From owner-rapd@net.bio.net Sun Apr 03 23:00:00 1994 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: e-mail address of C.D. Nelson or W.. Nance? Date: 3 Apr 1994 17:25:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 4 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199404040026.UAA07402@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net Could somebody please send me the e-mail address of C.D. Nelson or W.L. Nance of USDA Forest Service, Gulfport, MS. Many thanks Muhammad Lodhi From owner-rapd@net.bio.net Mon Apr 04 23:00:00 1994 Path: biosci!UCS.ORST.EDU!barkerr From: barkerr@UCS.ORST.EDU (Reed Barker) Newsgroups: bionet.molbio.rapd Subject: Re: AMOVA- where?, how? Date: 5 Apr 1994 15:27:42 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 32 Sender: daemon@net.bio.net Distribution: bionet Message-ID: References: <2n13f6$k67@mserv1.dl.ac.uk> NNTP-Posting-Host: net.bio.net Julian, Sorry it took so long to reply to your request, you may already have it. If not, you can get WINAMOVA by anonymous FTP to acasun1.unige.ch and it is found in the pub/amova directory. The file is WINAMOVA.ZIP. This archive contains several files, including WINAMOVA.EXE that runs as a Windows applicaiton. Reed R.E. Barker barkerr@ucs.orst.edu Corvallis, OR 97331-7102 (503) 750-8736 FAX:(503) 750-8750 On 26 Mar 1994, Julian Paul Robinson wrote: > Dear all, > I've heard that a program called AMOVA is > available. > Does any know how to get hold of it ? I am > able to use ftp to get it if it is available. > > Many thanks, > > Julian > > e-mail:jpr@st-and.ac.uk > > > From owner-rapd@net.bio.net Tue Apr 05 23:00:00 1994 Path: biosci!uoguelph.ca!lgood From: lgood@uoguelph.ca (Liam E Good) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 6 Apr 1994 13:18:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 4 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net subscribe From owner-rapd@net.bio.net Thu Apr 07 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!swrinde!gatech!newsxfer.itd.umich.edu!gumby!wupost!spool.mu.edu!torn!utnut!utcsri!newsflash.concordia.ca!sifon!VM1.MCGILL.CA From: "LECLERC-POTVIN,CAROLE,MS" Subject: RAPD marker conversion to single-copy marker Message-ID: <08APR94.19327746.0158@VM1.MCGILL.CA> Lines: 8 Sender: usenet@MUSICB.MCGILL.CA Organization: McGill University Date: Fri, 8 Apr 1994 22:53:45 GMT Hello I would like to hear from any one with experience in the conversion of multi-copy RAPD markers in single-copy RAPD markers. I would be very greatful for any advice or even references. Thank you in advance, Carole xp7x@musicb.mcgill.ca From owner-rapd@net.bio.net Thu Apr 07 23:00:00 1994 Path: biosci!UCS.ORST.EDU!barkerr From: barkerr@UCS.ORST.EDU (Reed Barker) Newsgroups: bionet.molbio.rapd Subject: Gel analysis (fwd) Date: 7 Apr 1994 20:09:03 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 34 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Will this one get through? I got the address wrong the first time, sorry. Reed. R.E. Barker barkerr@ucs.orst.edu Corvallis, OR 97331-7102 (503) 750-8736 FAX:(503) 750-8750 ---------- Forwarded message ---------- Date: Wed, 6 Apr 1994 17:21:53 -0700 (PDT) From: Reed Barker To: RAPD List Subject: Gel analysis Does anyone have experience with or information on video documentation of gels for image analysis? What kind of camera systems are best and what resolutions, filters, etc. are needed? I can analyze a gel on a computer image using NIH Image if there is an effective was to digitize the image. We are using a flatbed scanner now, but would like to use a direct camera system. There are stand alone products on the market now, but they are expensive and I do not know their effectiveness. Any input would be appreciated. Reed. R.E. Barker barkerr@ucs.orst.edu Corvallis, OR 97331-7102 (503) 750-8736 FAX:(503) 750-8750 From owner-rapd@net.bio.net Thu Apr 07 23:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!pipex!bnr.co.uk!corpgate!news.utdallas.edu!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd Subject: Re: Gel analysis (fwd) Date: 8 Apr 1994 14:35:33 -0400 Organization: R.S.M.A.S. Lines: 39 Distribution: bionet Message-ID: <2o485l$7fl@oj.rsmas.miami.edu> References: NNTP-Posting-Host: oj.rsmas.miami.edu In article , Reed Barker wrote: > >Does anyone have experience with or information on video documentation of >gels for image analysis? What kind of camera systems are best and what >resolutions, filters, etc. are needed? I can analyze a gel on a computer >image using NIH Image if there is an effective was to digitize the >image. We are using a flatbed scanner now, but would like to use a >direct camera system. There are stand alone products on the market now, >but they are expensive and I do not know their effectiveness. Any input >would be appreciated. > I have been using the Imaging Tech Inc. Vision Plus-AT for a couple of years to digitize gels and photos of gels. A camera based system is very flexible since you can zoom and crop physically, play with filters tilt, stretch, squeeze and otherwise distort a gel to straighten lanes. But you pay for the flexibility with the loss of resolution in most affordable camera systems. Our frame grabber/camera combination is built to match the VGA resolution, 640x480 pixels. If your flat bed is 400 dpi with a 1:1 conversion you are getting considerably better resolution for anything but a very small gel on the scanner. A 20x20 cm or 8x8 in gel can get 3200x3200 pixels from the flat bed, but 640x480 with the camera system. I am sure the newer systems with higher resolutions and pixels/dollar ratios are out there now. If increasing resolution is the goal though make sure you can beat the scanner befrore spending the money. And you will need LOTS of money to use a camera for high resolution of large (e.g. sequencing) gels. It is nice to have both so you can digitize on the scanner for computer analysis of the image with high resolution. And then use the camera system for combining gels with other things in the same picture, such as rulers, plates, apparatus, annotations, etc. Our camera system is used much more for such scientific art work than gel analysis now. Jack Kramer Tropical Research Center University of Florida From owner-rapd@net.bio.net Thu Apr 07 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!swrinde!gatech!newsxfer.itd.umich.edu!gumby!wupost!spool.mu.edu!torn!utnut!utcsri!newsflash.concordia.ca!sifon!VM1.MCGILL.CA From: "LECLERC-POTVIN,CAROLE,MS" Subject: single-copy RAPD markers Message-ID: <08APR94.19507909.0158@VM1.MCGILL.CA> Lines: 8 Sender: usenet@MUSICB.MCGILL.CA Organization: McGill University Date: Fri, 8 Apr 1994 23:03:46 GMT Hello, I would like to hear from anyone with experience in the conversion of a multi-copy RAPD marker into a single-copy marker. I would be very greatful for any advice or even reference on the subject. Thankyou in advance, Carole xp7x.@musicb.mcgill.ca From owner-rapd@net.bio.net Fri Apr 08 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!howland.reston.ans.net!xlink.net!wega.fibronics.de!neutron!jui From: jui@neutron.nacamar.de (Uwe Harmening) Subject: Multiple alignment software seeked Organization: NACAMAR Development Center Date: Thu, 7 Apr 1994 13:48:13 GMT X-Newsreader: TIN [version 1.2 PL2] Message-ID: <1994Apr7.134813.6410@neutron.nacamar.de> Lines: 23 Hallo world, I am looking for a software that basically does the same as GCG or Genius Husar (in Heidelberg). We want to do multiple alignment of DNA-sequences at university. Is this possible with PC-Gene or DNAsis? We have got Windows-PCs (486-33). I heard abaout Macawwin (or so), has anybody experiences with that? Thanx Uwe Harmening -- Tschoe Jui ----Emptiness is Form; Form is Emptiness---- Fidonet: 2:244/1370.11 Internet: jui@neutron.nacamar.de Snailnet: no way! From owner-rapd@net.bio.net Sun Apr 10 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!news.intercon.com!panix!zip.eecs.umich.edu!newsxfer.itd.umich.edu!news.cic.net!magnus.acs.ohio-state.edu!tcjones From: tcjones@magnus.acs.ohio-state.edu (Thomas C Jones) Newsgroups: bionet.molbio.rapd Subject: Negative controls Date: 11 Apr 1994 22:09:38 GMT Organization: The Ohio State University Lines: 7 Message-ID: <2ochr2$1mj@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: bottom.magnus.acs.ohio-state.edu Keywords: Sterility, Laminar flow hoods Hello, Can anyone recommend a relatively inexpensive, yet effective, hood to prevent contamination in PCR tubes? Are laminar flow hoods in fact the best way to go? Negative control bands are a problem, as you may have guessed. Thank you, T.J. From owner-rapd@net.bio.net Mon Apr 11 23:00:00 1994 Path: biosci!GPU.SRV.UALBERTA.CA!ryang From: ryang@GPU.SRV.UALBERTA.CA (Rong Yang) Newsgroups: bionet.molbio.rapd Subject: Mapping of RAPD loci using MAPMAKER Date: 12 Apr 1994 13:45:30 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Dear All, I am a novice user of MAPMAKER and am seeking for advices on the following question. We have a data set consisting of 70 haploid megagametophytes of a conifer species, each assayed for 282 RAPD loci. Our objective is to map these RAPD loci into linkage groups using MAPMAKER. I have been able to map 134 RAPD loci into 13 linkage groups, but with ONLY 524 cM total map distance. In other words, only closely linked (<17 cM) loci are mapped and in fact the majority of adjacent locus pairs are less than 10 cM apart. Therefore, the total mapped distance is shorter than those reported in the literature for the same number of loci. I follow the MAPMAKER procedure entitled "Dealing with Larger Data Sets". Could anyone tell me how to incorperate those loci that far aprt into the existing or new linkage groups. Thank you in advance for your help. Rong-Cai Yang University of Alberta From owner-rapd@net.bio.net Mon Apr 11 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!news.intercon.com!udel!news2.sprintlink.net!news.sprintlink.net!indirect.com!nike From: nike@indirect.com (Laurence Canter) Newsgroups: bionet.molbio.rapd,brasil.computacao.paralela Subject: Green Card Lottery- Final One? Date: 12 Apr 1994 07:44:30 GMT Organization: Canter & Siegel Lines: 34 Message-ID: <2odjgu$2df@herald.indirect.com> NNTP-Posting-Host: id1.indirect.com Green Card Lottery 1994 May Be The Last One! THE DEADLINE HAS BEEN ANNOUNCED. The Green Card Lottery is a completely legal program giving away a certain annual allotment of Green Cards to persons born in certain countries. The lottery program was scheduled to continue on a permanent basis. However, recently, Senator Alan J Simpson introduced a bill into the U. S. Congress which could end any future lotteries. THE 1994 LOTTERY IS SCHEDULED TO TAKE PLACE SOON, BUT IT MAY BE THE VERY LAST ONE. PERSONS BORN IN MOST COUNTRIES QUALIFY, MANY FOR FIRST TIME. The only countries NOT qualifying are: Mexico; India; P.R. China; Taiwan, Philippines, North Korea, Canada, United Kingdom (except Northern Ireland), Jamaica, Domican Republic, El Salvador and Vietnam. Lottery registration will take place soon. 55,000 Green Cards will be given to those who register correctly. NO JOB IS REQUIRED. THERE IS A STRICT JUNE DEADLINE. THE TIME TO START IS NOW!! For FREE information via Email, send request to cslaw@indirect.com -- ***************************************************************** Canter & Siegel, Immigration Attorneys 3333 E Camelback Road, Ste 250, Phoenix AZ 85018 USA cslaw@indirect.com telephone (602)661-3911 Fax (602) 451-7617 From owner-rapd@net.bio.net Mon Apr 11 23:00:00 1994 Path: biosci!LIFSCI.SDSU.EDU!SOBRAL From: SOBRAL@LIFSCI.SDSU.EDU Newsgroups: bionet.molbio.rapd Subject: Oil in thermocyclers Date: 12 Apr 1994 10:39:59 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 42 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <940412103817.202000c4@LIFSCI.SDSU.EDU> NNTP-Posting-Host: net.bio.net In response to: #4 12-APR-1994 10:11:16.23 NEWMAIL From: SMTP%"BIOSCI-REQUEST%NET.BIO.NET@CMSA.BERKELEY.EDU" To: rapd@net.bio.net CC: Subj: mineral oil on thermocycler block To: rapd@net.bio.net From: MCDORNEL%tuvira.ciagri.usp.br%NET.BIO.NET%CMSA.BERKELEY.EDU@Sdsc.Edu ( "Marcelo Carnier Dornelas") Subject: mineral oil on thermocycler block Date: 12 Apr 1994 09:34:54 -0700 Sender: daemon@net.bio.net Message-ID: NNTP-Posting-Host: net.bio.net Hi netters I've seen people using mineral oil on thermocycler block to increase the heat transfer efficiency with the tubes. Is it really necessary? Doesn't it reduce the lifetime of the thermocycler? Thanks for any comments I would say that we ALWAYS use mineral oil to make sure the contact between the tube and the machine is good. This is particularly important with some brands of tubes, which have small plastic protrusions on the bottom (from the mold) and therefore sit up in the well, not allowing uniform heating across samples. We have been doing this for 3 years now, with no observable\ effect on the cyclers (both Perkin-Elmer and MJ Research). Sincerely, Bruno WS Sobral Staff Scientist California Institute of Biological Research (CIBR) 11099 N. Torrey Pines Road, Suite 300 La Jolla CA USA 92037 (619)535-5483 (office) (619)535-5491 (lab) E-mail: sobral@lifsci.sdsu.edu or brunosfly@aol.com (619)535-5472, 535-5490 or 535-5481 (faxes) From owner-rapd@net.bio.net Mon Apr 11 23:00:00 1994 Path: biosci!TUVIRA.CIAGRI.USP.BR!MCDORNEL From: MCDORNEL@TUVIRA.CIAGRI.USP.BR ("Marcelo Carnier Dornelas") Newsgroups: bionet.molbio.rapd Subject: mineral oil on thermocycler block Date: 12 Apr 1994 09:34:54 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Hi netters I've seen people using mineral oil on thermocycler block to increase the heat transfer efficiency with the tubes. Is it really necessary? Doesn't it reduce the lifetime of the thermocycler? Thanks for any comments Marcelo Carnier Dornelas University of Sao Paulo "Luiz de Queiroz" School of Agronomy From owner-rapd@net.bio.net Mon Apr 11 23:00:00 1994 Path: biosci!TUVIRA.CIAGRI.USP.BR!MCDORNEL From: MCDORNEL@TUVIRA.CIAGRI.USP.BR ("Marcelo Carnier Dornelas") Newsgroups: bionet.molbio.rapd Subject: gelatin in PCR reactions Date: 12 Apr 1994 09:33:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Hi netters I've heard about using gelatin in PCR buffer. Is it so, or am I dreaming ? Which concentration is currently used? What is it for? Thanks a lot for any comments. Marcelo Carnier Dornelas Genetics Department University of Sao Paulo "Luiz de Queiroz" School of Agronomy From owner-rapd@net.bio.net Mon Apr 11 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd Subject: Re: Oil in thermocyclers Date: 12 Apr 1994 16:35:05 -0400 Organization: R.S.M.A.S. Lines: 21 Distribution: bionet Message-ID: <2of0lp$aa5@oj.rsmas.miami.edu> References: <940412103817.202000c4@lifsci.sdsu.edu> NNTP-Posting-Host: oj.rsmas.miami.edu In article <940412103817.202000c4@lifsci.sdsu.edu>, wrote: > > I've seen people using mineral oil on thermocycler block to >increase the heat transfer efficiency with the tubes. Is it really >necessary? Doesn't it reduce the lifetime of the thermocycler? > Thanks for any comments > >I would say that we ALWAYS use mineral oil to make sure the contact between >the tube and the machine is good. This is particularly important with some >brands of tubes, which have small plastic protrusions on the bottom (from >the mold) and therefore sit up in the well, not allowing uniform heating >across samples. We have been doing this for 3 years now, with no observable\ Glycerin will do the same thing with the advantage that it is water soluable which makes handling the tubes after cycling and cleaning the heating block easier. In the MJR I don't use anything in the block. If you get a batch of tubes with the nipples on the bottom, a finger nail clipper fixes them very quickly and easily. From owner-rapd@net.bio.net Tue Apr 12 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!swrinde!emory!news.cc.emory.edu!news.cc.emory.edu!not-for-mail From: vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) Newsgroups: bionet.molbio.rapd Subject: Hot-Start Date: 13 Apr 1994 12:22:07 -0400 Organization: Emory University Lines: 1 Message-ID: <2oh67f$q03@emoryu1.cc.emory.edu> NNTP-Posting-Host: emoryu1.cc.emory.edu X-Newsreader: TIN [version 1.2 PL2] From owner-rapd@net.bio.net Tue Apr 12 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!uhog.mit.edu!news.kei.com!yeshua.marcam.com!zip.eecs.umich.edu!newsxfer.itd.umich.edu!nntp.cs.ubc.ca!alberta!quartz.ucs.ualberta.ca!tribune.usask.ca!canopus.cc.umanitoba.ca!newsflash.concordia.ca!sifon!VM1.MCGILL.CA From: "LECLERC-POTVIN,CAROLE,MS" Subject: RE: RAPD marker conversion to single-copy marker Message-ID: <12APR94.18150591.0111@VM1.MCGILL.CA> Lines: 26 Sender: usenet@MUSICB.MCGILL.CA Organization: McGill University References: <08APR94.19327746.0158@VM1.MCGILL.CA> Date: Tue, 12 Apr 1994 21:48:21 GMT In article <08APR94.19327746.0158@VM1.MCGILL.CA> "LECLERC-POTVIN,CAROLE,MS" writes: >Hello >I would like to hear from any one with experience in the conversion >of multi-copy RAPD markers in single-copy RAPD markers. I would be >very greatful for any advice or even references. >Thank you in advance, >Carole >xp7x@musicb.mcgill.ca > I would like to clarify on my request. We identified some RAPD markers that are tightly linked and flanking a resistance gene. Those RAPD markers were cloned and the cloned probes can be used on a Southern of the amplification products. We know that some markers contain repeatitive DNA. I would like to transform these RAPD markers into codominant markers and use them as single-copy probes on PFGE Southerns. We also speculate that the polymorphism giving rise to the marker lie in the binding site of the primer, therefore we would loose the ability to detect polymorphism if an internal probe was used. How can we generate a probe (single-copy?) that will overlap with the primer binding site? I have read Paran & Michelmore's(1993) paper: is anybody aware of other methodology which could be used? Thank you in advance, Carole From owner-rapd@net.bio.net Tue Apr 12 23:00:00 1994 Path: biosci!daresbury!not-for-mail From: Louis van de Zande Newsgroups: bionet.molbio.rapd Subject: Re: Green Card Lottery- Final One? Date: 13 Apr 1994 10:24:58 +0100 Organization: Department of Biology, RUGroningen Lines: 40 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ogdpa$rs1@mserv1.dl.ac.uk> Reply-To: ZANDELPW@BIOL.RUG.nl Original-To: rapd@dl.ac.uk > Green Card Lottery 1994 May Be The Last One! > THE DEADLINE HAS BEEN ANNOUNCED. > > The Green Card Lottery is a completely legal program giving away a > certain annual allotment of Green Cards to persons born in certain > countries. The lottery program was scheduled to continue on a > permanent basis. However, recently, Senator Alan J Simpson > introduced a bill into the U. S. Congress which could end any future > lotteries. THE 1994 LOTTERY IS SCHEDULED TO TAKE PLACE > SOON, BUT IT MAY BE THE VERY LAST ONE. > > PERSONS BORN IN MOST COUNTRIES QUALIFY, MANY FOR > FIRST TIME. > > The only countries NOT qualifying are: Mexico; India; P.R. China; > Taiwan, Philippines, North Korea, Canada, United Kingdom (except > Northern Ireland), Jamaica, Domican Republic, El Salvador and > Vietnam. > > Lottery registration will take place soon. 55,000 Green Cards will be > given to those who register correctly. NO JOB IS REQUIRED. > > THERE IS A STRICT JUNE DEADLINE. THE TIME TO START IS > NOW!! > > For FREE information via Email, send request to > cslaw@indirect.com > > > -- > ***************************************************************** > Canter & Siegel, Immigration Attorneys > 3333 E Camelback Road, Ste 250, Phoenix AZ 85018 USA > cslaw@indirect.com telephone (602)661-3911 Fax (602) 451-7617 > > Sometimes this discussion group is just like real RAPD's. A lot of background that not many are interested in. Louis v d Zande From owner-rapd@net.bio.net Wed Apr 13 23:00:00 1994 Path: biosci!daresbury!imbb1.imbb.forth.gr!imbb1!louis From: louis@myia.imbb.forth.gr (Kitsos Louis) Newsgroups: bionet.molbio.rapd Subject: POSTDOC POSITIONS Followup-To: bionet.molbio.rapd Date: Thu, 14 Apr 94 17:21:07 GMT Organization: IMBB Lines: 17 Message-ID: NNTP-Posting-Host: 139.91.116.6 X-Newsreader: VersaTerm Link v1.1.1 INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY FOUNDATION FOR RESEARCH AND TECHNOLOGY, HELLAS HERAKLION, CRETE, GREECE FOUR POSTDOCTORAL POSITIONS, FUNDED BY THE HCM PROGRAMME OF THE EUROPEAN UNION AND THE MACARTHUR FOUNDATION, ARE AVAILABLE AT THE INSECT MOLECULAR GENETICS GROUP OF THE IMBB (C. DELIDAKIS, F.C. KAFATOS, K. LOUIS, B. SAVAKIS). THE RESEARCH TOPICS AT THE GROUP INCLUDE GENOME ANALYSIS, DEVELOPMENTAL GENETICS AND GENE STRUCTURE AND FUNCTION STUDIES OF DROSOPHILA, THE MALARIA MOSQUITO ANOPHELES GAMBIAE, AND THE MEDFLY CERATITIS CAPITATA. APPLICANTS SHOULD SEND A CV AND ARRANGE FOR 2 LETTERS OF RECOMMENDATION TO BE SENT TO K. LOUIS OR B. SAVAKIS: IMBB, FORTH, BOX 1527, 711 10 HERAKLION, CRETE, GREECE FAX: +30-81-231308 E-MAIL: LOUIS@MYIA.IMBB.FORTH.GR SAVAKIS@NEFELI.CC.UCH.GR From owner-rapd@net.bio.net Thu Apr 14 23:00:00 1994 Path: biosci!DEAKIN.EDU.AU!huangbx From: huangbx@DEAKIN.EDU.AU Newsgroups: bionet.molbio.rapd Subject: Strange agarose gel ethidium bromide staining fading Date: 14 Apr 1994 19:33:34 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 32 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199404150232.MAA05879@sol.ccs.deakin.edu.au> NNTP-Posting-Host: net.bio.net Hello Netters, First of all, I have to say sorry to you if you receive 2 copy of my question due to the computer problem. I am encountered an extraordinary strange phenomenon agarose gel ethidium bromide staining fading. Initially I ran my agarose gel in 1 x TAE buffer and stained the gel in ethidium bromide water solution. The images in the gels could last long enough for me to record the results and take photographes. Suddenly from my 55th PCR run, the gel once was puton the UV box, the images in the gel was fading so quickly that it was not long enough for me to do the necessary recording. What I did was to clean the container and make up another new solution. This new step would not help it at all. Then I switched to 1 x TBE buffer, the ethidium bromide solution was made up with 1 x TBE buffer as well. This measure slowered the fading. Again from my 85th PCR run, the fading is so quick. Does anyone have this sort of experience? Could you kindly give me some advice? Or any comments will be very appreciated! Thank you very much for your time. Bixing Huang huangbx@deakin.edu.au From owner-rapd@net.bio.net Thu Apr 14 23:00:00 1994 Path: biosci!COLUMBIA.EDU!jcm19 From: jcm19@COLUMBIA.EDU (Juan Carlos Morales) Newsgroups: bionet.molbio.rapd Subject: Re: Strange agarose gel ethidium bromide staining fading Date: 15 Apr 1994 05:40:11 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 36 Sender: daemon@net.bio.net Distribution: bionet Message-ID: References: <199404150232.MAA05879@sol.ccs.deakin.edu.au> NNTP-Posting-Host: net.bio.net On 14 Apr 1994 huangbx@deakin.edu.au wrote: > > Hello Netters, > > First of all, I have to say sorry to you if you receive 2 copy of my > question due to the computer problem. > > I am encountered an extraordinary strange phenomenon agarose gel ethidium > bromide staining fading. > > Initially I ran my agarose gel in 1 x TAE buffer and stained the gel in > ethidium bromide water solution. The images in the gels could last long > enough for me to record the results and take photographes. Suddenly from my > 55th PCR run, the gel once was puton the UV box, the images in the gel was > fading so quickly that it was not long enough for me to do the necessary > recording. What I did was to clean the container and make up another new > solution. This new step would not help it at all. > > Then I switched to 1 x TBE buffer, the ethidium bromide solution was made > up with 1 x TBE buffer as well. This measure slowered the fading. Again > from my 85th PCR run, the fading is so quick. > > Does anyone have this sort of experience? Could you kindly give me some > advice? Or any comments will be very appreciated! > > Thank you very much for your time. > > Bixing Huang > huangbx@deakin.edu.au > > > Try to keep your gel and EtBr solution in the dark as much as possible. That always help to maximize fluorescence. From owner-rapd@net.bio.net Fri Apr 15 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!europa.eng.gtefsd.com!emory!news.cc.emory.edu!news.cc.emory.edu!not-for-mail From: vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) Newsgroups: bionet.molbio.rapd Subject: DNA&RNA Extraction kits Date: 15 Apr 1994 18:08:03 -0400 Organization: Emory University Lines: 8 Message-ID: <2on383$24l@emoryu1.cc.emory.edu> X-Newsreader: TIN [version 1.2 PL2] I am looking for a kit that would allow fast and efficient RNA and DNA extraction from small tissue samples for subsequent RAPDs. I have seen some (like the TRI REAGENT from MRC). I would like to hear from any one who has evaluated or used these commercial kits. Thanks. From owner-rapd@net.bio.net Mon Apr 18 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!warwick!bham!bcs88.bham.ac.uk!user From: A.C.Hilton@bham.ac.uk (Anthony Hilton) Newsgroups: bionet.molbio.rapd Subject: RNA in RAPD Followup-To: bionet.molbio.rapd Date: 19 Apr 1994 11:46:56 GMT Organization: University of Birmingham Lines: 10 Distribution: world Message-ID: NNTP-Posting-Host: bcs88.bham.ac.uk Hi fellow RAPDers, Does anyone know if RNA interferes with RAPD. I can't see how it can as workers have performed RAPD on broth cultures of bacteria without making any attempt to remove RNA, and they claim to get adequate results. I currently use RNAase in my DNA preps but is it really worth while? Any comments welcome Wig. From owner-rapd@net.bio.net Mon Apr 18 23:00:00 1994 Path: biosci!cornell.edu!MAL5 From: MAL5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: RNA in RAPD Date: 19 Apr 1994 09:26:32 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199404191626.MAA26962@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net I don't think that RNA interferes with amplification process. However, it might bind some primers and amplify some portion of DNA but these wouldn't be RAPD products. If you run a RAPD reaction on gels which was amplified with DNA not treated with RNAse you will see a smear leading the RAPD products. Though I never had any problem with RNA (thats probably the only thing that has been friendly with RAPDs) even then I prefer to treat it with RNAse. It does not take much to do so, 10-15 minutes. Muhammad A. Lodhi >Hi fellow RAPDers, > >Does anyone know if RNA interferes with RAPD. I can't see how it can as >workers have performed RAPD on broth cultures of bacteria without making >any attempt to remove RNA, and they claim to get adequate results. I >currently use RNAase in my DNA preps but is it really worth while? > >Any comments welcome > >Wig. From owner-rapd@net.bio.net Mon Apr 18 23:00:00 1994 Path: biosci!BCRSSU.AGR.CA!WILLIS From: WILLIS@BCRSSU.AGR.CA Newsgroups: bionet.molbio.rapd Subject: Re: RNA in RAPD Date: 19 Apr 1994 13:53:02 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 50 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01HBD77L8NK2002C8D@GW.AGR.CA> NNTP-Posting-Host: net.bio.net RNA will interfere with PCR rxn's if present in sufficient amounts. See Suppression of PCR Amplification by High Levels of RNA, Biotechniques Vol. 14, No. 1 (1993). I imagine it would interfere with RAPD reactions as well. -----------------------------------------(=)----------------------------------- | Les Willis / | | Agriculture Canada Research Station (=) | | Summerland, British Columbia / | | Canada (=) | | V0H 1Z0 / | | Email Address: WILLIS@BCRSSU.AGR.CA (=) | ------------------------------------------/------------------------------------ >I don't think that RNA interferes with amplification process. However, it >might bind some primers and amplify some portion of DNA but these wouldn't >be RAPD products. If you run a RAPD reaction on gels which was amplified >with DNA not treated with RNAse you will see a smear leading the RAPD >products. Though I never had any problem with RNA (thats probably the only >thing that has been friendly with RAPDs) even then I prefer to treat it >with RNAse. It does not take much to do so, 10-15 minutes. >Muhammad A. Lodhi >Hi fellow RAPDers, > >Does anyone know if RNA interferes with RAPD. I can't see how it can as >workers have performed RAPD on broth cultures of bacteria without making >any attempt to remove RNA, and they claim to get adequate results. I >currently use RNAase in my DNA preps but is it really worth while? > >Any comments welcome > >Wig. Return-path: Received: from net.bio.net by GW.AGR.CA (PMDF #3062 ) id <01HBCY5LRDU8002A2X@GW.AGR.CA>; Tue, 19 Apr 1994 12:32:36 EST Received: (from daemon@localhost) by net.bio.net (8.6.8.1/8.6.6) id JAA09962 for rapd-list; Tue, 19 Apr 1994 09:26:38 -0700 Received: (from news@localhost) by net.bio.net (8.6.8.1/8.6.6) id JAA09958 for rapd-arpanet; Tue, 19 Apr 1994 09:26:36 -0700 Date: 19 Apr 1994 09:26:32 -0700 From: MAL5@cornell.edu (Muhammad A. Lodhi) Subject: RE: RNA in RAPD Sender: daemon@net.bio.net To: rapd@net.bio.net Message-id: <199404191626.MAA26962@postoffice.mail.cornell.edu> Content-transfer-encoding: 7BIT NNTP-Posting-Host: net.bio.net From owner-rapd@net.bio.net Mon Apr 18 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: RNA in RAPD Date: 19 Apr 1994 06:12:11 -0700 Organization: University of Arkansas Lines: 25 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <1DF8C01F0C@uamercury.uark.edu> NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: A.C.Hilton@bham.ac.uk (Anthony Hilton) >Subject: RNA in RAPD >Date sent: 19 Apr 1994 11:46:56 GMT >Hi fellow RAPDers, > >Does anyone know if RNA interferes with RAPD. I can't see how it can as >workers have performed RAPD on broth cultures of bacteria without making >any attempt to remove RNA, and they claim to get adequate results. I >currently use RNAase in my DNA preps but is it really worth while? > >Any comments welcome > >Wig. In our hands large amounts of small RNAs in DNA preps can lead to smearing of RAPD reactions. One also needs to be careful about underestimating the concentration of DNA due to massive RNA contamination. I believe that the small RNAs can act as random primers for Taq, just like they can in a sequencing reaction. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Tue Apr 19 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: Dissappearing DNA and RAPD's Date: 20 Apr 1994 05:50:34 -0700 Organization: University of Arkansas Lines: 27 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <359C7F006E@uamercury.uark.edu> NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: "Christopher J. S. Bolch" >Subject: Dissappearing DNA and RAPD's >Date sent: Wed, 20 Apr 1994 02:13:57 GMT >Anyone out there help with a perplexing problem. I am working on toxic >cyanobacteria doing some RAPDs. At least two of my strains are somewhat >awkward to extract DNA from. I now have twenty or so good extracts which >look fine on an agarose gel- heaps of DNA in a nice tight high mol weight >band. Our lab recently commissioned a new Hoeffer DNA fluorometer and I >have been checking my DNA concentration for the rapds. Fine for all my >exrtracts until I get to two isolates which look fine on agarose but the >fluormeter says ther is virtually zilch-zero-none DNA in the sample. >Curiously these samples have not been amplifying with the primers that >work very well for my other strains. I would say there was no DNA there >except there appears to be heaps of it when EtBr stained on Agarose. > >Any suggestions on what's going on, either with the fluorometer or the >rapd's. > >Cheers and thanks in advance. > > Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Tue Apr 19 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!daresbury!trane.uninett.no!sunic!EU.net!howland.reston.ans.net!europa.eng.gtefsd.com!gatech!swrinde!ihnp4.ucsd.edu!munnari.oz.au!newsroom.utas.edu.au!ml.csiro.au!mac_1303.ml.csiro.au!chris.bolch From: Christopher J. S. Bolch Subject: Dissappearing DNA and RAPD's Message-ID: <1994Apr20.021357.12871@ml.csiro.au> X-Xxmessage-Id: X-Xxdate: Wed, 20 Apr 94 01: 15:59 GMT Sender: news@ml.csiro.au Organization: CSIRO Division of Fisheries, Hobart, Tasmania X-Useragent: Version 1.1.3 Date: Wed, 20 Apr 1994 02:13:57 GMT Lines: 16 Anyone out there help with a perplexing problem. I am working on toxic cyanobacteria doing some RAPDs. At least two of my strains are somewhat awkward to extract DNA from. I now have twenty or so good extracts which look fine on an agarose gel- heaps of DNA in a nice tight high mol weight band. Our lab recently commissioned a new Hoeffer DNA fluorometer and I have been checking my DNA concentration for the rapds. Fine for all my exrtracts until I get to two isolates which look fine on agarose but the fluormeter says ther is virtually zilch-zero-none DNA in the sample. Curiously these samples have not been amplifying with the primers that work very well for my other strains. I would say there was no DNA there except there appears to be heaps of it when EtBr stained on Agarose. Any suggestions on what's going on, either with the fluorometer or the rapd's. Cheers and thanks in advance. From owner-rapd@net.bio.net Tue Apr 19 23:00:00 1994 Path: biosci!cornell.edu!MAL5 From: MAL5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: Dissappearing DNA and RAPD's Date: 20 Apr 1994 05:18:55 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199404201218.IAA21012@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net Hi Chris I observed something similar in grape DNA. I used to determine DNA concentration on spectophotometer. One time I tried to compare results with fluorometer and gel. I found enormous difference especially between spec. reading and fluorometer. What I found, I believe, was high amounts of polysaccharides. That glowed nicely on gel and gives good reading on spec but nothing on fluorometer. I started using NaCl (2.5 M) in DNA extraction after Fang et al. 1992. Biotechniques 13:52-56. For info you can read our DNA extraction protocol in the latest issue of Plant Mol Biol Repr Lodhi et al.1994.12:6-13. After that I did not have many problems with DNA. Hope this will help. Greetings Muhammad Lodhi >Anyone out there help with a perplexing problem. I am working on toxic >cyanobacteria doing some RAPDs. At least two of my strains are somewhat >awkward to extract DNA from. I now have twenty or so good extracts which >look fine on an agarose gel- heaps of DNA in a nice tight high mol weight >band. Our lab recently commissioned a new Hoeffer DNA fluorometer and I >have been checking my DNA concentration for the rapds. Fine for all my >exrtracts until I get to two isolates which look fine on agarose but the >fluormeter says ther is virtually zilch-zero-none DNA in the sample. >Curiously these samples have not been amplifying with the primers that >work very well for my other strains. I would say there was no DNA there >except there appears to be heaps of it when EtBr stained on Agarose. > >Any suggestions on what's going on, either with the fluorometer or the >rapd's. > >Cheers and thanks in advance. From owner-rapd@net.bio.net Tue Apr 19 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!howland.reston.ans.net!ee.und.ac.za!csir.co.za!hippo.ru.ac.za!mirk From: mirk@hippo.ru.ac.za (Prof R Kirby) Subject: Fingerprinting Conference - India - December Message-ID: Keywords: Fingerprinting Organization: Rhodes University, Grahamstown, South Africa Date: Wed, 20 Apr 1994 09:21:49 GMT Lines: 8 I've have been told that there is a DNA Fingerprinting Conference in India in December 1994. I cannot find any information on this. Any help out there. Reply to mirk@hippo.ru.ac.za. Ralph Kirby -- Prof Ralph Kirby - Department of Microbiology - Rhodes University Internet: mirk@hippo.ru.ac.za Telephone: (0461) 22023 xt 441 From owner-rapd@net.bio.net Tue Apr 19 23:00:00 1994 Path: biosci!DEAKIN.EDU.AU!huangbx From: huangbx@DEAKIN.EDU.AU (Bixing Huang) Newsgroups: bionet.molbio.rapd Subject: Summary of the strange ethidium bromide stain fast fading Date: 19 Apr 1994 23:23:07 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 84 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199404200622.QAA16740@sol.ccs.deakin.edu.au> NNTP-Posting-Host: net.bio.net Hello Netters, Many thanks to those who have replied to my question. I found that I am not only one to meet the fast fading problem, therefore I compile and post the replies for your reference. Again,thank you very much to everyone. Bixing Huang Dear Bixing, the only thing that I can imagine is alkaline somewhere along the lines which will denature your DNA within the gel and therefore destain it. I wouldn't have a clou where it could come from, maybe the bottle you store your ethidium is from glass and has had NaOH in it in the past? Why don't you add ethidium to your gels anyway? cheers I can't offer an explanation; only the comfort that I too have experienced the same phenomenon. I look forward to somebody providing an answer. rapd@net.bio.net Try to keep your gel and EtBr solution in the dark as much as possible. That always help to maximize fluorescence. #To: rapd@net.bio.net I don't know if this will be of any help but here goes. I stain gels with EtBr in water, wash the gels well in water and leave to destain in 1mM magnesium sulphate for about 15-20 minutes. As far as I know we have had no problems with fading. Another thing we run TAE gels and not TBE but there should be no problems. good luck That is very strange, I've never encountered a problem with fading on the UV box but if the Ethidium solution is old it might not be staining optimally. We use a drop of approximately 20microlitres of a 10mg/ml ethidium bromide solution in our stain and use it fresh every time, rather than storing the staining water solution itself. We stain for 20 mis and then do two washes in 10-15 mins each. Good luck solving your problem, Hi Bixing We had the same problem with our gels. We tried several different things nothing helped at all. Finally thing got ok by itself, or by something that we could not figure out. Please forward mails in response to this message. I'll appreciate. Thanks To: methods-and-reagents@net.bio.net I've seen it before. A rapid fade in flourescents on exposure of the gel to UV. Fortunately, it went away. Didn't have a chance to try anything. To: huangbx@DEAKIN.EDU.AU I read your post from Bionet newsgroup regarding the fading of EtBr before you could do anything. We used to have the same problem after we reused EtBr staining solution for long time. However, you mentioned 55th PCR or 85th PCR which I don't understand. Is that the time that you reuse your agarose gel? If it is the case, how do you recycle it? We reuse agarose for about 10-15 times and it getting brittle and turbid and we have to throw it away. Thanks. From owner-rapd@net.bio.net Tue Apr 19 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: Dissappearing DNA and RAPD's Date: 20 Apr 1994 05:54:35 -0700 Organization: University of Arkansas Lines: 38 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <35AE1933A0@uamercury.uark.edu> NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: "Christopher J. S. Bolch" >Subject: Dissappearing DNA and RAPD's >Date sent: Wed, 20 Apr 1994 02:13:57 GMT >Anyone out there help with a perplexing problem. I am working on toxic >cyanobacteria doing some RAPDs. At least two of my strains are somewhat >awkward to extract DNA from. I now have twenty or so good extracts which >look fine on an agarose gel- heaps of DNA in a nice tight high mol weight >band. Our lab recently commissioned a new Hoeffer DNA fluorometer and I >have been checking my DNA concentration for the rapds. Fine for all my >exrtracts until I get to two isolates which look fine on agarose but the >fluormeter says ther is virtually zilch-zero-none DNA in the sample. >Curiously these samples have not been amplifying with the primers that >work very well for my other strains. I would say there was no DNA there >except there appears to be heaps of it when EtBr stained on Agarose. > >Any suggestions on what's going on, either with the fluorometer or the >rapd's. > >Cheers and thanks in advance. > Sorry about the previous post. I sent the file before I knew what I was doing. Are you sure that what you see on the gel is DNA. There are other macromolecules that can bind Ethidium Bromide and fluoresce. I would recommend you check the UV spectrum (230 to 300 nm) on the suspect preps. If you don't have a nice peak at 260 then you have probably isolated some modified carbohydrate. If you don't have access to a UV spec then just try digesting a microgram with a 6base cutter. That should turn your high MW band into a smear unless it isn't DNA. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Wed Apr 20 23:00:00 1994 Path: biosci!agate!ihnp4.ucsd.edu!swrinde!sgiblab!sgigate.sgi.com!olivea!decwrl!decwrl!usenet.coe.montana.edu!Msu.oscs.montana.edu!uvsmr From: uvsmr@Msu.oscs.montana.edu (Matt Rognlie) Newsgroups: bionet.molbio.rapd Subject: What about RAPD on an organism with field contaminants? Date: Thu, 21 Apr 1994 08:16:45 Organization: Montana State University Lines: 13 Message-ID: <0097D477.3BAB7C40@Msu.oscs.montana.edu> Reply-To: uvsmr@Msu.oscs.montana.edu (Matt Rognlie) NNTP-Posting-Host: trex.oscs.montana.edu I have not seen this point much in the literature, but I assume it must be a problem. What does one do if one wants to do RAPDs on, say, invertebrates from the field (whole organism DNA prep due to size)? We are interested in doing RAPDs on snails that we would expect to also contain: virus, bacteria, algae, fungi, protozoa, oligochetes, etc. etc. Wouldn't this cause the RAPD to produce inconsistent results at best and erroneous data at worst? Any comments appreciated. Matthew Rognlie | Veterinary Molecular Biology | uvsmr@trex.oscs.montana.edu Marsh Lab | phone: 406-994-6379 Montana State University | FAX: 406-994-4303 Bozeman, MT 59717-0360 | From owner-rapd@net.bio.net Wed Apr 20 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: RAPD reactions Date: 21 Apr 1994 13:36:16 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9404212036.AA26337@phibred.phibred.com> NNTP-Posting-Host: net.bio.net Dear Netters, I have a couple of questions for you. 1) I have decreased the volume of my RAPD reaction, from 25 to about 15 ul. I have noticed a decreases in the band number and increases in other cases. Could the decrease be due to less product and increases due to an increase in annealing of primers (scale reduction of the reactio) and better thermal conduction? 2) There are variations in the banding pattern between the same DNA lines. Contamination is eliminated. Do viral, bacterial and other sources of DNA interfere? The material from which the DNA was extraced from was obtained from cotyledons (germinated from surface serilized seed in a sterile petri dish). 3) Can RAPD polymorphic products be cut out, cloned and made into larger primers? Thanks. From owner-rapd@net.bio.net Thu Apr 21 23:00:00 1994 Path: biosci!UNAMVM1.DGSCA.UNAM.MX!carvalho From: carvalho@UNAMVM1.DGSCA.UNAM.MX (Carvalho Torres Alexandro cassio-UACPYP) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD reactions Date: 21 Apr 1994 21:05:27 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: bionet Message-ID: References: <9404212036.AA26337@phibred.phibred.com> NNTP-Posting-Host: net.bio.net On 21 Apr 1994, IAN MURRAY wrote: > Dear Netters, > > I have a couple of questions for you. > > 1) I have decreased the volume of my RAPD reaction, from 25 to about 15 ul. > I have noticed a decreases in the band number and increases in other cases. > Could the decrease be due to less product and increases due to an increase in > annealing of primers (scale reduction of the reactio) and better thermal > conduction? > > 2) There are variations in the banding pattern between the same DNA lines. > Contamination is eliminated. Do viral, bacterial and other sources of DNA > interfere? The material from which the DNA was extraced from was obtained from > cotyledons (germinated from surface serilized seed in a sterile petri dish). > > 3) Can RAPD polymorphic products be cut out, cloned and made into larger > primers? > > Thanks. > Dear Murray, Have you ever decreased all the products of your pcr in the same proportion. A small number of taq, primers and DNTps?. Try do it. I observed that some bands are more thinner in small vol then a big one. Alexandro Carvalho Instituto Nacional de la Nutricion S. Zubiran. Mexico, DF. From owner-rapd@net.bio.net Thu Apr 21 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!netnews.nwnet.net!owl.csrv.uidaho.edu!raven.csrv.uidaho.edu!knaebel From: knaebel@raven.csrv.uidaho.edu (Knaebel David B) Newsgroups: bionet.molbio.rapd Subject: Re: Dissappearing DNA and RAPD's Date: 22 Apr 1994 02:42:58 GMT Organization: University of Idaho, Moscow, Idaho Lines: 33 Distribution: world Message-ID: <2p7dji$gf7@owl.csrv.uidaho.edu> References: <1994Apr20.021357.12871@ml.csiro.au> NNTP-Posting-Host: raven.csrv.uidaho.edu X-Newsreader: TIN [version 1.2 PL2] With cyanobacteria, could there be any odd phenolics produced as metabolites that co-purify with the DNA? This type of material has been a problem with extracting DNA from microbes in soil samples. It usually migrates on a gel at the same location as RNA, but some other people have told me that some of there samples has very large humic-DNA complexes that migrate with the large (genomic) DNA. The other side of this is that the humics (phenolics) can shut down the PCR / RAPD reactions. The complex also absorbs at near 260, so it is difficult to determine if it is DNA, humics, or DNA+humics. If you could separate the materials from each other by HPLC, or TLC, but I don't have any methods for doing that. Good luck Dave Knaebel MMBB / University of Idaho Christopher J. S. Bolch (chris.bolch@ml.csiro.au) wrote: : Anyone out there help with a perplexing problem. I am working on toxic : cyanobacteria doing some RAPDs. At least two of my strains are somewhat : awkward to extract DNA from. I now have twenty or so good extracts which : look fine on an agarose gel- heaps of DNA in a nice tight high mol weight : band. Our lab recently commissioned a new Hoeffer DNA fluorometer and I : have been checking my DNA concentration for the rapds. Fine for all my : exrtracts until I get to two isolates which look fine on agarose but the : fluormeter says ther is virtually zilch-zero-none DNA in the sample. : Curiously these samples have not been amplifying with the primers that : work very well for my other strains. I would say there was no DNA there : except there appears to be heaps of it when EtBr stained on Agarose. : Any suggestions on what's going on, either with the fluorometer or the : rapd's. : Cheers and thanks in advance. From owner-rapd@net.bio.net Thu Apr 21 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: RAPD primer eqaution Date: 22 Apr 1994 12:23:58 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 51 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9404221919.AA06393@phibred.phibred.com> NNTP-Posting-Host: net.bio.net Hello, Around November, ther was a post concerning a theoretical RAPD primer equation from Clark and Lanigan (1993, Mol Biol Evol 10: 1096-1111) that predicted the number number of bands that should be observed. V= expected # of fragments between length s1 and s2 a= 0.25^n n= number of bases in the primer V= (1-2a)^(s1-1) - (1-2a)^(s1) N= size of the genome Exp# of bands= N/(2/a) Exp # of bands between lengths s1 and s2 = V* Exp# of bands --------------------------------------- Has anyone verified this as yet or have any comments? Could the equation be modified to account for variations in the GC content? Could kinetics of the RAPD reaction be tdetermined and incorporated? On another note............ I have used the 96 well plate MJ PTC100 thermocycler for a long time now, constantly ((6mo). It now does not work and the reair service said that there was nothing wrong with it. The same reagents work on another thermocycler that we use (Ericomp using 0.5 ul tubes) to yield RAPD products. Simultaneous experiments showed that the MJ just did not work for RAPD and PCR. Does anyone have an idea of what is wrong?? Furthermore....... D the primers in RAPD compete for binding sites and does this become a limiting factor when the RAPD volumes are scaled down?? Thanks, sorry for the typo Ian Murray Murrayian@phibred.com From owner-rapd@net.bio.net Thu Apr 21 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Louis van de Zande Newsgroups: bionet.molbio.rapd Subject: Re: RAPD reactions Date: 22 Apr 1994 14:37:21 +0100 Organization: Department of Biology, RUGroningen Lines: 34 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2p8juh$c5@mserv1.dl.ac.uk> Reply-To: ZANDELPW@BIOL.RUG.nl Original-To: rapd@dl.ac.uk > Dear Netters, > > I have a couple of questions for you. > > 1) I have decreased the volume of my RAPD reaction, from 25 to about 15 ul. > I have noticed a decreases in the band number and increases in other cases. > Could the decrease be due to less product and increases due to an increase in > annealing of primers (scale reduction of the reactio) and better thermal > conduction? > > 2) There are variations in the banding pattern between the same DNA lines. > Contamination is eliminated. Do viral, bacterial and other sources of DNA > interfere? The material from which the DNA was extraced from was obtained from > cotyledons (germinated from surface serilized seed in a sterile petri dish). > > 3) Can RAPD polymorphic products be cut out, cloned and made into larger > primers? > > Thanks. > As for questions 1. and 2.: There is NOTHING known on the kinetics of RAPD reactions. So all guesses on what happens are equally good until something is examined properly (but who will do this?) Question 3: Yes. Tricky but possible. Try cutting the desired fragment with "sticky end" enzymes first. This will make life and cloning a lot easier (although not the complete fragment will be cloned). Louis v.d. Zande From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!VTVM1.CC.VT.EDU!RUSSULA From: RUSSULA@VTVM1.CC.VT.EDU (Jack Murphy) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 24 Apr 1994 18:17:32 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199404250117.SAA17781@net.bio.net> NNTP-Posting-Host: net.bio.net Dear RAPD people; Here's something for you to think about next time you're mixing up your PCR cocktail. DNA exists as a helix which makes about one coil about every ten base pairs. Unwinding this coil during replication and transcription is accomplished in-vivo by helicases and gyrases. We don't put any helicase or gyrase in our pcr mixes, so . . . what? If you think about it, a 1kb fragment should have 100 complete turns in it. Run this through 35 cycles of amplification and it should give you 2-to-the-35th copies. If the DNA doesnt uncoil, then imagine the mess!! If it does uncoil, then, no mess, but does it or does it not uncoil in our little plastic tubes??? I guess the question boils down to (no pun intended), does denaturing at 95C uncoil the molecule? Yours, Jack Murphy Mycology Lab Virginia Tech Russula@vtvm1.cc.vt.edu From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: mRNA display Date: 25 Apr 1994 11:34:15 -0700 Organization: University of Arkansas Lines: 28 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net >Date sent: Mon, 25 Apr 1994 17:31:40 +0000 (GMT) >From: "Thomas F. Gallagher " >Subject: Re: mRNA display >To: Doug Rhoads >Organization: University College Dublin >Doug >What I mean by self-priming is the presence of bands when the >oligo dTMN primer is omitted from the RT- step. Amplification of this >material with an upstream primer and an oligo dTMN primer gives about >25 bands in our case. This is OK if the bands are constant with each >primer combinbation. The number of bands might obscure interesting >differences in some cases. > >Tommy Gallagher I am not sure that I agree with your last statement. The amplifications that are only from the upstream primer represent legitimate amplicons. Just because only one primer is used don't discount the product. If the band produced shows up in both samples then it is not differentially expressed. You just didn't need both primers. Now, if you have significant genomic DNA contamination then you could be actually doing RAPDs on that contaminating DNA. We have been concerned about this since we have successfully run RAPDs on sub nanogram amounts of genomic DNA. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: compatible mini-preps Date: 25 Apr 1994 11:28:38 -0700 Organization: University of Arkansas Lines: 27 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: HACHEY@abrsle.agr.ca (john hachey) >Subject: compatible mini-preps >Date sent: 25 Apr 1994 10:06:15 -0700 >Hello, > >I am in the process of screening F2 plants for the segregation of my linked >RAPD marker in tomato. Is there a reliable mini-prep compatible with RAPDs >or am I not in Kansas anymore? The idea of hundreds of med-preps is not very >appealing to me > >Thanks, > >John. Check out Zhu, etal (1993) Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. NAR 21(22):5279-5280. We have also had luck with mild homogenization of tissue, boiling water bath in TE+ 0.1% triton, spin and use the supernate for direct addition to RAPD. We have used this for detection of fungal DNAs in infection sites. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!ABRSLE.AGR.CA!HACHEY From: HACHEY@ABRSLE.AGR.CA (john hachey) Newsgroups: bionet.molbio.rapd Subject: compatible mini-preps Date: 25 Apr 1994 10:06:15 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01HBLCXHFSJ6003E6Z@GW.AGR.CA> NNTP-Posting-Host: net.bio.net Hello, I am in the process of screening F2 plants for the segregation of my linked RAPD marker in tomato. Is there a reliable mini-prep compatible with RAPDs or am I not in Kansas anymore? The idea of hundreds of med-preps is not very appealing to me Thanks, John. -------- ,VV, : (OO) : /..\ : hachey@abrsle.agr.ca (~--~) : ---^^--- From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!agate!dog.ee.lbl.gov!ihnp4.ucsd.edu!swrinde!emory!europa.eng.gtefsd.com!news.umbc.edu!haven.umd.edu!purdue!mozo.cc.purdue.edu!inet.d48.lilly.com!inet!roblogan Newsgroups: bionet.molbio.rapd Subject: RAPD faqs? Message-ID: From: roblogan@lilly.com (Rob Logan) Date: Mon, 25 Apr 94 10:51:02 GMT Distribution: world Nntp-Posting-Host: phyloterm.d50.lilly.com X-Newsreader: VersaTerm Link v1.1 Lines: 6 I've only recently discovered this discussion group and have yet to see any references to a faq list(assuming it exists). Could someone please send me a location where I could ftp to? Any help would be greatly appreciated. Rob Logan From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!UNR.EDU!hoelzer From: hoelzer@UNR.EDU (Guy Hoelzer) Newsgroups: bionet.molbio.rapd Subject: Unsubscribe Date: 25 Apr 1994 08:44:27 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net To Whom it may Concern: Please unsubscribe me from the RAPD listserver. I can now read all postings on the news service (i.e. bionet.molbio.rapd). Thanks, Guy Hoelzer hoelzer@unr.edu From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: mRNA display Date: 25 Apr 1994 05:50:55 -0700 Organization: University of Arkansas Lines: 26 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: "Thomas F. Gallagher " >Subject: mRNA display >Date sent: 25 Apr 1994 09:11:23 +0100 >Has anyone encountered a problem with self priming in messenger RNA display? We > have been using this technique to look at mRNA profiles from juvenile and adu > lt oak. We use total RNA in the 1st strand cDNA step. We get good profiles in > the PCR reactions > > >Tommy Gallagher & Pat O'Mahoney >Botany Dept >Univ College Dublin. > We have been doing differential display in yeasts but have seen no evidence of self priming, if what you mean is bands that are present in all lanes regardless of the 'upstream' primer. We too have used only total RNA for first strand synthesis. Perhaps Gallagher and O'Mahoney would comment on WHY they are concerned?? Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!internet!biosci!not-for-mail From: kristoff (David Kristofferson) Newsgroups: bionet.molbio.rapd Subject: IMPORTANT BIOSCI INFORMATION Date: 25 Apr 1994 02:00:19 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 244 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199404250900.CAA02607@net.bio.net> NNTP-Posting-Host: net.bio.net Three important items follow: BIOSCI archive searching by e-mail, the BIOSCI FAQ, and the BIOSCI User Address Directory form. If you have not yet listed yourself in our e-mail address directory, please take a few minutes to complete and return the form below. If your address information has changed since you listed yourself, please send us an updated form. 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Thanks again for your cooperation! --------------- please cut here and return portion below --------------- New information or Update to old record (enter N or U): date (DD-MM-YY): first name: middle initial: family name: job title: e-mail address: e-mail network: phone number: FAX number: institution: address1: address2: address3: city: state/province: country: postal code: research interest: research interest: comment: comment: comment: comment: comment: From owner-rapd@net.bio.net Sun Apr 24 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: "Thomas F. Gallagher " Newsgroups: bionet.molbio.rapd Subject: mRNA display Date: 25 Apr 1994 09:11:23 +0100 Organization: University College Dublin Lines: 9 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2pftvb$69@mserv1.dl.ac.uk> Original-To: rapd@dl.ac.uk Has anyone encountered a problem with self priming in messenger RNA display? We have been using this technique to look at mRNA profiles from juvenile and adu lt oak. We use total RNA in the 1st strand cDNA step. We get good profiles in the PCR reactions Tommy Gallagher & Pat O'Mahoney Botany Dept Univ College Dublin. From owner-rapd@net.bio.net Mon Apr 25 23:00:00 1994 Path: biosci!ABRSLE.AGR.CA!DEMEKE From: DEMEKE@ABRSLE.AGR.CA Newsgroups: bionet.molbio.rapd Subject: Combination of decamers to generate more polymorphisms Date: 26 Apr 1994 14:24:42 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01HBN0336IN6003K7I@GW.AGR.CA> NNTP-Posting-Host: net.bio.net We are using descamers to generate polymorphism between near-isogenic lines differing for disease resistance in wheat. We have not been successful in getting polymorphism in some of our lines. Combining decamers produced the desired polymorphism in Hessian fly resistance in wheat (Abstract- Plant Genome II Meeting). Does any one have experience on this? If so what is the rationale? Tigst Demeke Lethbridge Research Station DEMEKE@ABRSLE.AGR.CA From owner-rapd@net.bio.net Tue Apr 26 23:00:00 1994 Path: biosci!agate!dog.ee.lbl.gov!ihnp4.ucsd.edu!swrinde!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!moe.ksu.ksu.edu!netserv.unmc.edu!netserv.unmc.edu!not-for-mail From: dchakrav@netserv.unmc.edu (Dhruba Chakravarti) Newsgroups: bionet.molbio.rapd Subject: Re: Summary of the strange ethidium bromide stain fast fading Date: 27 Apr 1994 13:25:16 -0500 Organization: University of Nebraska Medical Center, Omaha, NE, USA Lines: 9 Distribution: bionet Message-ID: <2pmamc$qr8@netserv.unmc.edu> References: <199404200622.QAA16740@sol.ccs.deakin.edu.au> NNTP-Posting-Host: netserv.unmc.edu I think that your problem is the UV transilluminator. Is it short wave UV (254nm?). Short wave UV bleaches EtBr stained DNA and would result in disappearing DNA band. I recommend using a longwave tansiulluminator (300nm) preferably of low-energy. Hope that helps. Dhruba Chakravarti. From owner-rapd@net.bio.net Wed Apr 27 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!howland.reston.ans.net!news.cac.psu.edu!news.tc.cornell.edu!travelers.mail.cornell.edu!cornell!batcomputer!ghost.dsi.unimi.it!genes!heitor From: heitor@genes.icgeb.trieste.it (Heitor Luiz da C. Coutinho) Subject: Reproducibility problems Message-ID: <1994Apr28.140233.4483@genes.icgeb.trieste.it> Summary: Enquiry about other experiences with RAPD problems of reproducibility Keywords: RAPD/reproducibility Organization: ICGEB Date: Thu, 28 Apr 1994 14:02:33 GMT Lines: 30 Hi netters, I have been facing one of the main problems of RAPDs: reproducibility between labs. I have used RAPDs very successfully to fingerprint strains of rhizobia and bradyrhizobia in a HYBAID thermo-cycler using superTaq (produced by HT Technology in the UK). The banding patterns obtained were pretty sharp and informative ( Coutinho et al., Let.Appl.Microb. 1993, 17, 282-284; Kay etal. Microbiology, 1994, in press) Using the same primers, but different machine and enzyme (Perkin-Elmer 9600, and Promega Taq, respectively), I'm not being able to obtain the same results. The banding patterns are now diffuse, with one predominant band while the others are very faint. I had to change some cycling parameters in order to get any results from the Perkin-Elmer machine, due to the higher heat-transfer efficiency. Basically I reduced the annealing temperatures and included a ramp of 1 minute to reach the extension temperature. This made me get some patterns (at last). But I still have the problem with smeary and diffused, with one or two predominant bands, fingerprints. Did any of you face similar problems when moving to another lab and trying to obtain RAPDs under different conditions? I am thinking of trying different enzymes. Are any of you working with the Stoffel fragment? How about the electrophoresis conditions? Or even the transilluminator? I suspected the quality of the water used to prepare the TBE buffer. Is this a reasonable suspicion? I also have doubts about the transilluminator, which is very old (I can even see the UV bulbs inside). I appreciate very much any idea, suggestions or comments from you. Regards, Heitor From owner-rapd@net.bio.net Wed Apr 27 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!ihnp4.ucsd.edu!munnari.oz.au!newsroom.utas.edu.au!ml.csiro.au!mac_1303.ml.csiro.au!chris.bolch From: Christopher J. S. Bolch Subject: Re: Dissappearing DNA and RAPD's Message-ID: <1994Apr28.064449.13323@ml.csiro.au> X-Xxmessage-Id: X-Xxdate: Thu, 28 Apr 94 05: 46:52 GMT Sender: news@ml.csiro.au Organization: CSIRO Division of Fisheries, Hobart, Tasmania X-Useragent: Version 1.1.3 References: <35AE1933A0@uamercury.uark.edu> Date: Thu, 28 Apr 1994 06:44:49 GMT Lines: 16 In article <35AE1933A0@uamercury.uark.edu> Doug Rhoads, DRHOADS@MERCURY.UARK.EDU writes: If you don't have access to a UV spec then just >try digesting a microgram with a 6base cutter. That should turn your high >MW band into a smear unless it isn't DNA. Just did it. Cut it with Ava I. Result: nice tight band disappears and get a nice smear. This means it is DNA?? Any other ideas guys?? Could it be some tertiary structure of the DNA that inhibits dye binding and RAPD primer annealing?? Seems unlikely after 4 minutes at 94!C before Cycling begins. Should I extend the Pre-cycling melting time?? Christopher Bolch From owner-rapd@net.bio.net Sat Apr 30 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!europa.eng.gtefsd.com!emory!news.cc.emory.edu!news.cc.emory.edu!not-for-mail From: vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) Newsgroups: bionet.molbio.rapd Subject: Degraded DNA sample Date: 1 May 1994 12:42:17 -0400 Organization: Emory University Lines: 11 Message-ID: <2q0m59$eur@emoryu1.cc.emory.edu> NNTP-Posting-Host: emoryu1.cc.emory.edu X-Newsreader: TIN [version 1.2 PL2] I do RAPDs on human DNA samples and some of them are from Paraffin embedded tissues. The DNA in these samples is partially degraded (about half the DNA is 500bp or less and the rest is upto10KB or so). Although these samples are fine for regular PCR, reproducibility is a problem with RAPDs compared to high mol wt DNA samples. Do others have the same problem with partially degraded samples? If so can this be overcome by isolating the high mol DNA by, say, cutting it out of preparatory agarose gel?? If there are nicks even in high mol wt DNA, do they cause problems with reproducibility??? Would appreciate any input on this. From owner-rapd@net.bio.net Sat Apr 30 23:00:00 1994 Path: biosci!news.cs.umb.edu!hsdndev!dartvax.dartmouth.edu!saturn.caps.maine.edu!maine.maine.edu!io01072 Organization: University of Maine System Date: Sat, 30 Apr 1994 12:13:23 EDT From: Hugo Volkaert Message-ID: <94120.121323IO01072@MAINE.MAINE.EDU> Newsgroups: bionet.plants,bionet.molbio.rapd Subject: TA microsatellite Lines: 29 Xref: biosci bionet.plants:3105 bionet.molbio.rapd:545 Hi bionetters I am doing research on the occurrence and polymorphism of microsatellites in co nifers. I haven't found a lot of polymorphisms in GT/CA repeats or CT/GA. Howev er, I have an AT/AT microsatellite that shows a high rate of variability. There are a few problems with it though. 1) I originally isolated it as a CA-repeat that was followed by a stretch of TA s. I amplified it from the genomic DNA and found that all the products I obtain ed are shorter than the original. Then I cloned the PCR amplified fragments and sequenced them. To my surprise, the CA/GT microsatellite was not present. What was left was a stretch of TAs. Am I amplifying a microsatellite family and is the CA/GT + TA member that I cloned only a minority???? I am not so sure since I haven't obtained more than 2 alleles from a single tree up to now. 2) When I PCR the plasmids containing the different AT stretches, I obtain two or more distinct bands as a result. Instability of the TA repeat in the plasmid /bacterial host is almost ruled out. I get a nice sequencing gel, upon isolatio n of plasmid from single colonies I always get two identical bands in several i solates, I get distinct bands and not smears. I assume that something is going on during the PCR amplification of the TA repeats. It cannot be a random proces s, because I get distinct bands, not a smear. What can it be????? Has anyone se en such results before? Any ideas on what might be going on are welcome. If you need more info, please E-mail meor if it is relevant to the whole net, post it. Thanks Hugo Volkaert Dept of Forest Environment Science, Univ of Maine 5755 Nutting Hall, Orono, ME 04473 207 581 2819 IO01072@maine.maine.edu