From owner-rapd@net.bio.net Wed Jan 05 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!warwick!bham!bcs88.bham.ac.uk!user From: A.C.Hilton@UK.AC.BHAM (Anthony Hilton) Newsgroups: bionet.molbio.rapd Subject: Test-Please Ignore!! Followup-To: bionet.molbio.rapd Date: 6 Jan 1994 12:36:54 GMT Organization: University of Birmingham Lines: 2 Distribution: world Message-ID: NNTP-Posting-Host: bcs88.bham.ac.uk Just a quick test to see if I can post out on this network.. Wig. From owner-rapd@net.bio.net Thu Jan 06 22:00:00 1994 Path: biosci!hubcap!darwin.sura.net!haven.umd.edu!news.umbc.edu!europa.eng.gtefsd.com!howland.reston.ans.net!pipex!uknet!bhamcs!bham!bcs88.bham.ac.uk!user From: A.C.Hilton@UK.AC.BHAM (Anthony Hilton) Newsgroups: bionet.molbio.rapd Subject: GelManager-Biosystematica Followup-To: bionet.molbio.rapd Date: 7 Jan 1994 10:08:44 GMT Organization: University of Birmingham Lines: 13 Distribution: world Message-ID: NNTP-Posting-Host: bcs88.bham.ac.uk Hello, I have recently acquired a demonstration disk of GelManager for windows from Biosystematica with a view to constructing a database of RAPD Fingerprints for later comparisons. The demo disk was limited in the facilities that it allowed you to use (as expected!) but at the same time was sufficient to make me enthusiastic that the software may meet my requirements. As Biosystematica is based in the Czech Republic and the package costs £895 I am eager to hear from anyone that has used this or a similar software package and has any recommendations as to its ease of use and limitations. Thanks in anticipation, A.Hilton. From owner-rapd@net.bio.net Thu Jan 06 22:00:00 1994 Path: biosci!hubcap!darwin.sura.net!howland.reston.ans.net!pipex!uknet!bhamcs!bham!bcs88.bham.ac.uk!user From: A.C.Hilton@UK.AC.BHAM (Anthony Hilton) Newsgroups: bionet.molbio.rapd Subject: GelManager-Biosystematica Followup-To: bionet.molbio.rapd Date: 7 Jan 1994 10:18:54 GMT Organization: University of Birmingham Lines: 10 Distribution: world Message-ID: NNTP-Posting-Host: bcs88.bham.ac.uk Hello, I have recently acquired a copy of GelManager demo-disk from BioSystematica with a view to creating a database of RAPD profiles for later comparison. The demo was limited in the applications available for use (as expected!) and so I am interested in the views of any users of gel scan software with ant recommendations / limitations experienced. Thankyou in anticipation, A. Hilton. From owner-rapd@net.bio.net Mon Jan 17 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: "Marcelo Carnier Dornelas" Newsgroups: bionet.molbio.rapd Subject: Band analysis: Software available? Date: 18 Jan 1994 14:27:42 -0000 Lines: 41 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2hgrku$o3c@mserv1.dl.ac.uk> Received-Date: Tue, 18 Jan 94 12:25:24 -0300 Original-To: rapd@net.bio.net X-Pmrqc: 1 Posted-Date: Tue, 18 Jan 1994 12:27:01 Hello there! I'm always confused when I have to analyse my RAPD gels because I generally don't know which bands are suitable (I mean SHARP and STRONG enough) and which are not (too much faint) to considerate. So, does anybody out there knows if there is a Software that could decide this for me, I mean, that could decide which bands are so faint that shouldn't be used? Does anybody has experience on this? Is there any trick I should know that would make this "band- discarting" decision easier? Thanks Marcelo -------------------------------------------------------------------- *** Marcelo Carnier Dornelas * . . * Genetics Department * * "Luiz de Queiroz" School of Agronomy * \___/ * University of Sao Paulo * * MCDORNEL@tuvira.ciagri.usp.br *** FAX 0194 22 3087 SMILE, tomorrow will be worse! (This logo was created by H.Wessig) **ALL OPINIONS EXPRESSED HEREIN ARE ALL MINE, NOT MY BOSS'S ** (THAT'S WHAT SHE PAYS ME FOR) _____________________________________________________________________ From owner-rapd@net.bio.net Mon Jan 17 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Louis van de Zande Newsgroups: bionet.molbio.rapd Subject: Re: Band analysis: Software available? Date: 18 Jan 1994 14:57:43 -0000 Organization: Department of Biology, RUGroningen Lines: 18 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2hgtd7$peo@mserv1.dl.ac.uk> Reply-To: ZANDELPW@BIOL.RUG.nl X-pmrqc: 1 Original-To: rapd@dl.ac.uk > Hello there! > > I'm always confused when I have to analyse my RAPD gels > because I generally don't know which bands are suitable (I mean SHARP > and STRONG enough) and which are not (too much faint) to considerate. > So, does anybody out there knows if there is a Software that could > decide this for me, I mean, that could decide which bands are so > faint that shouldn't be used? Does anybody has experience on this? > Is there any trick I should know that would make this "band- > discarting" decision easier? > Thanks > > Marcelo > Trust yourself, not a machine!!! Louis From owner-rapd@net.bio.net Mon Jan 17 22:00:00 1994 Path: biosci!CS.Arizona.EDU!organpipe.uug.arizona.edu!uunet!cs.utexas.edu!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd,bionet.molbio.evolution,bionet.software Subject: supercomputer access Date: 18 Jan 1994 09:53:50 -0500 Organization: R.S.M.A.S. Lines: 19 Message-ID: <2hgt5u$rg3@oj.rsmas.miami.edu> NNTP-Posting-Host: oj.rsmas.miami.edu Xref: biosci bionet.molbio.rapd:386 bionet.molbio.evolution:1350 bionet.software:6994 I haven't seen any recent solitations for free access to any of the national supercomputer centers lately, so: Does anyone know of any free access available at any supercomputer center where PHYLIP is or could be used? I have large data sets of of 300+ characters for 6000+ species for the discrete character programs. MIX takes several weeks to complete on a DEC5000/Sun 4 class machine and usurps memory and processor resources while running. These jobs are rapidly wearing out their welcome on all the hosts I have access to currently. I would also be interested in hearing others approaches to processing LARGE collections of RAPDs data. Jack Kramer University of Florida Tropical Research and Education Center Homestead, FL From owner-rapd@net.bio.net Wed Jan 19 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!moe.ksu.ksu.edu!pst.pp.ksu.edu!user From: pst@matt.ksu.ksu.edu (Paul St. Amand) Newsgroups: bionet.molbio.rapd Subject: Some batches of light mineral oil can kill your PCR or RAPD reactions! Followup-To: bionet.molbio.rapd Date: Thu, 20 Jan 1994 11:56:09 -0600 Organization: USDA & KSU Agronomy dept. Lines: 16 Message-ID: NNTP-Posting-Host: pst.pp.ksu.edu We have recently found that a BRAND NEW gallon of light mineral oil was somehow preventing our PCRs and RAPDs from working. Has anyone else had this happen? We autoclaved the bad batch and some working oil and found that autoclaving did not restore the bad batch, nor did it cause the working batch to fail. Can anyone suggest why light mineral oil could prevent PCRs or RAPDs from working? Possible reasons: contamination, oxidation, light, or others? Paul St. Amand, pst@matt.ksu.ksu.edu, Voice: (913) 532-6168 USDA & KSU Agronomy, 122 Throckmorton Hall, Manhattan KS 66506 From owner-rapd@net.bio.net Wed Jan 19 22:00:00 1994 Newsgroups: bionet.molbio.rapd,bionet.molbio.evolution,bionet.software Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!fzestabr From: fzestabr@chip.ucdavis.edu () Subject: Re: supercomputer access Message-ID: Followup-To: bionet.molbio.rapd,bionet.molbio.evolution,bionet.software Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] References: <2hgt5u$rg3@oj.rsmas.miami.edu> Date: Thu, 20 Jan 1994 00:38:46 GMT Lines: 27 Xref: biosci bionet.molbio.rapd:388 bionet.molbio.evolution:1355 bionet.software:7008 Jack Kramer (kramer@oj.rsmas.miami.edu) wrote: : I haven't seen any recent solitations for free access to any of the : national supercomputer centers lately, so: : Does anyone know of any free access available at any supercomputer : center where PHYLIP is or could be used? I have large data sets of I currently have access to the supercomputer facilities in Pittsburgh. Basically all I do is use the programs for sequence analysis. But I do think that they allow people to do other research activities and they have a large cluster of software packages. I don't know specifically about PHYLIP though. I have an account there and I got it by writing a very basic proposal. The proposals are written more for the required red tape because NSF provides funding for the facilities. So you fill out a proposal and request for a specific amount of supercomputer use time, no money required. The only address I have is: Pittsburgh Supercomputing Center Mellon Institute Buidling 4400 Fifth Avenue Pittsburgh, PA 15213 If you need more information just ask Elizabeth From owner-rapd@net.bio.net Wed Jan 19 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!uunet!dziuxsolim.rutgers.edu!gandalf.rutgers.edu!schaffnr From: schaffnr@gandalf.rutgers.edu (Donald Schaffner) Newsgroups: bionet.molbio.rapd,bionet.molbio.evolution,bionet.software Subject: Re: supercomputer access Message-ID: Date: 20 Jan 94 17:53:30 GMT References: <2hgt5u$rg3@oj.rsmas.miami.edu> Followup-To: bionet.molbio.rapd Organization: Rutgers Univ., New Brunswick, N.J. Lines: 9 Xref: biosci bionet.molbio.rapd:390 bionet.molbio.evolution:1356 bionet.software:7012 Another supercomputer question: I am looking for a supercomputer that has the GENSTAT statistics software on it. The analyses we are currently running on the PC version of the software can take days. Does anyone know of a supercomputer site that has GENSTAT? Thanks! From owner-rapd@net.bio.net Thu Jan 20 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Elizabeth BOULDING Newsgroups: bionet.molbio.rapd Subject: PCR machine by MJ Research model PTC-100 Date: 21 Jan 1994 14:42:47 -0000 Lines: 14 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2hopl7$4dl@mserv1.dl.ac.uk> Original-To: rapd@net.bio.net I have just purchased a PCR machine made by MJ Research (model PTC-100). I have read an a 1993 article in PCR methods and Applications which suggested that the MJ Research Machine was unable to amplify DNA fragments larger than 1.5 kb with a given set of RAPD primers whereas the Perkin-Elmer and the Techne machines amplified 3 fragments larger than 1.5 kb. I know there are people in this group who own MJ Research PCR machines and I am curious whether they have been consistantly successful at amplifying large fragments. Elizabeth Boulding. boulding@uoguelph.ca From owner-rapd@net.bio.net Thu Jan 20 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!library.ucla.edu!europa.eng.gtefsd.com!uunet!utcsri!newsflash.concordia.ca!canopus.cc.umanitoba.ca!tribune.usask.ca!skyfox.usask.ca!lees From: lees@skyfox.usask.ca Newsgroups: bionet.molbio.rapd Subject: TEST Date: 21 JAN 94 21:23:25 GMT Organization: University of Saskatchewan Lines: 1 Message-ID: <21JAN94.21232581@skyfox.usask.ca> NNTP-Posting-Host: sask.usask.ca please ignore this message, this is a test. From owner-rapd@net.bio.net Thu Jan 20 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: Some batches of light mineral oil can kill your PCR or RAPD reactions! Date: 21 Jan 1994 07:49:38 -0000 Lines: 22 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ho1ei$eh6@mserv1.dl.ac.uk> X-Sender: mal5@postoffice.mail.cornell.edu Original-To: pst@matt.ksu.ksu.edu (Paul St. Amand) Paul St. Amand wrote, "We have recently found that a BRAND NEW gallon of light mineral oil was somehow preventing our PCRs and RAPDs from working. Has anyone else had this happen? We autoclaved the bad batch and some working oil and found that autoclaving did not restore the bad batch, nor did it cause the working batch to fail." I have no direct experience with bad mineral oil but a visiting scientist in our lab. had it. After a short training in our lab. he went back to his country and started running RAPD reactions on grape DNA. For about 2 months he could not get the amplification. We suggsted him to change everything else except mineral oil till he ran out of the older bottle and bought a new one. Everything started working. It seemed like that the mineral oil had some chemical contamination. But the actual cause could never be traced as nothing was left to test. Muhammad A. Lodhi Cornell University Geneva, NY 14456 From owner-rapd@net.bio.net Thu Jan 20 22:00:00 1994 Path: biosci!lhc!darwin.sura.net!howland.reston.ans.net!news.intercon.com!panix!cmcl2!ACFcluster.NYU.EDU!LEFEVRE From: lefevre@ACFcluster.NYU.EDU Newsgroups: bionet.molbio.rapd Subject: Primer analysis and design: software available? Date: 20 Jan 1994 22:52:14 GMT Organization: New York University, NY, NY Lines: 3 Message-ID: <2hn1uu$ol4@cmcl2.NYU.EDU> Reply-To: lefevre@ACFcluster.NYU.EDU NNTP-Posting-Host: axp1.acf.nyu.edu I am trying to fing a program which will compare genes for areas of homology. Does anyone know of such Software? Comparison by Amino acid is OK too. I hope to be able to go directly from Genebank. From owner-rapd@net.bio.net Thu Jan 20 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: "" Newsgroups: bionet.molbio.rapd Subject: MINERAL OIL Date: 21 Jan 1994 17:05:04 -0000 Lines: 7 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2hp200$aku@mserv1.dl.ac.uk> Original-To: RAPD@NET.bio.net Re possible mineral oil contamination. When I first started running RAPD reaction I made the mistake of using off the shelf oil instead of reagent grade. The potential contaminant was vitamin E used as a stabilizer. I'm not completely sure that's what caused a problem but there is a phenol ring in the chemical. R. Wilkerson From owner-rapd@net.bio.net Sun Jan 23 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!warwick!uknet!EU.net!howland.reston.ans.net!spool.mu.edu!uwm.edu!msuinfo!harbinger.cc.monash.edu.au!yarrina.connect.com.au!warrane.connect.com.au!pandora.connect.com.au!user From: karyn@warrane.connect.com.au (Karyn Davis) Newsgroups: bionet.molbio.rapd Subject: Software for RAPD data analysis? Followup-To: bionet.molbio.rapd Date: Mon, 24 Jan 1994 17:12:29 +1000 Organization: Connect.com.au P/L, Sydney, Australia Lines: 10 Distribution: world Message-ID: NNTP-Posting-Host: pandora.connect.com.au Hi, I am just starting out with RAPDs. I'm wondering what software people are using for the analysis of their results. Please email any recommendations to me and I will post a summary of the replies. Thanks Karyn Davis From owner-rapd@net.bio.net Wed Jan 26 22:00:00 1994 Path: biosci!CS.Arizona.EDU!math.arizona.edu!news.Arizona.EDU!hamblin.math.byu.edu!sol.ctr.columbia.edu!howland.reston.ans.net!cs.utexas.edu!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd,bionet.software Subject: GelCompar software? Date: 27 Jan 1994 10:02:31 -0500 Organization: R.S.M.A.S. Lines: 9 Message-ID: <2i8l27$q4a@oj.rsmas.miami.edu> NNTP-Posting-Host: oj.rsmas.miami.edu Xref: biosci bionet.molbio.rapd:397 bionet.software:7052 We are getting ready to buy additional software to augment our image analysis system which will help with gel band pattern manipulation and comparison. I would be interested in any comments on GelCompar by Applied Maths in Kortrijk, Belgium. Their literature looks fantastic and that scares me. Jack Kramer kramer@fiu.edu From owner-rapd@net.bio.net Thu Jan 27 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Newsgroups: bionet.molbio.rapd Subject: DNA concentration Date: 28 Jan 1994 13:28:04 -0000 Lines: 15 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ib3t4$5c1@mserv1.dl.ac.uk> Apparently-To: rapd@net.bio.net Original-To: rapd@net.bio.net Reply to a question by Kari Kammiovirta concerning DNA concentration for RAPDs: We also find it necessary to measure concentration of each DNA sample and adjust all the samples to equal concentration. We use an intercalating dimeric dye YoPro (available from Molecular Probes Inc.). The dye and some ribonuclease are added to an aliquot of the DNa sample, and after 30 min (to give the RNase time to act), fluorescence is measured. This is set up easily in a 96-well plate. A set of dilutions of a DNA concentration standard (salmon sperm, or whatever) is also measured, and a calibration curve is constructed. The whole process can be automated using a pipetting robot (we use Beckman Biomek). I believe I posted a more detailed protocol here a while ago. Antoni Rafalski DuPont Molecular Breeding From owner-rapd@net.bio.net Thu Jan 27 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Newsgroups: bionet.molbio.rapd Subject: DNA concentration Date: 28 Jan 1994 18:42:50 -0000 Organization: Brigham Young University Lines: 58 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ibmba$jah@mserv1.dl.ac.uk> X-Vms-To: IN%"rapd@net.bio.net" Original-To: rapd@net.bio.net >Hello! > >I'm quite new in this newsgroup ( @ 1 month) and I'm curious about DNA >concentration in RAPD. There haven't been much discussion about that issue. >I've been doing RAPD's for 2 yrs and I have always checked the concentration >in EtBr-plate and made dilution 10ng/ul. Usually folks have only given some >dilution ratio ( 1/100 or 1/1000). I have noticed that my DNA extractions >yield large variation of concentrations depending of strain(working mostly >with fungi but also plants). So if I use only some standard dilution ratio, >I would have huge variation in DNA concentration in the final reaction and I' >ve been thinking that it also affects to the banding pattern.Can somebody >please explain is concentration check necessary orjust my own imagination. > >Thanks in advance. > >Kari Kammiovirta,Dept. of Plant Biology, Helsinki Univ. > >PS. Sorry about my not-so-fluent english. I titrated template concentration (Drosophila melanogaster) in order to attempt to choose a concentration that is in the middle of a plateau so far as reproducibility of bands is concerned. The plateau turned out to be narrower than I had hoped, but workable. The banding pattern at 40 and 80 ng/mcl were very similar. The bands present were the same, although one band was more intense at the higher concentration. The other concentrations were unsatisfactory (see below). ng/mcl results 10 no amplification 20 about half as many bands as at the higher concentrations 100 same bands, less amplification than at 20 ng/mcl 200 no amplification I chose to use 60 ng/mcl as my standard concentration. Since I am amplifying DNA in crude extracts from single flies, I cannot estimate the DNA concentration directly. I know about how much DNA is in a single fly and make dilutions accordingly, assuming that I get about the same amount of DNA from each fly. Since I know that this will not always be correct, I need to be on a plateau to give me some leeway and still produce reliable amplification. To check the assumption that I am on such a plateau, I have also titrated the crude template extracts. The result is that I can go about a factor of two in template concentration in either direction and get the same amplification pattern. James L. Farmer Department of Zoology 571 WIDB Brigham Young University Provo, Utah 84602, USA (801) 378-2153 (OFFICE) (801) 378-7499 (FAX) FARMERJ@YVAX.BYU.EDU FARMERJ@BYUVAX.BITNET From owner-rapd@net.bio.net Thu Jan 27 22:00:00 1994 Path: biosci!daresbury!keele!uknet!pipex!sunic!news.funet.fi!klaava!MMKAB-01208.pc.Helsinki.FI!kkammiov From: kkammiov@viikki21.helsinki.fi (KARI KAMMIOVIRTA (MMKAB)) Newsgroups: bionet.molbio.rapd Subject: DNA-concentration Date: Fri, 28 Jan 1994 12:24:18 GMT Organization: University of Helsinki Lines: 18 Message-ID: NNTP-Posting-Host: mmkab-01208.pc Hello! I'm quite new in this newsgroup ( @ 1 month) and I'm curious about DNA concentration in RAPD. There haven't been much discussion about that issue. I've been doing RAPD's for 2 yrs and I have always checked the concentration in EtBr-plate and made dilution 10ng/ul. Usually folks have only given some dilution ratio ( 1/100 or 1/1000). I have noticed that my DNA extractions yield large variation of concentrations depending of strain(working mostly with fungi but also plants). So if I use only some standard dilution ratio, I would have huge variation in DNA concentration in the final reaction and I' ve been thinking that it also affects to the banding pattern.Can somebody please explain is concentration check necessary orjust my own imagination. Thanks in advance Kari Kammiovirta,Dept. of Plant Biology, Helsinki Univ. PS. Sorry about my not-so-fluent english. From owner-rapd@net.bio.net Fri Jan 28 22:00:00 1994 Path: biosci!rutgers!biosci!CS.Arizona.EDU!math.arizona.edu!news.Arizona.EDU!hamblin.math.byu.edu!sol.ctr.columbia.edu!howland.reston.ans.net!cs.utexas.edu!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd,bionet.software Subject: GelCompar software? Message-ID: <2i8l27$q4a@oj.rsmas.miami.edu> Date: 27 Jan 94 15:02:31 GMT Organization: R.S.M.A.S. Lines: 9 NNTP-Posting-Host: oj.rsmas.miami.edu Xref: biosci bionet.molbio.rapd:405 bionet.software:7104 We are getting ready to buy additional software to augment our image analysis system which will help with gel band pattern manipulation and comparison. I would be interested in any comments on GelCompar by Applied Maths in Kortrijk, Belgium. Their literature looks fantastic and that scares me. Jack Kramer kramer@fiu.edu From owner-rapd@net.bio.net Fri Jan 28 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd,bionet.software Subject: GelCompar software? Date: 27 Jan 1994 10:02:31 -0500 Organization: R.S.M.A.S. Lines: 9 Message-ID: <2i8l27$q4a@oj.rsmas.miami.edu> NNTP-Posting-Host: oj.rsmas.miami.edu Xref: biosci bionet.molbio.rapd:401 bionet.software:7086 We are getting ready to buy additional software to augment our image analysis system which will help with gel band pattern manipulation and comparison. I would be interested in any comments on GelCompar by Applied Maths in Kortrijk, Belgium. Their literature looks fantastic and that scares me. Jack Kramer kramer@fiu.edu From owner-rapd@net.bio.net Fri Jan 28 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Newsgroups: bionet.molbio.rapd Subject: DNA concentration Date: 28 Jan 1994 18:42:50 -0000 Organization: Brigham Young University Lines: 58 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ibmba$jah@mserv1.dl.ac.uk> X-Vms-To: IN%"rapd@net.bio.net" Original-To: rapd@net.bio.net >Hello! > >I'm quite new in this newsgroup ( @ 1 month) and I'm curious about DNA >concentration in RAPD. There haven't been much discussion about that issue. >I've been doing RAPD's for 2 yrs and I have always checked the concentration >in EtBr-plate and made dilution 10ng/ul. Usually folks have only given some >dilution ratio ( 1/100 or 1/1000). I have noticed that my DNA extractions >yield large variation of concentrations depending of strain(working mostly >with fungi but also plants). So if I use only some standard dilution ratio, >I would have huge variation in DNA concentration in the final reaction and I' >ve been thinking that it also affects to the banding pattern.Can somebody >please explain is concentration check necessary orjust my own imagination. > >Thanks in advance. > >Kari Kammiovirta,Dept. of Plant Biology, Helsinki Univ. > >PS. Sorry about my not-so-fluent english. I titrated template concentration (Drosophila melanogaster) in order to attempt to choose a concentration that is in the middle of a plateau so far as reproducibility of bands is concerned. The plateau turned out to be narrower than I had hoped, but workable. The banding pattern at 40 and 80 ng/mcl were very similar. The bands present were the same, although one band was more intense at the higher concentration. The other concentrations were unsatisfactory (see below). ng/mcl results 10 no amplification 20 about half as many bands as at the higher concentrations 100 same bands, less amplification than at 20 ng/mcl 200 no amplification I chose to use 60 ng/mcl as my standard concentration. Since I am amplifying DNA in crude extracts from single flies, I cannot estimate the DNA concentration directly. I know about how much DNA is in a single fly and make dilutions accordingly, assuming that I get about the same amount of DNA from each fly. Since I know that this will not always be correct, I need to be on a plateau to give me some leeway and still produce reliable amplification. To check the assumption that I am on such a plateau, I have also titrated the crude template extracts. The result is that I can go about a factor of two in template concentration in either direction and get the same amplification pattern. James L. Farmer Department of Zoology 571 WIDB Brigham Young University Provo, Utah 84602, USA (801) 378-2153 (OFFICE) (801) 378-7499 (FAX) FARMERJ@YVAX.BYU.EDU FARMERJ@BYUVAX.BITNET From owner-rapd@net.bio.net Fri Jan 28 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Newsgroups: bionet.molbio.rapd Subject: DNA concentration Date: 28 Jan 1994 13:28:04 -0000 Lines: 15 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ib3t4$5c1@mserv1.dl.ac.uk> Apparently-To: rapd@net.bio.net Original-To: rapd@net.bio.net Reply to a question by Kari Kammiovirta concerning DNA concentration for RAPDs: We also find it necessary to measure concentration of each DNA sample and adjust all the samples to equal concentration. We use an intercalating dimeric dye YoPro (available from Molecular Probes Inc.). The dye and some ribonuclease are added to an aliquot of the DNa sample, and after 30 min (to give the RNase time to act), fluorescence is measured. This is set up easily in a 96-well plate. A set of dilutions of a DNA concentration standard (salmon sperm, or whatever) is also measured, and a calibration curve is constructed. The whole process can be automated using a pipetting robot (we use Beckman Biomek). I believe I posted a more detailed protocol here a while ago. Antoni Rafalski DuPont Molecular Breeding From owner-rapd@net.bio.net Fri Jan 28 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!sunic!news.funet.fi!klaava!MMKAB-01208.pc.Helsinki.FI!kkammiov From: kkammiov@viikki21.helsinki.fi (KARI KAMMIOVIRTA (MMKAB)) Newsgroups: bionet.molbio.rapd Subject: DNA-concentration Date: Fri, 28 Jan 1994 12:24:18 GMT Organization: University of Helsinki Lines: 18 Message-ID: NNTP-Posting-Host: mmkab-01208.pc Hello! I'm quite new in this newsgroup ( @ 1 month) and I'm curious about DNA concentration in RAPD. There haven't been much discussion about that issue. I've been doing RAPD's for 2 yrs and I have always checked the concentration in EtBr-plate and made dilution 10ng/ul. Usually folks have only given some dilution ratio ( 1/100 or 1/1000). I have noticed that my DNA extractions yield large variation of concentrations depending of strain(working mostly with fungi but also plants). So if I use only some standard dilution ratio, I would have huge variation in DNA concentration in the final reaction and I' ve been thinking that it also affects to the banding pattern.Can somebody please explain is concentration check necessary orjust my own imagination. Thanks in advance Kari Kammiovirta,Dept. of Plant Biology, Helsinki Univ. PS. Sorry about my not-so-fluent english. From owner-rapd@net.bio.net Sun Jan 30 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: mal5@cornell.edu (Muhammad Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: PCR machine by MJ Research model PTC-100 Date: 31 Jan 1994 08:38:33 -0000 Lines: 8 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2iig29$a5o@mserv1.dl.ac.uk> X-Sender: mal5@postoffice.mail.cornell.edu Original-To: Elizabeth BOULDING I think this report about MJ machine is not correct as we have been using our MJ machine for RAPD and PCR for small or large bands for last two years without any problem. I don't see any reason why it shouldn't amplify larger bands, at least thats what my experience says. Muhammad A. Lodhi Cornell University From owner-rapd@net.bio.net Sun Jan 30 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: mal5@cornell.edu (Muhammad Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: DNA-concentration Date: 31 Jan 1994 08:40:32 -0000 Lines: 27 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2iig60$a8s@mserv1.dl.ac.uk> X-Sender: mal5@postoffice.mail.cornell.edu Original-To: kkammiov@viikki21.helsinki.fi (KARI KAMMIOVIRTA (MMKAB)) >Hello! > >I'm quite new in this newsgroup ( @ 1 month) and I'm curious about DNA >concentration in RAPD. There haven't been much discussion about that issue. >I've been doing RAPD's for 2 yrs and I have always checked the concentration >in EtBr-plate and made dilution 10ng/ul. Usually folks have only given some >dilution ratio ( 1/100 or 1/1000). I have noticed that my DNA extractions >yield large variation of concentrations depending of strain(working mostly >with fungi but also plants). So if I use only some standard dilution ratio, >I would have huge variation in DNA concentration in the final reaction and I' >ve been thinking that it also affects to the banding pattern.Can somebody >please explain is concentration check necessary orjust my own imagination. > >Thanks in advance Kari I have seen that DNA conc. does not make much difference in the RAPD amplification within a certain range in Vitis (grape) DNA but beyond that range it makes difference. Please see our article "Inheritance and reliability of RAPD markers" in Proc. of the Symp. Applications of RAPD Technology to Plant Breeding, held in November 1992 in Minneopolis, MN. However, we found that its always a good idea to quantify DNA so that if something goes wrong it will be easy to track it down. Muhammad A. Lodhi Cornell University From owner-rapd@net.bio.net Sun Jan 30 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Louis van de Zande Newsgroups: bionet.molbio.rapd Subject: DNA concentration Date: 31 Jan 1994 08:20:31 -0000 Organization: Department of Biology, RUGroningen Lines: 11 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2iif0f$9g7@mserv1.dl.ac.uk> Reply-To: ZANDELPW@BIOL.RUG.nl X-pmrqc: 1 Original-To: rapd@dl.ac.uk You will find that the "degrees of freedom" in the DNA concentration for a reproducible RAPD pattern strongly depend on what kind of polymerase is used for the reaction, or indeed what kind of genome. I think nobody can afford to just apply a rule of thumb and get away with it. So always use replicate reactions to be sure of the reproducibility of the reaction. Louis v.d. Zande From owner-rapd@net.bio.net Mon Jan 31 22:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!swrinde!sgiblab!sgigate.sgi.com!olivea!news.bu.edu!taco!tangent From: tangent@unity.ncsu.edu (Phillip Wilcox) Subject: Re: PCR machine by MJ Research model PTC-100 Message-ID: <1994Jan31.235646.9697@ncsu.edu> Originator: tangent@forbt2.nrrc.ncsu.edu Sender: news@ncsu.edu (USENET News System) Reply-To: tangent@unity.ncsu.edu (Phillip Wilcox) Organization: North Carolina State University Computing Center References: <2hopl7$4dl@mserv1.dl.ac.uk> Distribution: bionet Date: Mon, 31 Jan 1994 23:56:46 GMT Lines: 57 In article <2hopl7$4dl@mserv1.dl.ac.uk>, Elizabeth BOULDING writes: > Path: taco!biosci!daresbury!not-for-mail > From: Elizabeth BOULDING > Newsgroups: bionet.molbio.rapd > Subject: PCR machine by MJ Research model PTC-100 > Date: 21 Jan 1994 14:42:47 -0000 > Lines: 14 > Sender: daemon@mserv1.dl.ac.uk > Distribution: bionet > Message-ID: <2hopl7$4dl@mserv1.dl.ac.uk> > Original-To: rapd@net.bio.net > > I have just purchased a PCR machine made by MJ Research > (model PTC-100). I have read an a 1993 article in PCR methods and > Applications which suggested that the MJ Research Machine was unable to > amplify DNA fragments larger than 1.5 kb with a given set of RAPD > primers whereas the Perkin-Elmer and the Techne machines amplified > 3 fragments larger than 1.5 kb. > I know there are people in this group who own MJ Research PCR machines > and I am curious whether they have been consistantly successful at > amplifying large fragments. > Elizabeth Boulding. > boulding@uoguelph.ca > > > > Our lab at NCSU (Forest Biotechnology Group) has eight MJ Research machines, five of which are the PTC 100 version. Our experience with these is that we can regularly and repeatably amplify fragments in excess of 1500bp. We have not however compared the MJs with the PEC and Techne machines, so I'm afraid I cannot help on that issue. I can say however that there is high repeatability among and within MJ machines - which is why we have eight of them. We did test a PEC 9600 machine a couple of years ago (before getting the 96 well MJs), but it didn't perform too well, and handling all of those tubes was a lot less ergonomically pleasing and time efficient than using Falcon plates. Also the price tag on the PEC seemed a little exorbitant. The tech assistance from MJ has been good also, and they've also loaned machines free of charge for things like classes and substitute machines when we've needed to service a machine. In addition, they offered to help get Perkin Elmer off our backs after PEC inferred that we were violating a patent by not using their machines. PEC eventually backed down on that one, but it left an unpleastant aftertaste that meant we would avoid using their products unless legally required to do so. The net result is that our lab has MJ machines at less cost and more convenience as well as more pleasant relations with our suppliers. Even IF it costs less amplification of larger fragments in the MJs, the result is still a net benefit to us. Hope this helps. Phillip Wilcox Forest Biotech. Dept. Forestry, NCSU. From owner-rapd@net.bio.net Mon Jan 31 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: gy11@cornell.edu (Guangning Ye) Newsgroups: bionet.molbio.rapd Subject: subscribe Date: 1 Feb 1994 16:46:31 -0000 Lines: 2 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2im117$6bc@mserv1.dl.ac.uk> X-Sender: gy11@postoffice.mail.cornell.edu Original-To: RAPD@net.bio.net From owner-rapd@net.bio.net Mon Jan 31 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Priscilla Bettini Newsgroups: bionet.molbio.rapd Subject: nonradioactive DNA sequencing Date: 1 Feb 1994 16:20:30 -0000 Organization: Dipartimento di Biologia Animale e Genetica Lines: 15 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ilvge$4u9@mserv1.dl.ac.uk> Original-To: rapd@dl.AC.UK Does anyone have informations/suggestions/comments/hints....on nonradioactive DNA sequencing techniques ? Thanks in advance ! Prisci Bettini Dept. Animal Biology & Genetics Univ. Florence ------------------------------------------- UNIVERSITA' DEGLI STUDI DI FIRENZE Dipartimento di Biologia Animale e Genetica via Romana, 17 - 50125 Firenze (Italy) tel : 055-222448 fax : 055-222565 E-MAIL: DIBAGEF1@VM.IDG.FI.CNR.IT -------------------------------------------