury!yama.mcc.ac.uk!bofh.dot!thor.cf.ac.uk!news From: Nick Jacobsen Subject: Clontech epidermal cDNA libraries Sender: news@cf.ac.uk (USENET News System) Message-ID: Date: Mon, 3 Jun 1996 09:31:23 GMT X-Nntp-Posting-Host: d053.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics Cardiff Lines: 12 Hi there, Can anyone out there give me advice about Clontech human cDNA libraries. I am thinking of buying a human epidermal cDNA library (cat. no. HL1112b). I have heard various reports about the quality of some of Clontech's libraries. Has anyone ever used this one and if so is it worth the money, many thanks Nick Jacobsen From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.ageing,bionet.molbio.bio-matrix,bionet.molbio.evolution,bionet.molbio.gene-linkage,bionet.molbio.gene-org,bionet.molbio.methds-reagnts Path: biosci!daresbury!yama.mcc.ac.uk!bofh.dot!thor.cf.ac.uk!news From: Nick Jacobsen Subject: Re: DNA ligation help Sender: news@cf.ac.uk (USENET News System) Message-ID: Date: Mon, 3 Jun 1996 09:21:04 GMT To: xcl@med.unc.edu X-Nntp-Posting-Host: d053.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii References: <4opneu$nke@newz.oit.unc.edu> Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics Cardiff Lines: 14 Xref: biosci bionet.molbio.ageing:2744 bionet.molbio.bio-matrix:732 bionet.molbio.evolution:4576 bionet.molbio.gene-linkage:1080 bionet.molbio.methds-reagnts:45236 Hi, I find that the majority of problems with DNA ligation inefficiency are due to ATP degradation in the reaction buffer. If in doubt order new buffer and freeze in small aliquots. You can also order Pharmacia ligase - you have to order separate 10mM ATP with it to add to the reaction as required. You have to be fairly accurate with the amount of ATP added as too much is as bad as too little. I hope this is of help to you cheers Nick Jacobsen. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Newsgroups: bionet.molbio.ageing,bionet.molbio.bio-matrix,bionet.molbio.evolution,bionet.molbio.gene-linkage,bionet.molbio.gene-org,bionet.molbio.methds-reagnts Path: biosci!daresbury!yama.mcc.ac.uk!bofh.dot!thor.cf.ac.uk!news From: Nick Jacobsen Subject: Re: DNA ligation help Sender: news@cf.ac.uk (USENET News System) Message-ID: Date: Mon, 3 Jun 1996 09:20:58 GMT To: xcl@med.unc.edu X-Nntp-Posting-Host: d053.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii References: <4opneu$nke@newz.oit.unc.edu> Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics Cardiff Lines: 14 Xref: biosci bionet.molbio.ageing:2743 bionet.molbio.bio-matrix:731 bionet.molbio.evolution:4575 bionet.molbio.gene-linkage:1079 bionet.molbio.methds-reagnts:45235 Hi, I find that the majority of problems with DNA ligation inefficiency are due to ATP degradation in the reaction buffer. If in doubt order new buffer and freeze in small aliquots. You can also order Pharmacia ligase - you have to order separate 10mM ATP with it to add to the reaction as required. You have to be fairly accurate with the amount of ATP added as too much is as bad as too little. I hope this is of help to you cheers Nick Jacobsen. From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!news.dbtech.net!dialup1.mtwo.com!user From: forte@mtwo.com (Michael Forte) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: StratagenQuikChange Date: 3 Jun 1996 20:58:39 GMT Organization: db Technology Lines: 29 Message-ID: References: NNTP-Posting-Host: dialup1.mtwo.com In article , Tori wrote: > Has anyone used the Stratagene Quik Change protocol? I just started > fooling around with it and wanted to know if anyone had had any successes > or failures. In my most recent attempt I got lots and lots of colonies. I > have to do minipreps to see if they are real but at first glance it seems > as though I have too many positives. > > Post replys if you choose but also please e-mail me directly. > twilliam@biomed.med.yale.edu > > Thanks much, > Tori > > *************************************************************************** > Tori D. Williams > twilliam@biomed.med.yale.edu > > DST SK DK PHD2B SCI 0(+> LIVELONG... AGGIEPRIDE MAC = ME!!!! :) > *************************************************************************** My lab just switched to this systems and in our hands, it works very well. Lots of mutants that we were never able to make with pALTER were generated easily and abundantly with this system. It may not work quite as well as advertized, but it is the best we have found. Mike Forte From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!sdd.hp.com!hamblin.math.byu.edu!acs2.byu.edu!news.cuny.edu!msvax.mssm.edu!MING From: ming@msvax.mssm.edu Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.molbio.recombination,bionet.molbio.yeast Subject: Re: WE NEED YOUR INPUT Date: 3 Jun 1996 20:37:11 GMT Organization: Mount Sinai Medical Center, New York City Lines: 26 Message-ID: <4ovidn$pmh@news.cuny.edu> References: <4otll5$3ni$6@mhade.production.compuserve.com> Reply-To: ming@msvax.mssm.edu NNTP-Posting-Host: msvax.mssm.edu Xref: biosci bionet.molbio.methds-reagnts:45262 bionet.molbio.proteins:8027 bionet.molbio.recombination:194 bionet.molbio.yeast:5359 In article <4otll5$3ni$6@mhade.production.compuserve.com>, Survey Administration <74750.1341@CompuServe.COM> writes: >Please excuse this brief intrusion, but we are trying to locate >any and all holders of academic degrees conferred within the last >ten (10) years. > >The Kitty Knight Corporation, Boston, MA, is currently searching >for qualified individuals to participate in a ***PAID*** study >focusing on post-secondary, graduate, and professional education >in the United States. > >Holders of ALL types of degrees in ALL fields of study are needed. > We would like to hear from you as long as your degree was earned >at an accredited institution in the United States. > >After an initial screening, qualified participant will be asked to >complete a questionaire of approx 150 questions. Everyone who >completes the survey will receive a $100 stipend. Naturally, >*ALL* information will be held in the strictest confidence. > >To express interest, please send your name, mailing address, and >photocopies of **ALL** degrees you have earned to: The Kitty >Knight Corporation, Attn: Study 96-3H, Back Bay Annex, P O Box >546, Boston, MA 02117. (If not obvious from the degree, please >indicate the field of study.) > >Thank You Very Much! From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!news.eecs.umich.edu!newshub.tc.umn.edu!lynx.unm.edu!bubba.NMSU.Edu!dkim From: dkim@nmsu.edu (DANIEL Y KIM) Newsgroups: bionet.molbio.methds-reagnts Subject: Precipitating bactriophage Date: 3 Jun 1996 19:02:37 GMT Organization: New Mexico State University, Las Cruces, NM Lines: 21 Message-ID: <4ovcsd$75a@bubba.NMSU.Edu> NNTP-Posting-Host: verdi.nmsu.edu Hello: I have been trying to construct a filamentous phage display of a protein fused to M13 gene III. In my plasmid/phagemid, the gene III fusion is interrupted by an Amber stop codon. I imagine there is a fair amount of protein being produced which is not fused to gene III and which will be secreted into the growth medium, instead of being incorporated into a phage particle. Is there a way to precipitate the phage without the free protein? It seems that PEG will precipitate both phage and free protein (60 kDa) out of the medium (positive Western signal in the absence of helper phage). If not, is there a convenient way to filter out the larger phage from the free protein? Thanks Daniel Kim dkim@nmsu.edu From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!lll-winken.llnl.gov!fnnews.fnal.gov!unixhub!news.Stanford.EDU!not-for-mail From: ladasky@leland.Stanford.EDU (John Ladasky) Newsgroups: bionet.molbio.ageing,bionet.molbio.bio-matrix,bionet.molbio.evolution,bionet.molbio.gene-linkage,bionet.molbio.gene-org,bionet.molbio.methds-reagnts Subject: Re: DNA ligation help Followup-To: bionet.molbio.methds-reagnts Date: 3 Jun 1996 10:47:29 -0700 Organization: Stanford University, CA 94305, USA Lines: 23 Message-ID: <4ov8fh$7gm@elaine35.Stanford.EDU> References: <4opneu$nke@newz.oit.unc.edu> NNTP-Posting-Host: elaine35.stanford.edu Xref: biosci bionet.molbio.ageing:2745 bionet.molbio.bio-matrix:733 bionet.molbio.evolution:4577 bionet.molbio.gene-linkage:1081 bionet.molbio.methds-reagnts:45260 Followups have been limited to bionet.molbio.methds-reagnts. In article <4opneu$nke@newz.oit.unc.edu>, chunlin xin wrote: >Hi, there, I have trouble in DNA ligation, the case below: > Insert Fragment: 16.3kb SalI Frag. > Vector: Bscrip-KS(-) SalI cut plasmid or other > salI cut expression vector > It looks like no ligation problem, but in fact, I cannot >success to ligate them and transfer them into DH5a no matter I use >chemical or electroparation method, Have anybody ever meet the same problem? >How can I solve them? Any suggestion is appreciated! Thanks for you attention! A 16.3 Kb insertion is quite large. DH5-alpha, and most other normal strains of E. coli, cannot reliably maintain plasmids larger than 10 Kb. (I'm not sure why this is so.) So your ligation and transformation may both be successful, but then you are not getting any ampicillin-resistant colonies. Can you subclone this fragment? -- Unique ID : Ladasky, John Joseph Jr. Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???) Location : Stanford University, Dept. of Structural Biology, Fairchild D-105 Keywords : immunology, music, running, Green From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!chi-news.cic.net!newsxfer2.itd.umich.edu!tank.news.pipex.net!pipex!usenet.eel.ufl.edu!bofh.dot!warwick!lyra.csx.cam.ac.uk!news From: Paul Digard Newsgroups: bionet.molbio.methds-reagnts Subject: Re: expressing truncated proteins Date: Mon, 03 Jun 1996 18:57:41 +0000 Organization: University of Cambridge, England Lines: 25 Message-ID: <31B335A5.2212@mole.bio.cam.ac.uk> References: <4ov44i$6eu@cronkite.ocis.temple.edu> <4ov52n$6eu@cronkite.ocis.temple.edu> NNTP-Posting-Host: mac08.vrl.path.cam.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Macintosh; I; 68K) Richard Waring wrote: > > We need to express truncated versions of an already small protein (137aa) > In E. coli and have read that it is better to fuse them to another protein > like DHFR. > Does anyone know of any references or have any experience in trying this? > Thioredoxin fusions are meant to be good for this purpose. The reference I have is LaValle et al, Biotechnology 11, p187-193. I'm pretty sure the plasmids are commercially available. The address on the paper is Genetics Institute, 87 Cambridge Park Drive, Cambridge, MA 02140. I've never tried the system myself, and I should think it's probably much like every other Ecoli expression system; pot luck for eukaryotic proteins! Cheers Paul Digard Division of Virology Department of Pathology University of Cambridge From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!cs.utk.edu!gaia.ns.utk.edu!jcox2.utmem.edu!user From: sghosh@utmem1.utmem.edu (Sourav Ghosh) Newsgroups: bionet.molbio.methds-reagnts Subject: Mouse epithelial cell library in pJG4-5 Info Request. Date: 3 Jun 1996 18:57:05 GMT Organization: UT-Memphis Lines: 6 Message-ID: NNTP-Posting-Host: jcox2.utmem.edu Hi, I would appreciate any info on availibility of mouse cDNA libraries, preferably epithelial cell libraries, cloned in pJG4-5 vector (Dr. Brent's vector) for Yeast two-hybrid screens. Thanks. Regards, Sourav From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!rutgers!uwm.edu!newsspool.doit.wisc.edu!news.doit.wisc.edu!news From: mmgosink@facstaff.wisc.edu Newsgroups: bionet.molbio.methds-reagnts Subject: Nuclear translocation. How fast does it happen? Date: Mon, 03 Jun 1996 13:49:36 -0600 Organization: University of Wisconsin, Madison Lines: 18 Message-ID: <31B341D0.20C3@facstaff.wisc.edu> NNTP-Posting-Host: 128.104.66.136 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0b3Gold (Macintosh; I; PPC) Dear Netters: Does anyone know how fast protein translocation occurs in a cell. I just need an approximate value i.e. seconds, minutes, or hours... (I am primarily interested in translocation of a nuclear protein injected into the cytoplasm of frog oocytes.) I am co-injecting two proteins into the cytoplasm of a frog oocytes. They must interact for a period of time in order for me to see my expected phenotype. However, one of the pair contains what looks like a nuclear localization domain in addition a homolog of my protein IS nuclearly localized. I would like to know if localization occurs at such a high rate, that if my protein is translocated, would I ever see the expected phenotype. Thanks in advance, Mark From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!MAIL.MED.CORNELL.EDU!hmmoss From: hmmoss@MAIL.MED.CORNELL.EDU (Heidi Moss) Newsgroups: bionet.molbio.methds-reagnts Subject: immunoscope software Date: 3 Jun 1996 10:35:05 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 4 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hey netters! Does anyone know of the company that sells immunoscope software?? many thanks!!! --Heidi From owner-methds-reagnts@net.bio.net Sun Jun 02 23:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!fconvx.ncifcrf.gov!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.ums.edu!umabnet.ab.umd.edu!umabnet.ab.umd.edu!mremingt From: "Mary P. Remington" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Uni-directional deletions Date: Mon, 3 Jun 1996 12:07:45 -0400 Organization: University of Maryland at Baltimore Lines: 1 Message-ID: References: <31A52158.4317@ion.bpmf.ac.uk> NNTP-Posting-Host: umabnet.ab.umd.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: I've had great success with Stratagene's Exo/Mung Bean Deletion Kit. Mary From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!sgigate.sgi.com!nntp.coast.net!fu-berlin.de!cs.tu-berlin.de!uni-erlangen.de!rznews.rrze.uni-erlangen.de!news From: Heinz-Juergen Schaefers Newsgroups: bionet.molbio.methds-reagnts Subject: Re: (no subject) Date: Tue, 04 Jun 1996 11:46:04 -0700 Organization: University of Erlangen-Nuernberg, Med. IV, Nephrology Research Lab Lines: 30 Message-ID: <31B4846C.D0F@erlangen.netsurf.de> References: <9606031232.AA15493@ussunk.glaxo.com> NNTP-Posting-Host: 10.10.91.5 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Win16; I) Tim Lansing wrote: > > Does anyone have the sequence of the vector pRK-5 that they could send > me? My E-mail address is lansing~tl@glaxo.com. Many thanks in advance! > > Tim Lansing > Glaxo Wellcome R & D Hi! Goto: http://www.ebi.ac.uk/srs/wgetz select EMBL and EMBLNEW, deselect SWISSPROT type in: pRK-5 click on DO QUERY and you will get the sequence.... bye Heinz ----------------------------------------------------------- Dipl.Biol. Heinz-Juergen Schaefers E-mail: h-j.schaefers@erlangen.netsurf.de Friedrich-Alexander-Universitaet Erlangen-Nuernberg Medizinische Klinik IV Nephrologische Forschungslaboratorien Loschgestr. 8 D-91054 Erlangen Germany From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!tank.news.pipex.net!pipex!usenet1.news.uk.psi.net!uknet!EU.net!sun4nl!rivm!lwli281 From: Karel.Wernars@rivm.nl (Karel Wernars) Subject: Which bacterial species support pUC18/19 replication? Message-ID: <4p1s1r$10pk_001@lwli281.rivm.nl> Sender: news@rivm.nl Organization: RIVM X-Newsreader: News Xpress Version 1.0 Beta #4 Date: Tue, 4 Jun 1996 17:33:47 GMT Lines: 18 Dear netters, A few days ago I was asked by a colleague if I had any information on the hostrange of pUC18/19 plasmids. Can these replicate in bacteria other then E. coli?. To my idea various enterobacteriaceae can support replication e.g. Salmonella and Vibrio. Digging through literature didn't help me much to confirm this, and I only came across lots of reports on modifications of pUC18/19 to prepare shuttle vectors that would replicate in a pacticular bacterial host of interest. Can anyone help me with this question and are there any studies available on the host range of (unmodified) pUC18/19?. Thanks, Karel Wernars (Karel.Wernars@rivm.nl) From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!daresbury!lyra.csx.cam.ac.uk!rw200 From: rw200@cus.cam.ac.uk (R. Woodward) Newsgroups: bionet.molbio.methds-reagnts Subject: microsomes Date: 4 Jun 1996 11:36:26 GMT Organization: University of Cambridge, England Lines: 16 Message-ID: <4p173q$lko@lyra.csx.cam.ac.uk> NNTP-Posting-Host: ursa.cus.cam.ac.uk X-Newsreader: NN version 6.5.0 #2 (NOV) Dear All, Can anyone tell me the likely speed I would have to spin an invitro translation spin at in order to pellet microsomes. Also can I buy sources of microsomes from an origin other than canine? I saw some where that perpole us quale oviduct as the source of their microsomal prep. Robert R Woodward Email rw200@cus.cam.ac.uk oi From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!in2.uu.net!EU.net!Norway.EU.net!nntp.uio.no!nntp.uib.no!usenet From: Oivnd Enger Newsgroups: bionet.molbio.methds-reagnts Subject: Help: Alternative to Amersham/USB kit for DNA/RNA isolation Date: 4 Jun 1996 11:23:01 GMT Organization: University of Bergen, Norway Lines: 12 Message-ID: <4p16al$j2f@ugress.uib.no> NNTP-Posting-Host: brumle.im.uib.no Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 32bit) Hi there I've been using the Amersham/USB kit for simultaneous isolation of DNA and RNA from environmental bacteria. However, in my application the bottle with the sample buffer goes empty way long before the rest of the kit. Amersham on their side refuse to sell this buffer separately and also to give me the recipe. Could any of you please help me out by proposing an alternative kit or even better an alternative buffer. thanks in advance -oivind From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!IMPSAT1.COM.AR!ppadula From: ppadula@IMPSAT1.COM.AR Newsgroups: bionet.molbio.methds-reagnts Subject: (no subject) Date: 4 Jun 1996 03:40:02 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <31B3C29B.6F1F@impsat1.com.ar> NNTP-Posting-Host: net.bio.net Does anybody know why it is often difficult for me to make a PCR from a PCR product using exactly the same primers I use the first PCR, I mean like a nested but without changing primers?. I get cero band. From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!rutgers!uwm.edu!newsfeed.internetmci.com!newsxfer2.itd.umich.edu!tank.news.pipex.net!pipex!usenet1.news.uk.psi.net!uknet!EU.net!sun4nl!rivm!lwli281 From: Karel.Wernars@rivm.nl (Karel Wernars) Subject: Which bacterial species support pUC18/19 replication? Message-ID: <4p1jgh$lg8_001@lwli281.rivm.nl> Sender: news@rivm.nl Organization: RIVM, p.o.box 1, NL-3720 BA Bilthoven, the Netherlands X-Newsreader: News Xpress Version 1.0 Beta #4 Date: Tue, 4 Jun 1996 15:08:01 GMT Lines: 17 Dear netters, A few days ago I was asked by a colleague if I had any information on the hostrange of pUC18/19 plasmids. Can these replicate in bacteria other then E. coli?. To my idea various enterobacteriaceae can support replication e.g. Salmonella and Vibrio. Digging through literature didn't help me much to confirm this, and I only came across lots of reports on modifications of pUC18/19 to prepare shuttle vectors that would replicate in a pacticular bacterial host of interest. Can anyone help me with this question and are there any studies available on the host range of (unmodified) pUC18/19?. Thanks, Karel Wernars (Karel.Wernars@rivm.nl) From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Newsgroups: bionet.molbio.methds-reagnts Path: biosci!daresbury!yama.mcc.ac.uk!thor.cf.ac.uk!news From: Nick Jacobsen Subject: Re: pcr problem with gc rich sequences Sender: news@cf.ac.uk (USENET News System) Message-ID: Date: Tue, 4 Jun 1996 09:20:02 GMT X-Nntp-Posting-Host: d053.mg.cf.ac.uk Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii References: <4ot046$7d5@athena.ulaval.ca> Mime-Version: 1.0 X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Organization: Institute of Medical Genetics Cardiff Lines: 12 Hi, I had similar problems with a very GC rich sequence. I solved the problem by first designing primers with high Tms (about 80 C). I then used NEB Deep Vent exo- (not designed for PCR but works anyway). The reaction conditions were 98 C 5mins, (72 C 1min,98 C 1min)X30. This amplified the desired product from a gene that previously didn't want to know. hope this helps cheers nick From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!rutgers!uwm.edu!newsfeed.internetmci.com!howland.reston.ans.net!hole.news.pipex.net!pipex!tube.news.pipex.net!pipex!lade.news.pipex.net!pipex!newshost.zeneca.co.uk!usenet From: John Brennand Newsgroups: bionet.molbio.methds-reagnts Subject: Re: HELP Date: 4 Jun 1996 08:51:02 GMT Organization: zeneca.co.uk Lines: 11 Message-ID: <4p0tdm$m38@mailhost.zeneca.co.uk> References: <4p0q96$n3m@hermes.cair.du.edu> NNTP-Posting-Host: 156.71.144.158 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; PPC) To: tfurukaw@du.edu X-URL: news:4p0q96$n3m@hermes.cair.du.edu Tisha Buy, "Molecular Cloning - A Laboratory Manual", 2nd ed., eds Sambrook, Fritsch & Maniatis. Can order this via Sigma. It will help get your molecular approaches off on the right foot. john From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!rutgers!csn!nntp-xfer-1.csn.net!carbon!hermes.cair.du.edu!usenet From: Tisha Newsgroups: bionet.molbio.methds-reagnts Subject: HELP Date: 4 Jun 1996 07:57:26 GMT Organization: University of Denver Lines: 17 Message-ID: <4p0q96$n3m@hermes.cair.du.edu> NNTP-Posting-Host: jmpc5.mfh.du.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Windows; I; 16bit) I am hoping that there is someone that can point me in the right direction to finding the answers to the following questions: How might I go about substituting 32P-labelled adenine for cold adenine in a dsDNA? Gene X is purified as a cDNA. How, other than PCR, can I get enough of it to sequence, translate in vitro, etc...? Sea urchins secrete a protease similar to chymotrypsin, which is involved in hatching the blastula from its fertilization envelope. If an antibody to bovine chymotrypsin turns out to stop hatching, how could I use it to clone the gene for the sea urchin enzyme? Thank you in advance for anyone who might be able to point me in the direction to find the correct answers to these questions. From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uib.no!usenet From: Oivnd Enger Newsgroups: bionet.molbio.methds-reagnts Subject: Help: Alternative to Amersham/USB kit for DNA/RNA isolation Date: 4 Jun 1996 11:56:24 GMT Organization: University of Bergen, Norway Lines: 12 Message-ID: <4p1898$j2f@ugress.uib.no> NNTP-Posting-Host: brumle.im.uib.no Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 32bit) Hi there I've been using the Amersham/USB kit for simultaneous isolation of DNA and RNA from environmental bacteria. However, in my application the bottle with the sample buffer goes empty way long before the rest of the kit. Amersham on their side refuse to sell this buffer separately and also to give me the recipe. Could any of you please help me out by proposing an alternative kit or even better an alternative buffer. thanks in advance -oivind From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!newsfeed.internetmci.com!in2.uu.net!van-bc!unixg.ubc.ca!news From: John Brunstein Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Vertical Agarose gels, help Date: 4 Jun 1996 20:55:10 GMT Organization: The University of British Columbia Lines: 37 Message-ID: <4p27re$kci@nntp.ucs.ubc.ca> References: <4mpo5o$1f92@power.ci.uv.es> <4p1g70$gvi@mercury.hgmp.mrc.ac.uk> NNTP-Posting-Host: parvo.biochem.ubc.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: mlush@hgmp.mrc.ac.uk X-URL: news:4p1g70$gvi@mercury.hgmp.mrc.ac.uk OK :-)) Been there, seen those problems, and solved them! Here's how I handle them (this is for 1.5% agarose, 1% glycerol gels 1.5mm thick, about 20x20cm). (1) Slippage of the gel from between the plates (boy, is it ever a bummer to see your hard-won bandshift samples slide right out of the plates and into a sodden little lump an hour into the gel run!) Before pouring the gel, I run a bit of sequencing tape (the yellow electrical stuff) across the bottom of the gel just inside the spacer, in a sort of 'U' shape...(I'll attempt a bad ASCII art rendition below). Leave these on for the run, as the gel starts to slide it hits them and holds the gel in place..but since most of the bottom is unobstructed, current flow is not interfered with | | | | | | GEL | |<---- Spacer | | | | | |T T|<----- Tape runs down, around bottom, and | |T T| | partly up far side ------------------- (2) Removal of comb. Another really frustrating problem...but easily solved with a bit of practice. The problem is that as you remove the comb, a vacuum is created in the well and the gel collapses into it, causing tears. Solution: I use a thin spatula to pry at the glas plates as I ease the comb out little by little...you'll have to move the spatula around a bit to ensure that all wells get air into them as you work the comb out, so don't rush it...but after a few wrecked gels you'll get the hang of it. Hope these answers solve your problems, they took me a few weeks of trial and error to settle on as the final system. I use these gels routinely for bandshifts (my DNA is too large for acrylamide systems), and I no longer have any problems with preparing them. There is a bit of a problem with getting the wells really clean, bits of agarose tend to stay stick in there despite my best attempts to cut them out, and these can make the results messier than one might like..but the sytem works. From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!daresbury!lyra.csx.cam.ac.uk!drm21 From: drm21@mole.bio.cam.ac.uk (David Micklem) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA ligation help Date: Tue, 04 Jun 1996 21:49:36 +0000 Organization: Wellcome/CRC Institute Lines: 38 Message-ID: References: <4oq7qd$skl@newz.oit.unc.edu> <31B43D9D.2E3C@ukcc.uky.edu> NNTP-Posting-Host: drm-mac1.welc.cam.ac.uk X-Newsreader: Yet Another NewsWatcher 2.2.0b6 In article <31B43D9D.2E3C@ukcc.uky.edu>, kostya levay wrote: >Here are my 2 cents: > >Once I had very bad experience in cloning 12kb insert into Bluescript >with transformation into DH5a. Actually, all of purified plasmids >contained deletion of different size. Then I realised that the >products of leaking promotors? could be harmful for bacteria and lead >me into troubles. Interestingly, that DH5a doesn't have "lacIq" in its >genotype, i.e. overproduction of lac repressor protein, inhibiting >transcription from the lac promoter. Many other cells do have lacIq. >I transformed my ligation into JM101, got my construct from the first >hit and since then DH5a is on my black list (when I have tricky >situations). > Another trick for cloning large constructs is to use a low copy number plasmid. It may be that in some cases the deletion problem is not due to toxic leaking promotors but to the load on the bacteria of producing _huge_ quantities of a large plasmid. A plasmid that is maintained at only a few copies per cell is tolerated better - although its more of a hassle to get LOTS of DNA for subsequent steps. (But then how much do you actually need...) Good luck, David _____________________________________________________________ D.R.Micklem, Wellcome/CRC Institute, Time flies like an arrow... Tennis Court Road, Cambridge CB2 1QR Fruit flies like a banana. UK Tel: [+44] (0)1223 334129 Email:drm21@mole.bio.cam.ac.uk Fax: [+44] (0)1223 334089 _____________________________________________________________ From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!UACHIH.UACHNET.MX!jibave From: jibave@UACHIH.UACHNET.MX ("J. Ibave") Newsgroups: bionet.molbio.methds-reagnts Subject: Help about computers applied to Biology Date: 4 Jun 1996 13:51:47 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <64315.jibave@uachih.uachnet.mx> Reply-To: NNTP-Posting-Host: net.bio.net Hello, I'm working on developing an algorithim to determine the DNA sequence of a natural polypeptide based on a polypeptide which has suffered a mutation. I'm analyzing this kind of situations and can't figure out an efficient method to determine which was the mutation. (insertion, deletion or a change of nucleotides). If you are working on something similar, I will appreciate your help. Thanks a lot. Luis Arturo Medrano Soto Dr. Jose L. Ibave Facultad de Ciencias Agrotecnologicas P.O. Box 24 University of Chihuahua Chihuahua, Chih. Mexico 31000 tel. (voice) 0152+14+146615 (fax) 0152+14+134888 From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!newsfeed.internetmci.com!in1.uu.net!news.tufts.edu!opal.tufts.edu!kmorris1 From: kmorris1@opal.tufts.edu (KELLY THOME) Newsgroups: bionet.molbio.methds-reagnts Subject: Cell cycle analysis by FACS Date: 4 Jun 96 15:49:56 -0500 Organization: Tufts University - Medford, MA Lines: 14 Distribution: world Message-ID: <1996Jun4.154956@opal.tufts.edu> NNTP-Posting-Host: opal.tufts.edu I've done cell cycle analysis on a FACS before, but always with fresh cells. Now I'm setting up a time course (block HeLa cells with nocodazole 24 hours, release, 4 hour timepoints) and along with RNA and protein isolation I want to do cell cycle analysis. How do I prepare/store the cells at each time point so that I can do the analysis at a later time? This is sort of a last-minute addition to this experiment and I won't be able to get FACS time for at least a week or two (I hate crowded machines :-). Do I just freeze the cell pellet? If someone has experience with this sort of thing, I'd appreciate a protocol or some suggestions. Thank you very much! Kelly Thome Ph.D. kmorris1@opal.tufts.edu Pathology Department fax: 617-732-7449 Brigham and Women's Hospital Boston, MA 02115 From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!metro!metro!news From: simon Newsgroups: bionet.molbio.methds-reagnts Subject: Source of TGF-beta1 Date: Tue, 04 Jun 1996 10:36:35 +0000 Organization: Dept. of Biochem., Univ of Sydney Lines: 19 Distribution: inet Message-ID: <31B411B3.2A15@biochem.usyd.edu.au> NNTP-Posting-Host: sbepb.biochem.usyd.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) We are planning a series of experiments using TGF-beta1 and have been startled by the horrendous cost of it as a commercial product. Does anyone have a suitable recombinant source of it that they would be prepared to share? Thanks Simon -- _______________________________________________________________ Dr Simon B Easterbrook-Smith Phone: (+) 612 351 3905 Department of Biochemistry FAX: (+) 612 351 4726 University of Sydney Email: sbe@biochem.usyd.edu.au Sydney NSW 2006 AUSTRALIA _______________________________________________________________ Every complex question has a simple, straight-forward, easy to understand, wrong, answer. _______________________________________________________________ From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!sdd.hp.com!swrinde!newsfeed.internetmci.com!info.ucla.edu!nnrp.info.ucla.edu!wlheye.jsei.ucla.edu!hassane From: hassane@wlheye.jsei.ucla.edu (hassane mchaourab) Newsgroups: bionet.molbio.methds-reagnts Subject: pegasus Date: 4 Jun 1996 18:43:09 GMT Organization: Jules Stein Eye Institute, UCLA Lines: 9 Message-ID: <4p203t$ofc@uni.library.ucla.edu> NNTP-Posting-Host: wlheye.jsei.ucla.edu Hi, I am trying to buy an old RC-5 centrifuge. There is a company pegasus scientific that sells them refurbished for 6000$ with a one year labor and parts warranty. Anybody had any experience with there products, particularly, the centrifuges? Or is there other companies with competitive prices ? From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!newsfeed.internetmci.com!news.campus.mci.net!news.uky.edu!usenet From: kostya levay Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA ligation help Date: Tue, 04 Jun 1996 13:43:57 +0000 Organization: University of Kentucky, Lexington Lines: 18 Message-ID: <31B43D9D.2E3C@ukcc.uky.edu> References: <4oq7qd$skl@newz.oit.unc.edu> NNTP-Posting-Host: 128.163.192.145 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.01 (Macintosh; I; 68K) To: chunlin xin Here are my 2 cents: Once I had very bad experience in cloning 12kb insert into Bluescript with transformation into DH5a. Actually, all of purified plasmids contained deletion of different size. Then I realised that the products of leaking promotors? could be harmful for bacteria and lead me into troubles. Interestingly, that DH5a doesn't have "lacIq" in its genotype, i.e. overproduction of lac repressor protein, inhibiting transcription from the lac promoter. Many other cells do have lacIq. I transformed my ligation into JM101, got my construct from the first hit and since then DH5a is on my black list (when I have tricky situations). Cheers, Kostya Levay Plant Pathology U.of Kentucky, Lexington. From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!UQTR.UQuebec.ca!Louis_Croisetiere From: Louis_Croisetiere@UQTR.UQuebec.ca (Louis Croisetiere) Newsgroups: bionet.molbio.methds-reagnts Subject: Anacystis nidulans vs antibiotics Date: 4 Jun 1996 11:52:52 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <9606041848.AA37702@triton.UQTR.UQuebec.ca> NNTP-Posting-Host: net.bio.net Dear All, I would like to obtain references or informations regarding the use of antibiotics in a continuous culture of the cyanobacteria Anacystis nidulans (also called Synechococcus leopoliensis). Thanks in advance, Louis Croisetiere From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!news.mc.edu!usenet From: "Robert G. Hamilton" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Vertical Agarose gels, help Date: 4 Jun 1996 18:24:13 GMT Organization: Mississippi College Lines: 10 Message-ID: <4p1v0d$9o0@ox.mc.edu> References: <4mpo5o$1f92@power.ci.uv.es> <4p1g70$gvi@mercury.hgmp.mrc.ac.uk> NNTP-Posting-Host: mobo.mc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) mlush@hgmp.mrc.ac.uk (Mr. M.J. Lush) wrote: :-) does chilling the gel when removeing >the comb help? I have found that cooling the gel increases the chance of the gel slipping out. Dont make the wells any deeper than necessary is the best way to avoid problems getting the comb out...otherwise..BE PATIENT!!! (If you need to load a large sample, you need to invest some time getting the comb out of the well!) From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!galaxy.ucr.edu!ihnp4.ucsd.edu!news.service.uci.edu!rablab.biochem.uci.edu!user From: dmthomas@orion.oac.uci.edu (Didier Thomas) Newsgroups: bionet.molbio.methds-reagnts Subject: Swiss 3T3 transfection Date: Tue, 04 Jun 1996 11:30:32 +1000 Organization: UC Irvine Lines: 12 Message-ID: NNTP-Posting-Host: rablab.biochem.uci.edu Can someone tell me what is the best method to transfect Swiss 3T3. I have tried the calcium phosphate method, but it does not work. Thanks. -- Didier Thomas University of California, Irvine College of Medicine Dept. of Biological Chemistry Med. Sci. I, Room D214 Irvine, CA 92717 USA From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!ns1.faseb.org!lamarck.sura.net!news.mc.edu!usenet From: "Robert G. Hamilton" Newsgroups: bionet.molbio.methds-reagnts Subject: Re: DNA Sequencers Date: 1 Jun 1996 02:41:46 GMT Organization: Mississippi College Lines: 21 Message-ID: <4ooala$a5g@ox.mc.edu> References: <31AD920B.10F0@vims.edu> NNTP-Posting-Host: mobo.mc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) We have used the LiCor sequencer with great success. It routinely reads 800bp to 99%+ accuracy. Our main reason for purchasing the LiCor over the ALF (we faced the same choice) was maintenance cost. The LiCor uses a lower power laser, more inexpensive plates, and is a mechanically simpler machine. We believe that the LiCor will run longer without breaking down, and repairs will be cheaper if it does break down (we can repair many items ourselves due to the mechanical simplicity of the system). We did not buy the service contract, as we did not think it was needed, and so far, after a year and a half of reasonably heavy use by inexperienced undergraduate and high school student users, we have had no problems. Support is outstanding. There is always someone at the end of a phone to answer a question. I am also using it for RAPDs, and it is looking good so far (we are just starting with this analysis..but we are getting data). The machine is easy to use, and while it has a fully automatic setting, we are beginning to use a more manual reading method as we always check the auto reads..so why not read them ourselves in the first place! The autorad-like image is very easy to read manually. From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!MUSS.CIS.MCMASTER.CA!u9310871 From: u9310871@MUSS.CIS.MCMASTER.CA ("A.S. Sohal") Newsgroups: bionet.molbio.methds-reagnts Subject: (none) Date: 4 Jun 1996 11:10:07 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi! I am experimenting with some new site-directed mutagenesis protocols and was wondering if anybody has had any success. The ones I am talking about are the MORPH single-primer site-directed mutagenesis protocol from 5 prime 3 prime and the Chameleon protocol from Stratagene. I am having troubles isolating my "mutated" plasmid from the mut S cells although they have become amp resistant meaning that the transformation must have been successful. If someone could help me I would very grateful. Thanx From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!metro!metro!seagoon.newcastle.edu.au!usenet From: mdpjr@cc.newcastle.edu.au (Phil Robinson) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Acrylamide Gel setting too quickly -- IDEAS?? Date: Tue, 04 Jun 1996 15:59:34 GMT Organization: The University of Newcastle Lines: 26 Message-ID: <4ovqoo$cgg@seagoon.newcastle.edu.au> References: <4oqlfi$8iu@dfw-ixnews9.ix.netcom.com> NNTP-Posting-Host: jh-rob.newcastle.edu.au X-Newsreader: Forte Free Agent 1.0.82 macnevin@ix.netcom.com (Brian MacNevin) wrote: >Well, when I mix up my acrylamide gel solution and begin pipetting it >between the glass plates for electrophoresis, I have found that it >starts to set before I can get it evenly dispersed in the plates. > My first thought is that this might be due to the temp of the >solution. It seems to me that I don't have this problem if the >solution is allowed to cool a little before adding the final solution. >I was wonderin if anyone else has had these problems and if it really >is due to the temp of the solution? Seems like too much APS and/or TEMED. Start by reducing the TEMED. Set up little test lots in eppendorf tubes and see how long they take to set. Something like 10-20 min should be fine. However, we approach the problem by cooling the acrylamide solutions on ice. We pour gradients and to prevent any chance of it setting in our gradient mixer or tubing, we and many others, find that keeping the gel solution on ice prior to pouring significantly slows down polymerisation rates. And old and well-known trick... Should not be necessary for single-percent acrylamide, although we do it there too - out of habit more than anything. Phil Newcastle Australia From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!STUDENTS.UWLAX.EDU!page_pm From: page_pm@STUDENTS.UWLAX.EDU (Page) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: (none) Date: 4 Jun 1996 11:07:52 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <48071.jibave@uachih.uachnet.mx> NNTP-Posting-Host: net.bio.net Jose, You have to send an unsubscribe message to "biosci-server@net.bio.net", not to the discussion group itself. Paul. > unsubscribe > > Dr. Jose L. Ibave > Facultad de Ciencias Agrotecnologicas > P.O. Box 24 > University of Chihuahua > Chihuahua, Chih. Mexico 31000 > tel. (voice) 0152+14+146615 > (fax) 0152+14+134888 > > From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!agate!nature.berkeley.edu!lhom From: lhom@nature.berkeley.edu (Louis Hom) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: Gedankenexperiment Date: 4 Jun 1996 17:47:15 GMT Organization: University of California, Berkeley Lines: 7 Message-ID: <4p1sr3$3fc@agate.berkeley.edu> References: NNTP-Posting-Host: nature.berkeley.edu I believe Nilabh Shastri at UC Berkeley has expressed an octapeptide in mammalian cells. Or maybe an octapeptide plus the ATG Met. -- _______________________________________________________________________________ Lou Hom >K '93 "If brevity be the soul of wit, lhom@nature.berkeley.edu play on!" http://www.ocf.berkeley.edu/~lhom From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!MUSS.CIS.MCMASTER.CA!u9310871 From: u9310871@MUSS.CIS.MCMASTER.CA ("A.S. Sohal") Newsgroups: bionet.molbio.methds-reagnts Subject: Help:Stratagene and MORPH mutagenesis kits Date: 4 Jun 1996 10:43:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi! My name is Avtar. I am experimenting with the MORPH single-primer site-directed mutagenesis kit and the Chameleon double-stranded site-directed mutagenesis kit from Stratagene. I was wondering if anybody has been able to get these protocols to work. I am having trouble isolating my plasmid after transformation into the mut S cells using a standard alkaline lysis mini-prep protocol. I hopt someone can help me. Thanx From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!rutgers!oitnews.harvard.edu!yale!gumby!newspump.wustl.edu!newsreader.wustl.edu!biodec.wustl.edu!graham From: graham@biodec.wustl.edu (James Graham) Newsgroups: bionet.molbio.methds-reagnts Subject: Re: TKO miniflourometer (long) Date: 4 Jun 1996 12:19:59 -0500 Organization: Washington University Biology, St. Louis, MO Lines: 132 Message-ID: References: <4nvdne$o4c@newz.oit.unc.edu> <4opn1m$g5l@elna.ethz.ch> NNTP-Posting-Host: @biodec.wustl.edu X-Newsreader: NN version 6.5.0 #1 (NOV) Alexander, In my opinion, Philips comments, while very valuable to those who have useable TK100's, fall a little short in terms of their ability to serve as a "FAQ" on the TK100. Firstly, he describes relatively minor problems (ie. 2X discrepency with UV absorbance quantitation) and an "overflourescence" problem, while what I have consitently seen is the tendency to intermitently read a particular sample as a "blank", which then showed excellent yield following agarose gel electrophoresis, along with the good utility described. Philip describes problems involving rather finer points in quantitation, rather than the outright failures described in the newsgroup archive. (I am personally familiar with 5 units, three that are unreliable to the point of being unusuable, and two which are in routine reportedly successful use across two departments I have been in.) The presence of dust, while certainly a confounding aspect (particularily when it is suggested that it is so difficult to exclude) may be related to some problems, but seems to me an unlikely "explanation", particularily when far more sensitive experiments are being carried out sucessfully in the same laboratories by the same hands. The assay procedure itself, so often blamed, is really quite simple acutally, in comparison to many routine laboratory procedures. I fail to find user error a likely explanation for the widespread failure of the intstrument reported in the newsgroup archives, barring a tenuous potential for proper beam alignment in terms of cuvette positioning or other unacceptable design problem . While signficant sensitivity to traces of contaminants may be at the heart of the problem(s), the maufuacturer and others have reported that the Hoest quantitation itself is relative insensitive to a range of common laboratory reagents likley encountered (eg. SDS, phenol, proteins, etc.). Finally, Phillipe's comments neglect to mention that the problem with the TK100 was acknowledged by Hoeffer to an extent at one time, at least in terms of a "reconditioning" of some kind offered to users at significantadditional relative expense. Any FAQ would have to include some reference to whatever this still unexplained recall involved. (Failing to observe the reported problem, is indeed negative evidence -a type of data usually found of limited utility in defining reported phenomena.) Regards, Jim J. Graham PhD Biology Department Washington University of St. Louis Philip wrote: I certainly can't shed much light on the problems of individual machines that I haven't personally used, but I have used 3 different TKO miniflourometers bought over a long period with I believe reasonable results i.e. the amounts of DNA we estimate by the TKO seem to roughly agree with what we see on a gel following EB staining. So why do some people have problems? Certainly one problem is dust. If you do not dispense the reagent from a closed container i.e. Repipette it is very difficult to get good results. Pipette tips can be a problem. They can be sterile and still have dust on them. The problem of dust interferring with any fluorescenct technique is well known. A second problem is that there can be chemical interference. The most dramatic example I know of I discovered myself recently. I wanted to follow the binding of DNA in an alkali miniprep to glass beads to see if binding was quantitative. I got suspiciously high readings for the amount of "DNA" left in the supernatant. As a control I mixed NaOH and SDS with the standard GET (or TEG as some call it) buffer used in alkali minipreps and then added the usual 5 M KOAc reagent to obtain the usual white precipitate which I spun out. I then assayed the amount of "DNA" in this supernatant. I emphasize that this was a completely mock miniprep. No cells were present. To my surprise I got very high fluorescence. So there it seems there is some chemical reaction between GET, NaOH, SDS, and KOAc that can masquerade as "DNA" byfluorescence. I only did this once, so I'd appreciate it if someone anyone wants to repeat this little experiment and see if they confirm my result. However, I tentatively conclude that a very common procedure can lead to false results. I hasten to add that after washing and elution of the DNA from the glass beads, I got a reasonable value for the amount of DNA. A third problem is that many common miniprep methods yield DNA that is probably contaminated with RNA. When you consider that there is about 10x more RNA than DNA in a cell, a procedure that gets rid of 90% of the RNA still leaves equal amounts of DNA and RNA. Since plasmid DNA is only a fraction of the total DNA there is plenty of room for error. When we first set up our automated sequencing facility we found that consistently the TKO gave lower readings than A260 for DNA content. Usually the difference was less than 2-fold, but in some preps the differences were as much as 5 or 10 fold. My impression (not a careful study!) is that as folks got better at making miniprep DNA the sequencing results improved and the discrepency between the fluorometer and A260 tended to get less. Many workers think that if they use RNase or CsCl the resulting DNA is free of RNA. Probably not so. EB is a very insensitive way of looking at RNA on a gel, and since pancreatic RNase only cleaves at U,C, polypurine runs will lead to rather large fragments of RNA contaminating your DNA. (I assume this is the material that is so nicely removed by the various ion exchange and glass methods.) Note that RNA oligos will not pellet in CsCl, so that large pellet of RNA you may see in the bottom of your tube does not mean there is no RNA contamination. I know of at least one paper in which the authors examined standard miniprep DNA by HPLC and found a large peak of A260 absorbing material that was not plasmid DNA. In some organisms like yeast, you can RNase and phenol extract and get rediculously large amounts of RNA in preps of chromosomal DNA which can only be removed by a second RNAse treatment after the bulk of protein and RNA are removed. So if you demand that the TKO give you results consistent with UV absorbance measurements, you are are probably not playing fair. Another problem is that one should not expect these fluorescence measurements to be +/- 10%-at least not without a lot of work. I wonder if people who estimate DNA concentration by running gels think that technique is +/- 10%. I hope not. I always rezero the machine just before taking a reading, and I'd say that results are usually +/- 20% or better. Do people really need better for most molecular biology experiments? I do not believe the electronics of the machines I have seen are unstable. If they were, do you think you would be able to get consistent results with the calf thymus DNA standards? Just look at the display with no sample in the cuvette. On the machines I use, it is quite stable for reasonable periods. Of course, if you let the DNA dye complex stay in the cuvette being bombarded by UV for many minutes, you will see the display drift, but this is probably fluorescent bleaching not instability of the machine. In summary, I find the TKO to be a great time saver in the lab. It has to be used with care and understanding like any highly sensitive technique. There are traps for the unwary which I have discussed, but maybe some of you ought to get your machines off the shelf one last time, and see if after considering some of the points I've raised it works for you. Usual disclaimers. Philip Carl Res. Assoc. Prof. of Pharmacology UNC Chapel Hill From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!ihnp4.ucsd.edu!munnari.OZ.AU!news.ecn.uoknor.edu!news.uoknor.edu!omrf From: frank@omrf.uokhsc.edu (BartFrank) Newsgroups: bionet.molbio.methds-reagnts Subject: IGNORE: Test Only Date: Tue, 04 Jun 1996 05:00:01 GMT Organization: The University of Oklahoma (USA) Lines: 1 Message-ID: <4p1q3d$io2@frazier.uoknor.edu> Reply-To: frank@omrf.uokhsc.edu (BartFrank) NNTP-Posting-Host: 157.142.200.175 test From owner-methds-reagnts@net.bio.net Mon Jun 03 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!elroy.jpl.nasa.gov!swrinde!tank.news.pipex.net!pipex!oleane!in2p3.fr!swidir.switch.ch!scsing.switch.ch!swsbe6.switch.ch!surfnet.nl!tudelft.nl!news From: Lesley Robertson Newsgroups: bionet.molbio.methds-reagnts,bionet.microbiology Subject: Re: Persistant phenotypic differences (serial subculture)? Date: 4 Jun 1996 16:43:51 GMT Organization: Delft University of Technology Lines: 61 Message-ID: <4p1p47$4v2@mo6.rc.tudelft.nl> References: <4p1ljj$l5g@masala.cc.uh.edu> NNTP-Posting-Host: kluyver2.stm.tudelft.nl Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Xref: biosci bionet.molbio.methds-reagnts:45313 bionet.microbiology:6261 benedik@uh.edu (Michael Benedik) wrote: >In article >graham@biodec.wustl.edu (James Graham) writes: > snip >> When one grows several broth batch cultures of a single bacterial >> strain inoculated from say an saturated overnight culture, a single >> colony, or from a frozen glycerol stock, one will notice a variety of >> differences between the two cultures, at least in terms of iducability of >> promoters, or perhaps the transformation efficiency of subsequently prepared >> calcium competent cells. Rumor has it that a two-dimensional PAGE >> analysis of identically cultured cellls prepared from two cultures >> originating from slightly different inocula will show considerable >> differences, far beyond that which could be attributed to differences >> among any nondividing cells originally introduced. >> snip >Good observations. I too have been wondering similar things. We notice >that we get very different levels of expression when we dilute an >overnight culture 10e3-fold compared to 10e6-fold. (Looking at >expression of extracellular nuclease from Serratia marcescens). And >these are washed cells so it isn't carry over of some growth medium >component. > >Obviously you should rule out differences in carry over of signal >molecules in the growth medium. > >My guess it is a very slow turnover of some intracellular signal. But >we have not spend any time looking at number of generations or of >rates. > This sounds very much like the changes in cultures that occur when they are grown in continuous cultures - for example, growing them for long periods at growth rates approaching mu max seems to select for "turbo-bacteria" with much higher mu max values than the original inoculum, growing them with low levels of antibiotics gradually produces strains that can tolerate higher levels of antibiotics, etc. I've always believed that its because any "pure culture" is actually a collection of not quite identical bacteria - there will be a lot of the type that most suit the particular growth conditions, but low numbers of others carrying properties that are not really needed. Change the conditions (e.g. by increasing the growth rate), and you favour one of the minority populations which then becomes dominant. With the continuous cultures, it's much more pronounced in heterotrophs than obligate autotrophs. Isn't there a similar phenomenon with pathogens losing their path