From owner-rapd@net.bio.net Tue Feb 01 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: nicholas Newsgroups: bionet.molbio.rapd Subject: Thermal cyclers Date: 2 Feb 1994 15:28:30 -0000 Lines: 16 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2iogqu$2fp@mserv1.dl.ac.uk> Original-To: rapd I have been reading the net for about two months and noticed that the MJ machines are liked. Does any out there have experience with the Hybaid P5102 or the PlateCyclers from Coy corporation. How do these machines compare. We are about to invest in a machine so advice is appreciated. Thanks Nicholas McLetchie School for Biological Sciences 101 Morgan Building University of Kentucky Lexington KY 40506 606 257-6786 ph 606 257 1717 fax mclet@ukcc.uky.edu From owner-rapd@net.bio.net Tue Feb 01 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Reamplification of RAPD products Date: 2 Feb 1994 15:22:32 -0000 Lines: 13 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2iogfo$20l@mserv1.dl.ac.uk> X-Sender: mal5@postoffice.mail.cornell.edu Original-To: RAPD@net.bio.net Dear Friends I have been trying to reamplify RAPD bands cut out of the agarose gel, with the same primers as they were amplified the first time in RAPD reaction. In many cases I get more than one amplified product (bands) when I use a single band as template. Eventually, I will be labelling these bands with digoxigenin once I am sure that I am getting only one product. Has anyone seen such an amplification (two bands from the amplification of a single band) and what he/she did to overcome this problem. Your suggestions will be highly appreciated. Thanks in advance Muhammad A. Lodhi Cornell University From owner-rapd@net.bio.net Tue Feb 01 22:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!news.ucdavis.edu!usenet From: caetano@fauna.ucdavis.edu Subject: Re: Reamplification of RAPD products Message-ID: Sender: usenet@ucdavis.edu (News Guru) Reply-To: caetano@fauna.ucdavis.edu Organization: Animal Science Dept, UC Davis References: <2iogfo$20l@mserv1.dl.ac.uk> Distribution: bionet Date: Wed, 2 Feb 1994 16:14:47 GMT Lines: 59 In article <2iogfo$20l@mserv1.dl.ac.uk>, mal5@cornell.edu (Muhammad A. Lodhi) writes: >Dear Friends >I have been trying to reamplify RAPD bands cut out of the agarose gel, with >the same primers as they were amplified the first time in RAPD reaction. >In many cases I get more than one amplified product (bands) when I use a >single band as template. Eventually, I will be labelling these bands with >digoxigenin once I am sure that I am getting only one product. Has anyone >seen such an amplification (two bands from the amplification of a single >band) and what he/she did to overcome this problem. Your suggestions will >be highly appreciated. Thanks in advance > >Muhammad A. Lodhi >Cornell University > What size are the "extra" bands you are getting? Are they smaller than the band you wish to reamplify? If so they could be originating from internal priming sites in your isolated band. |XXXXXXX-------------------XXXXXXXXXX------------XXXXXXXX| original XXXXXXX-------------------XXXXXXXXXX------------XXXXXXXXX XXXXXXX -------------------XXXXXXXXXX all 3 products XXXXXXX-----------XXXXXXX You might be able to overcome this if you were not amplifying the smaller bands in your original RAPD reaction. The apearence of the extra bands might be due to changes in buffer concentrations or template in your reamp reaction. Try to adjust for that. If the extra bands you are getting are larger than the one you want to reamp it might be that you are not getting good separation pf your products in the agarose gell. Run a long, higher concentration gell for as long as you can (overnight) so you can get better separation. Your bands are going to be difused so make sure you know exactly where your target band is. Good luck! ********************************************************************************* Alexandre R. Caetano | E-mail : "ARCAETANO@UCDAVIS.EDU" Univ. of California-Davis | Dept. Animal Science | ******************************************************************************** What's the meaning of life? Life has no meaning, it's only a consequence of the complexity of carbon chemistry... From owner-rapd@net.bio.net Tue Feb 01 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: murrayian@phibred.com (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: DNA extraction method using CTAB???? Date: 2 Feb 1994 18:33:01 -0000 Lines: 7 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2iorkt$agk@mserv1.dl.ac.uk> Original-To: "rapd@net.bio.net"@phibred.com I have been using the Edwards method (1991 mNucleic Acid Res. Vo 19 # 6 pp1349) which is a relativly simple procedure. I was wondering if anyone had tried replacing the SDS with CTAB. I wish to bulk the DNA. Thanks in advance Murrayian@phibred.com From owner-rapd@net.bio.net Wed Feb 02 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Newsgroups: bionet.molbio.rapd Subject: Re-amplification Date: 3 Feb 1994 01:35:36 -0000 Lines: 20 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ipkd8$sm8@mserv1.dl.ac.uk> Apparently-To: rapd@net.bio.net Original-To: rapd@net.bio.net In response to a question from Muhammad A. Lodhi concerning re-amplification of RAPD bands: Re-amplification is a very tricky process: - sometimes you get the desired band w/o problem - sometimes more then one band shows up - sometimes you get one band, but it is not the band you want Repeated plugging the desired band out and re-amplifying once again sometimes helps and sometimes not. Once you get a band that looks right, it is still necessary to check the identity of the band by hybridizing to a Southern blot of segregating RAPDs (RAPDs from individuals positive and negative for the band of interest). Alternatively, you may prefer to clone the band (of course the confirmation of band identity on a segregating population is still necessary). There is a discussion of this in the Williams et al MEthods in Enzymology paper (vol 218, pp 704-740). Antoni P.S. A limited number of reprints of the ME paper is available). From owner-rapd@net.bio.net Wed Feb 02 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: "Doug Rhoads" Newsgroups: bionet.molbio.rapd Subject: Re: Reamplification of RAPD products Date: 3 Feb 1994 15:19:40 -0000 Organization: University of Arkansas Lines: 42 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ir4mc$5nv@mserv1.dl.ac.uk> Original-To: rapd@net.bio.net >Dear Friends >I have been trying to reamplify RAPD bands cut out of the agarose gel, with >the same primers as they were amplified the first time in RAPD reaction. >In many cases I get more than one amplified product (bands) when I use a >single band as template. Eventually, I will be labelling these bands with >digoxigenin once I am sure that I am getting only one product. Has anyone >seen such an amplification (two bands from the amplification of a single >band) and what he/she did to overcome this problem. Your suggestions will >be highly appreciated. Thanks in advance > >Muhammad A. Lodhi >Cornell University I am not at all surprised that you see many of the original bands from the RAPD. Agarose gels do an adequate job of resolving DNAs of different sizes but the bands you see are only the median migration rate for all of the fragments of that size. Thus each band is present everywhere in the gel just at lower levels the further you move from its median migration point. Three suggestions for you that we have used (based on need) are: 1) Purify on acrylamide gels they produce sharper resolution. 2) Clone the fragment you want. 3) Purify the band twice on agarose gels. Option 3 is usually a gamble unless you have lots of DNA. Option 2 takes longer. I favor option 1 as it is not any longer than agarose electrophoresis. For reamplification all you need to do is cut the band out. Rinse with H2O a couple times. Soak the gel fragment O/N in 50 to 100 ul of 10 mM Tris 0.1 mM EDTA. Then use 2 ul in another RAPD. This has worked for us, hope it works for you. 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 Feb 02 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Louis van de Zande Newsgroups: bionet.molbio.rapd Subject: Re: Re-amplification Date: 3 Feb 1994 08:34:19 -0000 Organization: Department of Biology, RUGroningen Lines: 41 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2iqcub$fe7@mserv1.dl.ac.uk> Reply-To: ZANDELPW@BIOL.RUG.nl Original-To: rapd@dl.ac.uk > Forwarded by: server-daemon > Date sent: 3 Feb 1994 01:35:36 -0000 > Date forwarded: Thu, 3 Feb 94 0:38:34 UT > From: rafalski > Send reply to: rafalski > To: "bionet.molbio.rapd mail newsgroup" > Subject: Re-amplification > In response to a question from Muhammad A. Lodhi concerning > re-amplification of RAPD bands: > Re-amplification is a very tricky process: > - sometimes you get the desired band w/o problem > - sometimes more then one band shows up > - sometimes you get one band, but it is not the band you want > Repeated plugging the desired band out and re-amplifying once again > sometimes helps and sometimes not. > Once you get a band that looks right, it is still necessary to check the > identity of the band by hybridizing to a Southern blot of segregating > RAPDs (RAPDs from individuals positive and negative for the band of > interest). Alternatively, you may prefer to clone the band (of course the > confirmation of band identity on a segregating population is still > necessary). > There is a discussion of this in the Williams et al MEthods in Enzymology > paper (vol 218, pp 704-740). > > Antoni > > P.S. A limited number of reprints of the ME paper is available). > This is (unfortunately) all very true. But things are worse! Even when one band of the desired size is obtained, it sometimes will hybridize to MORE THAN ONE band in the original RAPD. No matter what stringency. Maybe, just maybe, digesting the fragment with restriction endonucleases will provide you with a probe that behaves the way you expect and gets rid of the disturbing sequences. But you will have to anticipater a lot of questions when presenting the results. Louis van de Zande From owner-rapd@net.bio.net Thu Feb 03 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!noc.near.net!das-news.harvard.edu!husc-news.harvard.edu!kuhub.cc.ukans.edu!mbcf!kitchingman Newsgroups: bionet.molbio.rapd Subject: Re: Reamplification of RAPD products Message-ID: <1994Feb3.080119.8654@mbcf> From: kitchingman@mbcf.stjude.org Date: 3 Feb 94 08:01:19 -600 References: <2iogfo$20l@mserv1.dl.ac.uk> Distribution: bionet Organization: St. Jude Children's Research Hospital Lines: 12 2In article <2iogfo$20l@mserv1.dl.ac.uk>, mal5@cornell.edu (Muhammad A. Lodhi) writes: > In many cases I get more than one amplified product (bands) when I use a > single band as template...... You should assume that every band on an agarose or acrylamide gel is contaminated with a small amount of every lower molecular weight band. While the low level of contamination will usually not show up if you just re-run a purified band on a gel, when you amplify by PCR the contaminating bands will make their presence known. A second round of gel electrophoresis will reduce the level of contamination of the band of interest, but short of cloning, there is no way that I know of to get around this problem. Geoff From owner-rapd@net.bio.net Sat Feb 05 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Eric Yang Newsgroups: bionet.molbio.rapd Subject: TA cloning system Date: 6 Feb 1994 08:41:45 -0000 Lines: 14 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2j2ag9$h0k@mserv1.dl.ac.uk> Original-To: rapd@net.bio.net Could anyone out there tell me something about the TA cloning system (Invitrogen) for cloning PCR products? What are it's advantages and disadvantages? Does it really work? Thanks. ****************************************************************************** Eric V. Yang, Ph.D. Developmental Biology Center e-mail: eyang@darwin.bio.uci.edu University of California Voice/Fax #: 714-856-5385 Irvine, California 92717 ****************************************************************************** From owner-rapd@net.bio.net Sat Feb 05 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Eric Yang Newsgroups: bionet.molbio.rapd Subject: subscribe Date: 6 Feb 1994 08:41:48 -0000 Lines: 3 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2j2agc$h13@mserv1.dl.ac.uk> Original-To: rapd@net.bio.net subscribe Eric Yang From owner-rapd@net.bio.net Sun Feb 06 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: "Jurg E. Frey" Newsgroups: bionet.molbio.rapd Subject: nonradioactive DNA sequencing Date: 7 Feb 1994 10:40:36 -0000 Lines: 38 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2j55r4$912@mserv1.dl.ac.uk> Original-To: rapd@dl.ac.uk in response to a question from Prisci Bettini: I would suggest the Silver Sequencing Kit by Promega. Although I have only used it three times so far, I think it is reasonable to say that you can read an average of about 200 bp per run. The protocol is not very difficult especially if you happen to have some experience with PAGE (polyacrylamid gel electrophoresis) and PCR. Then it is rather a combination of these two methods, expanded by silver staining. The protocol on silver staining is very similar to what was published by BJ Bassam and G Caetano-Anolles: Silver Staining of DNA in Polyacrylamide Gels. Applied Biochemistry and Biotechnology 42, 181-188. The main advantages of the Silver Sequencing: no use of radioactivity, fast (8-10 hours from start to the final sequence), and there is no need to label anything. It works with your unlabeled PCR primers. However, since I am new to sequencing altogether, I can not compare this protocol to the classical ones. On the other hand, since I was able to master the protocol, I guess it is managable for many other LABsters. Good luck. Juerg E. Frey, Federal Research Station, CH-8820 Waedenswil, Switzerland (e-mail FREY@FAW.ETHZ.CH) From owner-rapd@net.bio.net Mon Feb 07 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!spool.mu.edu!torn!utnut!utcsri!newsflash.concordia.ca!canopus.cc.umanitoba.ca!browns From: browns@cc.umanitoba.ca (Stuart Brown) Newsgroups: bionet.molbio.rapd Subject: Re: TA cloning system Date: 8 Feb 1994 17:50:51 GMT Organization: University of Manitoba, Winnipeg, Manitoba, Canada Lines: 39 Distribution: bionet Message-ID: <2j8jdr$qgi@canopus.cc.umanitoba.ca> References: <2j2ag9$h0k@mserv1.dl.ac.uk> NNTP-Posting-Host: antares.cc.umanitoba.ca In article <2j2ag9$h0k@mserv1.dl.ac.uk> Eric Yang writes: >Could anyone out there tell me something about the TA cloning system >(Invitrogen) for cloning PCR products? What are it's advantages and >disadvantages? Does it really work? >Eric V. Yang, Ph.D. I use the TA system on a regular basis for cloning and sequencing PCR fragments. I find the system has some odd quirks, but works adequately for my needs. It would be better to use restriction sites built into the primers, but that is not always possible. An odd thing about the TA system is that you get an assortment of blue, white, and white with a bit of blue in the center bacterial clones after transformation and growth O/N on Kan plates with X-gal. I turns out that the white-with-blue centers clones most often contain the inserts. I pick about 20 clones from each ligation for mini-preps and restriction digestion. I always get some odd paterns of inserts (bigger and smaller than the band that I was trying to clone), but almost always get a few proper inserts in my 20 clones. These odd sized inserts may have more to do with minor contaminants of my PCR band than an actual problem with the kit. I also get lots of clones with no insert, but I can ususally screen them out with a quick gel on crude mini-preps without restriction digest. Once the bands are cloned, sequencing is nice and easy with M13 universal and Reverse primers. My PCR fragments are about 400 base pairs, so I can get completely overlapping seq from both strands. In summary, the kit works, it is not too expensive, and you can count on having clones in about 3 days after running the PCR on a gel. -Stu -- Stuart M. Brown If you can remain cool when all U. of Manitoba, Dept. Plant Science Around you are in panic, Winnipeg, Manitoba, CANADA browns@ccu.umanitoba.ca Then you surely misunderstand the situation From owner-rapd@net.bio.net Mon Feb 07 22:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!sol.ctr.columbia.edu!news.kei.com!newsstand.cit.cornell.edu!cornell!uw-beaver!netnews.nwnet.net!ns1.nodak.edu!badlands!kistner From: kistner@badlands.NoDak.edu (Michele A Kistner) Subject: DNA extration using CTAB???? Sender: usenet@ns1.nodak.edu (Usenet login) Message-ID: Date: Mon, 7 Feb 1994 15:00:21 GMT Nntp-Posting-Host: badlands.nodak.edu Organization: North Dakota Higher Education Computing Network X-Newsreader: TIN [version 1.2 PL2] Lines: 9 We use a protocol that we got from Dr. Hegelson at the university of WI. that uses 2% (w/v) CTAB, 1.4 M NaCl, 0.2% (v/v) 2-mercaptoethanol, 0.5 M EDTA, and 100 mM Tris pH8.0 - we also added 1 mM 1,10 phenanthroline - which eliminates oxidation - we work with wild species of potato and were having oxidation problems. I don't bulk my DNA for RAPDs but a graduate student working in the same lab as me uses this extraction buffer and bulks for RFLPS with great sucess. I'm not sure of his e-mail address but, if you would like to correspond with him send e-mail to Bin Ye at my address kistner@badlands.nodak.edu. From owner-rapd@net.bio.net Tue Feb 08 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: Carvalho Torres Alexandro cassio-UACPYP Newsgroups: bionet.molbio.rapd Subject: When I use a primer? Date: 9 Feb 1994 05:13:04 -0000 Lines: 11 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2j9rd0$h80@mserv1.dl.ac.uk> Original-To: rapd@net.bio.net Dear Molecular Biologist, I have a very practical question. When should I use a primer to study a bacterial pathogen specie and which random primer or primers I need to use to study a special phenotypic characteristics? What is the most interesting strategy used? Is it the size of the primer, the G+C proportion or anything else? Which is the most practical choice I must to perform. Someone have some experience or sugestion with this doubt? Must I use a random choice or have some parameters to follow? Best regards, Alexandro Carvalho-INNSZ Mx FAX 655 96 75 From owner-rapd@net.bio.net Wed Feb 09 22:00:00 1994 Path: biosci!lhc!darwin.sura.net!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Carvalho Torres Alexandro cassio-UACPYP Newsgroups: bionet.molbio.rapd Subject: RE: When I use a primer? Date: 10 Feb 1994 07:30:38 -0000 Lines: 21 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2jcnqu$6if@mserv1.dl.ac.uk> Original-To: rapd@net.bio.net On Wed, 9 Feb 1994 rafalski@esvax.dnet.dupont.com wrote: > I suggest that you read some of the literature on the subject. > Our paper in Trends in Genetics (vol 9, August 1993, Issue #8, pages > 275-280) is a good place to start. > Antoni Rafalski > > Rafalski wrote but I need more information about question like that. If I will study a special pathogens with in a thousand random primer conbination how I/you design random primers for this specific microorganisms? Is there a specific strategy? Why some random primers used for Staph. aureus and other bacteria are longer than those used for other genus of bacteria. Should I prove a lot of different primers? Thank very much, Alexandro Carvalho From owner-rapd@net.bio.net Wed Feb 09 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: hemant@hoh.mbl.edu (Hemant Chickarmane) Newsgroups: bionet.molbio.rapd Subject: RE: When I use a primer? Date: 10 Feb 1994 15:27:21 -0000 Lines: 10 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2jdjop$c6v@mserv1.dl.ac.uk> Original-To: carvalho@unamvm1.dgsca.unam.mx, rapd@net.bio.net The primers are chosen by trail and error. You might try one of the primer kits from Operon or the primer set from Univ. british Columbia. The 10 bp primers designed according to criteria from the Dupont group do work for Staphylococcus aureus and other Gram-positive and Gram-negative bacteria. The longer primers that you are talking about are not necessary. Hemant Chikarmane Marine Biological Laboratory Woods Hole MA 02543 hemant@mbl.edu From owner-rapd@net.bio.net Thu Feb 10 22:00:00 1994 Path: biosci!biosci!not-for-mail From: ines@jouy.inra.fr (Ines Foulhouze) Newsgroups: bionet.general,bionet.molbio.evolution,bionet.jobs,bionet.population,bionet.molbio.rapd Subject: Postdoctoral position available at INRA - France Date: 10 Feb 1994 16:06:47 -0800 Organization: I.N.R.A. - JOUY Lines: 29 Sender: kristoff@net.bio.net Distribution: world Message-ID: <2jb5rv$db@jouy.jouy.inra.fr> Reply-To: wajnberg@antibes.inra.fr NNTP-Posting-Host: net.bio.net Xref: biosci bionet.general:7701 bionet.molbio.evolution:1448 bionet.jobs:3362 bionet.molbio.rapd:428 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Postdoctoral position available immediately at the National Institute for Agronomic Reseach of Antibes, France (Riviera). The project concerns the identification of molecular markers-quantitative trait loci (QTL) associations in a parasitic wasp. This is a 2 or 3-year full-time position at a salary of approximately 120,000 FF (i.e. about 23,000 USD) per year. Applicants should have a Ph.D or equivalent degree and experience or training in genetics, population genetics and molecular biology. Send curriculum vitae, a statement of research interests and 3 references to E. Wajnberg or F. Vanlerberghe, INRA, Laboratoire de biologie des invertebres, Unite biologie des populations, B.P. 2078, 06606 Antibes, France. wajnberg@antibes.inra.fr or fvl@antibes.inra.fr ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Eric Wajnberg Centre de Recherche d'Antibes o__ ___ Laboratoire de Biologie des Invertebres _.>/ _ ___ Unite de Biologie des Populations (_) \(_) I.N.R.A. ^`'~*-,\|/,-*~'`^ 37, Bld. du Cap. 06600 Antibes. France. Tel : (33) 93.67.88.92 Fax : (33) 93.67.88.98 e-mail : wajnberg@antibes.inra.fr ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ From owner-rapd@net.bio.net Fri Feb 11 22:00:00 1994 Path: biosci!NET.BIO.NET!biosci-help From: biosci-help@NET.BIO.NET (BIOSCI Administrator) Newsgroups: bionet.molbio.rapd Subject: test of rapd@net.bio.net Date: 12 Feb 1994 02:44:59 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: bionet Message-ID: Reply-To: biosci-help@net.bio.net NNTP-Posting-Host: net.bio.net test of rapd@net.bio.net From owner-rapd@net.bio.net Sat Feb 12 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!uwm.edu!msuinfo!harbinger.cc.monash.edu.au!yarrina.connect.com.au!warrane.connect.com.au!pandora.connect.com.au!user From: karyn@connect.com.au (Karyn Davis) Newsgroups: bionet.molbio.rapd Subject: Summary of analysis software request Followup-To: bionet.molbio.rapd Date: Mon, 14 Feb 1994 07:39:57 +1000 Organization: Connect.com.au P/L, Sydney, Australia Lines: 94 Message-ID: NNTP-Posting-Host: pandora.connect.com.au Here is the summary of the results ofmy posting requesting recommendations for software for the analysis of RAPD and RFLP data. From Muhammad Lodhi: (mal5@cornell.edu) suggested PAUP This is available from Dr. David Swofford. His email address is swofford@onyx.si.edu Peter Reinthal (bio_reinthal@emunix.emich.edu) suggested REAP. This is available from Doug McElroy Department of Biology Western Kentucky University Bowling Green, KY 42101 He asks that you send him a blank disk. Reference for REAP: Journal of Heredity, 1992 83 p157-158, McElroy, D.; Moran, P.; Bermingham, E. & Kornfield, I. "REAP: An integrated environment for the manipulation and phylogenetic analysis of restriction data" The Institute for Molecular Evolutionary Genetics has available MEGA (Molecular Evolutionary Genetisc Analysis) & RESTSITE. MEGA info is regularly posted to bionet.molbio.evolution. David Moon (DAVHMOON@pira.cena.usp.br) suggested NTSYS. This is available from Exeter Publishing Ltd. 100 North Country Road Setauket NY 11733 USA FAX: 516-751-3435 and MAPMAKER/QTL. This can be FTPed from genome.wi.mit.edu NTSYS also recommended by KARI KAMMIOVIRTA (kkammiov@viikki21.helsinki.fi) From David_Milton@UManitoba.CA: GelReader (display of scanned images of gel or autoradiogram, estimates of fragment length, standard curves for molecular weight and calculated averages) Double Digester for the construction of restriction maps Both packages can be FTPed (use ARCHIE to find the site closest to you). Anthony Hilton (hiltonac@sun1.bham.ac.uk) has found GelManager (by Biosystematica based in Czech) and GelCompar (by Applied Maths in Belgium) to be useful packages. The addresses for the companies are: Biosystematica Pod rovinou 688\16 140 00 Prague 4-Krc The Czech Republic. Applied Math Risquons-Toutstraat 38 B-8511 Kortrijk Belgium. Yecheng Wu (wyc@crsa.bu.edu) suggessted Scanalytics/CSPI has a product called RFLPscan, which is designed to do analysis on RAPD data and RFLP data. It process gel images to get band information, performs calibration, band matching and databasing. If you need more information, you can contact Mr. Chuck Vecoli at 508 663 7598 ext. 2300, who is the product manager. His email is cvecoli@cspi.com Doug Rhoads (DRHOADS@mercury.uark.edu) wrote: Our analyses have mostly been visual comparisons of gel photos scored for band sharing. Data either as binary character states or as % band sharing (Nei and Li) are then fed into Phyllip (usually Dollop method) for tree generation. Personally, I am very skeptical of turning gel comparisons over to a computer as multiple lanes must be assimilated and a cutoff established for when a faint band stops being scorable. I think an experienced person can do these comparisons fairly quickly whereas lane variation in background or yield requires a floating background subtraction for establishing peaks. Joe Felsenstein (joe@genetics.washington.edu) suggested PHYLIP (Phylogeny Inference Package version 3.5) This is a FREE package of programs for inferring phylogenies and carrying out certain related tasks. At present it contains 31 programs, which carry out different algorithms on different kinds of data. PHYLIP is avialable by anonymous FTP from evolution.genetics.washington.edu (128.95.12.41). A couple of packages that I recently found references for are: RAPDPLOT (calulates pairwise distance matrix, does cluster analysis and constructs dendrograms). reference: Journal of Medical Entomolgy 29 p939-945, Kambhampati, S; Black, WC & Karamjit, SR "Random amplified polymorphic DNA of mosquito species and populations (Diptera: Culicidae): Techniques, statistical analysis and applications" Available from William C. Black IV, Department of Environmental Health Colorado State University Fort Collins CO 80523 Another package I haven't yet seen is ODEN. Reference: Computer applications in biosciences 1994 10 p11, Ina, Y. "ODEN: A program for molecular evolutionary analysis . . ." Thanks Karyn Davis karyn@connect.com.au From owner-rapd@net.bio.net Sun Feb 13 22:00:00 1994 Path: biosci!news.cs.umb.edu!hsdndev!dartvax.dartmouth.edu!saturn.caps.maine.edu!maine.maine.edu!io20109 Organization: University of Maine System Date: Mon, 14 Feb 1994 13:11:40 EST From: Message-ID: <94045.131140IO20109@MAINE.MAINE.EDU> Newsgroups: bionet.molbio.rapd Subject: decontamination protocol References: Lines: 8 Hello, does anyone know any protocol for effectively REMOVING microrganisms contaminating field samples (for posterior RAPD analyses)? All I can find are protocols for axenic cultures, which will kill bacteria, etc, but not remove them (their DNA). In my case, field samples of seaweed have a lot of organisms stuck to the cell mucilages. Thank's! Ester From owner-rapd@net.bio.net Sun Feb 13 22:00:00 1994 Path: biosci!GPU.SRV.UALBERTA.CA!dchong From: dchong@GPU.SRV.UALBERTA.CA (Daniel Chong) Newsgroups: bionet.molbio.rapd Subject: Linkage analysis with unknown parental phase Date: 14 Feb 1994 21:04:47 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net RAPD markers have been obtained from a single full-sib cross between Populus deltoides and P. maximowiczii. I am doing linkage and mapping analysis on those markers using MAPMAKER. The underlined genetic model is testcross (or pseudotestcross) with unknown linkage-phase. Can anyone tell me how to code my data for MAPMAKER ? Are there programs dealing with phase unknown data? Thank you. Daniel Chong Dapartment of Forest Science University of Alberta From owner-rapd@net.bio.net Mon Feb 14 22:00:00 1994 Path: biosci!parc!barrnet.net!sgiblab!munnari.oz.au!ariel.ucs.unimelb.EDU.AU!ucsvc.ucs.unimelb.edu.au!wehi.edu.au!wehi.edu.au!finnin Newsgroups: bionet.molbio.rapd Subject: test post Message-ID: <1994Feb15.122319.1@wehi.edu.au> From: finnin@wehi.edu.au Date: 15 Feb 94 12:23:19 +1000 Organization: Walter & Eliza Hall Institute NNTP-Posting-Host: wehit.wehi.edu.au Lines: 2 test this is a test post From owner-rapd@net.bio.net Mon Feb 14 22:00:00 1994 Path: biosci!parc!barrnet.net!sgiblab!sdd.hp.com!vixen.cso.uiuc.edu!howland.reston.ans.net!europa.eng.gtefsd.com!uhog.mit.edu!news.kei.com!yeshua.marcam.com!zip.eecs.umich.edu!umn.edu!gaia.ucs.orst.edu!osshe.edu!news.uoregon.edu!netnews.nwnet.net!news.u.washington.edu!toby From: toby@u.washington.edu (Toby Bradshaw) Newsgroups: bionet.molbio.rapd Subject: Re: Linkage analysis with unknown parental phase Date: 14 Feb 1994 23:15:18 GMT Organization: University of Washington, Seattle Lines: 32 Distribution: bionet Message-ID: <2jp0m6$dng@news.u.washington.edu> References: NNTP-Posting-Host: stein.u.washington.edu In article , Daniel Chong wrote: > > RAPD markers have been obtained from a single full-sib cross between >Populus deltoides and P. maximowiczii. I am doing linkage and mapping >analysis on those markers using MAPMAKER. The underlined genetic model >is testcross (or pseudotestcross) with unknown linkage-phase. Can anyone >tell me how to code my data for MAPMAKER ? Are there programs dealing with >phase unknown data? Thank you. In a pseudotestcross mapping project you can run it through MAPMAKER as A/C and B/D, i.e. A is one homozygous null, C is the with-band counterpart. Use A and C where P.d. is null, and B/D where P.m. is null. You'll get two maps, one for each parent. Dominant markers linked in repulsion are so poorly cross-informative that you can't really hope to merge the maps without some codominant marker data. I've done a lot of mapping in Populus hyrids with RAPDs, RFLPs, and STSs. Some of the STSs could be used to merge your maps, since I know they work in P. deltoides and many work in P. maximowiczii. Send me your mailing address and I'll send you a copy of a mapping manuscript we have submitted to TAG that has all the gory details. Toby Bradshaw | Department of Biochemistry | Will make genetic linkage maps and College of Forest Resources | for food. University of Washington, Seattle | toby@u.washington.edu | From owner-rapd@net.bio.net Mon Feb 14 22:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: When I use a primer? Date: 15 Feb 1994 15:17:45 -0000 Organization: University of Arkansas Lines: 34 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net >Dear Molecular Biologist, >I have a very practical question. When should I use a primer to study a >bacterial pathogen specie and which random primer or primers I need to >use to study a special phenotypic characteristics? What is the most >interesting strategy used? Is it the size of the primer, the G+C >proportion or anything else? Which is the most practical choice I must to >perform. Someone have some experience or sugestion with this doubt? Must >I use a random choice or have some parameters to follow? >Best regards, >Alexandro Carvalho-INNSZ >Mx FAX 655 96 75 > In our experience trial and error is the best method. Certain factors influence decisions including: genome complexity, genome GC/AT ratio, and the number of bands you want per reaction. Based on a few radically disparate species we find that for larger genomes (>5x10^8 bp) the percentage of primers that give any bands goes up with increasing GC content in the primer. 90% of primers with 80% or greater GC/AT give one or more bands. For primers with 50% GC/AT only about 30-50% give one or more `scorable' bands. There also is a 3' end preference based on the target genome. We have seen a few genomes for which primers that end in AA, AT, or CT are more successful (i.e., give higher number of bands or more likely to give bands) than primers ending in C or G (unpublished observation). We are especially fond of the two base primers (see previous discussions) but they work best with eukaryotes rather than prokaryotes. 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 Feb 15 22:00:00 1994 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: Linkage analysis with unknown parental phase Date: 16 Feb 1994 02:32:16 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 40 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199402160232.AA28413@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net > RAPD markers have been obtained from a single full-sib cross between >Populus deltoides and P. maximowiczii. I am doing linkage and mapping >analysis on those markers using MAPMAKER. The underlined genetic model >is testcross (or pseudotestcross) with unknown linkage-phase. Can anyone >tell me how to code my data for MAPMAKER ? Are there programs dealing with >phase unknown data? Thank you. >Daniel Chong Hi Daniel I think you can code the data as following; 'A' for (-) bands 'H' for (+) bands '-' for missing data So if the genotype are, ++-+--++-+-+ it could be changed to, HHAHAAHHAHAH for analysis with MAPMAKER. Also to overcome the problem of missing phase each marker has to be duplicated to create dummy loci. For example, OPA-1 should be changed as following; OPA-1 +-+-+-+++-- OPA-1A HAHAHAHHHAA OPA-1B AHAHAHAAAHH In the initial analysis I will not include markers that are heterozygous in both parents (segregating 3:1). These markers will be used once the two parental linkage groups are estalished, to find homologous linkage groups in both the parents. For that purpose RFLP and isozyme markers could also be used. This results lot of junk in the form of duplicates of linkage groups but these can easily be identified and removed from the analysis. This strategy works very well when the phase is missing. We have used similar strategy in grape maps. At present, there is no software that can handle pseudotest crosses, to my knowledge. Some people recommend JOINMAP but I prefer MAPMAKER due to several reasons. Hope that it will be useful Muhammad A. Lodhi Cornell University From owner-rapd@net.bio.net Tue Feb 15 22:00:00 1994 Path: biosci!POPGEN.BIOLOGY.UMT.EDU!tmo From: tmo@POPGEN.BIOLOGY.UMT.EDU (Thomas Mitchell-Olds) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 16 Feb 1994 18:49:59 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 53 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9402161852.AA01629@popgen.biology.umt.edu> NNTP-Posting-Host: net.bio.net Postdoc in Plant Molecular Population Genetics: A postdoc is available to study molecular evolutionary genetics of Arabis fecunda, a wild relative of Arabidopsis. We are studying population differentiation and DNA sequence evolution at several loci. Regulatory genes controlling initiation of flowering (e.g., luminadependens) have pleiotropic effects on fecundity and age of first reproduction. Analysis of DNA sequence polymorphisms and mapping quantitative trait loci will permit inferences about factors affecting molecular evolution and its ecological consequences. This position entails laboratory work in molecular evolution during most of the year, and field studies of wild populations during the summer. Applicants should be experienced with methods of molecular biology and concepts of evolutionary genetics. Applicants should also be willing to conduct field work in mountainous areas of western Montana during the summer. This research is part of a long term analysis of ecological genetics in undisturbed natural plant populations. We have five years of field data on life histories, demography and natural selection in five wild populations. This postdoctoral position will examine DNA sequence variation at loci that may be responsible for observed variation in life histories. Application deadline: 15 April 1994, or until a suitable candidate is found Preferred starting date: 1 July 1994 For further information contact: Thomas Mitchell-Olds e-mail: tmo@popgen.biology.umt.edu phone: 607-275-0065 Permanent Address: Division of Biological Sciences University of Montana Missoula, MT 59812 Address for correspondence until 1 June 1994: Section of Genetics and Development Biotechnology Building Cornell University Ithaca, NY 14853 From owner-rapd@net.bio.net Tue Feb 15 22:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!daresbury!doc.ic.ac.uk!agate!howland.reston.ans.net!cs.utexas.edu!uunet!psinntp!nstn.ns.ca!dragon.acadiau.ca!dragon.acadiau.ca!901106c From: 901106c@dragon.acadiau.ca (Timothy Chipman) Subject: Inquiry: Anaylsis of RAPD PCR data.. simple data set... Message-ID: <1994Feb16.035909.8389@relay.acadiau.ca> Summary: i Sender: news@relay.acadiau.ca Nntp-Posting-Host: dragon.acadiau.ca Organization: Acadia University Date: Wed, 16 Feb 1994 03:59:09 GMT Lines: 37 This is an inquiry that is prompted partially by the previous posting on analysis of data, partially because of the fact that it is thesis time. I am curious if anyone can recommend a package that would be good for doing a simple comparison as follows: Generate indices of similarity for each sub-gp, pooled group: 3 groups of 5 related individuals. To be precise: I have used 2 10-mer primers to generate banding patterns for groups of 5 drone honeybees from 3 different hives. All the queens for the three hives are sisters. Thus, I have two seperate sets of data - one for each primer. All I want to do is get some quantitative index of similarity .. however, this is not quite as simple as I might have hoped. I have looked at a package called "Amova" which looks very snazzy; alas I think it might be akin to shooting a fly with a bazooka. However... does anyone have any recommendations for what might be able to quantify this data? Oops forgot to mention - I have scored the individuals for the presence/ absence of 10 different bands generated by each primer. Anyway. If anyone has any words of support, or suggestions of some use, I would greatly appreciate hearing them! Thanks! Tim Chipman 901106c@dragon.acadiau.ca . From owner-rapd@net.bio.net Wed Feb 16 22:00:00 1994 Path: biosci!GPU.SRV.UALBERTA.CA!dchong From: dchong@GPU.SRV.UALBERTA.CA (Daniel Chong) Newsgroups: bionet.molbio.rapd Subject: Linkage analysis with unknown phase: answer summary Date: 17 Feb 1994 23:22:23 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 154 Sender: daemon@net.bio.net Distribution: bionet Message-ID: References: <199402142149.AA17318@diemen.utas.edu.au> NNTP-Posting-Host: net.bio.net On Tue, 15 Feb 1994 R.Vaillancourt@bioch.utas.edu.au wrote: > Dear Daniel, > > I am embarking in a project where I will be mapping RAPD's in similar types > of crosses in Eucalyptus. I do not know the answer to your question but I > am interested in what you find out. A person that may be able to help you > is Muhammad Lodhi at Cornell University (mal5@cornell.edu (Muhammad A. > Lodhi)). He has analysed data from grape (Vitis) using pseudo-testcross. > Could you post a summary of your answers? > > Good luck > > Sincerely, > > Dr. Rene E. Vaillancourt, > Project Leader, Molecular Biology > CRC for Temperate Hardwood Forestry > Hobart, Tasmania 7005, AUSTRALIA > fax: 61 02 202703 > r.vaillancourt@bioch.utas.edu.au Dear Rene; Muhammd and Toby took their time answering my question. I appreciate very much. Thanks both of you. Their answer are included in this mail. Based on their answers, the following is what I did to code and analyze my data. Hope you find it understandable and useful. ******Coding RAPDs of pseudo-testcross for MAPMAKER******* Datatype f2 backcross xx xx xx symbols 1=H 0=A For each 1:1 segregating RAPD locus, score 1 for present, 0 for absent and - for missing, and inversely rescore the same locus (0 for present and 1 for absent) to create a dummy locus. For two loci you will have genotype AaBb aaBb Aabb aabb RAPD1-a 1 0 1 0 ...... 1 RAPD1-b 0 1 0 1 ...... 2 RAPD2-a 1 1 0 0 ...... 3 RAPD2-b 0 0 1 1 ...... 4 ********Analysis using MAPMAKER ********* For F2 backcross model, MAPMAKER 'thinks' HH/AA (11/00) in coupling and HA/AH (10/01) in repulsion phase. However MAPMAKER will do all pairwise comparisons for each locus including the dummy loci in its "two point" analysis. For the above example, there will be 6 pairwise comparisons, two for coupling; '1,3' and '2,4', two for repulsion; '2,3' and '1,4' and two self-comparisons; '1,2' and '3,4'. The two pairs of self-comparison will definitely not linked to each other. If the coupling is the true phase MAPMAKER will give you a higher LOD score for comaprisons '1,3' and '2,4'. Higher LOD score for '1,4' and '2,3' if they are in repulsion. By setting up stringent linkage criteria (e.g. 4, 25) for MAPMAKER you will screen out unlinked comparisons and select the phase which has the best LOD scores. In this way, for each linkage group, you will have two copies with identical LOD scores and map distances. Select either for furture ordering. By the way you will also know the phase of linked loci. If '1,3' and '2,4' have been selected RAPD1 and RAPD2 are in coupling and if '1,4' and '2,3' have been selected they are in repulsion. Happy Mapping............Daniel ******************************************************************* Question: > RAPD markers have been obtained from a single full-sib cross between >Populus deltoides and P. maximowiczii. I am doing linkage and mapping >analysis on those markers using MAPMAKER. The underlied genetic model >is testcross (or pseudotestcross) with unknown linkage-phase. Can anyone >tell me how to code my data for MAPMAKER ? Are there programs dealing with >phase unknown data? Thank you. >Daniel Chong Answer No.1 Hi Daniel I think you can code the data as following; 'A' for (-) bands 'H' for (+) bands '-' for missing data So if the genotype are, ++-+--++-+-+ it could be changed to, HHAHAAHHAHAH for analysis with MAPMAKER. Also to overcome the problem of missing phase each marker has to be duplicated to create dummy loci. For example, OPA-1 should be changed as following; OPA-1 +-+-+-+++-- OPA-1A HAHAHAHHHAA OPA-1B AHAHAHAAAHH In the initial analysis I will not include markers that are heterozygous in both parents (segregating 3:1). These markers will be used once the two parental linkage groups are estalished, to find homologous linkage groups in both the parents. For that purpose RFLP and isozyme markers could also be used. This results lot of junk in the form of duplicates of linkage groups but these can easily be identified and removed from the analysis. This strategy works very well when the phase is missing. We have used similar strategy in grape maps. At present, there is no software that can handle pseudotest crosses, to my knowledge. Some people recommend JOINMAP but I prefer MAPMAKER due to several reasons. Hope that it will be useful Muhammad A. Lodhi Cornell University > ********************************************************************** Daniel Chong wrote: > > RAPD markers have been obtained from a single full-sib cross between >Populus deltoides and P. maximowiczii. I am doing linkage and mapping >analysis on those markers using MAPMAKER. The underlyiing genetic model >is testcross (or pseudotestcross) with unknown linkage-phase. Can anyone >tell me how to code my data for MAPMAKER ? Are there programs dealing with >phase unknown data? Thank you. Answer No. 2 In a pseudotestcross mapping project you can run it through MAPMAKER as A/C and B/D, i.e. A is one homozygous null, C is the with-band counterpart. Use A and C where P.d. is null, and B/D where P.m. is null. You'll get two maps, one for each parent. Dominant markers linked in repulsion are so poorly cross-informative that you can't really hope to merge the maps without some codominant marker data. I've done a lot of mapping in Populus hyrids with RAPDs, RFLPs, and STSs. Some of the STSs could be used to merge your maps, since I know they work in P. deltoides and many work in P. maximowiczii. Send me your mailing address and I'll send you a copy of a mapping manuscript we have submitted to TAG that has all the gory details. Toby Bradshaw | Department of Biochemistry | Will make genetic linkage maps and College of Forest Resources | for food. University of Washington, Seattle | toby@u.washington.edu | ******************************************************** > From owner-rapd@net.bio.net Thu Feb 17 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: NTSYS-pc;HELP! Date: Fri, 18 Feb 1994 10:59:28 GMT Organization: University of Helsinki Lines: 11 Message-ID: NNTP-Posting-Host: mmkab-01208.pc Hi there! I have a problem with my NTSYS-datahandling. How could I make 3-dimensional view of data where similarity is already calculated? I know how to do it from 1/0 matrix, but if I want to calculate some similarities inside groups and between groups, it is impossible to me (I'm not a 'good' in math). Anyone who can solve this problem please, help. My eternal gratitude is yours. Greetings Kari From owner-rapd@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!YVAX.BYU.EDU!anderswr From: anderswr@YVAX.BYU.EDU (W. Ralph Andersen) Newsgroups: bionet.molbio.rapd Subject: DNA extracted from Penstemon Date: 18 Feb 1994 17:11:58 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01H90ZN6CQB6019TOD@yvax.byu.edu> NNTP-Posting-Host: net.bio.net We are having difficulty extracting DNA from wild Penstemon leaves. Anyone have suggestions. We have tried Thanks. Renae VanBuren From owner-rapd@net.bio.net Thu Feb 17 22:00:00 1994 Path: biosci!bcm!news.msfc.nasa.gov!europa.eng.gtefsd.com!howland.reston.ans.net!usc!yeshua.marcam.com!zip.eecs.umich.edu!umn.edu!gaia.ucs.orst.edu!osshe.edu!news.uoregon.edu!netnews.nwnet.net!news.u.washington.edu!toby From: toby@u.washington.edu (Toby Bradshaw) Newsgroups: bionet.molbio.rapd Subject: Re: Linkage analysis with unknown parental phase Date: 16 Feb 1994 16:17:29 GMT Organization: University of Washington, Seattle Lines: 48 Distribution: bionet Message-ID: <2jtgup$s6s@news.u.washington.edu> References: <199402160232.AA28413@postoffice.mail.cornell.edu> NNTP-Posting-Host: stein.u.washington.edu In article <199402160232.AA28413@postoffice.mail.cornell.edu>, Muhammad A. Lodhi wrote: >> RAPD markers have been obtained from a single full-sib cross between >>Populus deltoides and P. maximowiczii. I am doing linkage and mapping >>analysis on those markers using MAPMAKER. The underlined genetic model >>is testcross (or pseudotestcross) with unknown linkage-phase. Can anyone >>tell me how to code my data for MAPMAKER ? Are there programs dealing with >>phase unknown data? Thank you. >>Daniel Chong > >Hi Daniel >I think you can code the data as following; >'A' for (-) bands >'H' for (+) bands >'-' for missing data This is much better than the way I recommended in a previous post, i.e. coding as A/C and B/D. Actually, either will work in MAPMAKER but the A/H B/H is the correct genetic model. >So if the genotype are, ++-+--++-+-+ >it could be changed to, HHAHAAHHAHAH for analysis with MAPMAKER. >Also to overcome the problem of missing phase each marker has to be >duplicated to create dummy loci. For example, OPA-1 should be changed as >following; > OPA-1 +-+-+-+++-- > OPA-1A HAHAHAHHHAA > OPA-1B AHAHAHAAAHH > In the initial analysis I will not include markers that are heterozygous >in both parents (segregating 3:1). These markers will be used once the two >parental linkage groups are estalished, to find homologous linkage groups >in both the parents. For that purpose RFLP and isozyme markers could also >be used. > >This results lot of junk in the form of duplicates of linkage groups but >these can easily be identified and removed from the analysis. This >strategy works very well when the phase is missing. We have used similar >strategy in grape maps. At present, there is no software that can handle >pseudotest crosses, to my knowledge. Some people recommend JOINMAP but I >prefer MAPMAKER due to several reasons. Hope that it will be useful Toby Bradshaw | Department of Biochemistry | Will make genetic linkage maps and College of Forest Resources | for food. University of Washington, Seattle | toby@u.washington.edu | From owner-rapd@net.bio.net Fri Feb 18 22:00:00 1994 Path: biosci!CGNET.COM!N.HUANG From: N.HUANG@CGNET.COM Newsgroups: bionet.molbio.rapd Subject: AFLP Date: 19 Feb 1994 23:29:20 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01H93LC9W19C00080L@irri.cgnet.com> NNTP-Posting-Host: net.bio.net Dear Netter, I have a question. Someone told me that a new technique called AFLP is so useful in producing markers that it might be comparable with RAPD. Can anyone tell me how the technique works and how to obtain detailed information? Thanks. Ning Huang International Rice Research Institue PO Box 933 1099 Manila Philippines Nhuang@IRRI.CGNET.COM FAX 63-2-818-2087 Phone 63-2-818-1926 From owner-rapd@net.bio.net Sat Feb 19 22:00:00 1994 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: AFLP Date: 20 Feb 1994 01:52:11 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199402200152.AA03519@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net In response to Ning Huang's psoting of February 19 >Dear Netter, > >I have a question. Someone told me that a new technique called >AFLP is so useful in producing markers that it might be >comparable with RAPD. Can anyone tell me how the technique >works and how to obtain detailed information? Thanks. Hi Ning I am including a reference here that may be helpful in understanding AFLP. Also there was an interesting talk at Plant Genome II meeting at San Diego about applications of AFLP by Mark Zabeau from Keygene Holland. Unfortunately, I have neither his e-mail address nor the abstract, both are missing from the abstract. He will be a good person to talk to. Someone else may be able to help you on that. "Amplified fragment length polymorphisms of Meloidogyne spp. using oligonucleotide primers. Xue-B. Baillie-D-L. Webster-J-M. Fundamental and Applied Nematology.16 (6). 1993. 481-487." Muhammad A. Lodhi Cornell University From owner-rapd@net.bio.net Sat Feb 19 22:00:00 1994 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: estimation of genome size Date: 20 Feb 1994 16:35:45 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199402201636.AA12115@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net Hi Friends Could somebody suggest how can I estimate the total genome size of a genotype. Or how can I estimate about the %age of the genome size that I have covered in the genetic maps so far. At present, I have genetic linkage maps of grape (one for each parent) and analysis of DNA content. Your responses will be appreciated. Thanks Muhammad A. Lodhi Cornell Univeristy From owner-rapd@net.bio.net Sun Feb 20 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!usenet.ins.cwru.edu!lerc.nasa.gov!magnus.acs.ohio-state.edu!dsammata From: dsammata@magnus.acs.ohio-state.edu (Diana Sammataro) Newsgroups: bionet.molbio.evolution,bionet.molbio.rapd,bionet.population-bio,sc Subject: RAPDs and arthropods Date: 21 Feb 1994 21:22:22 GMT Organization: The Ohio State University Lines: 27 Distribution: bionet Message-ID: <2kb8me$8ks@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: top.magnus.acs.ohio-state.edu Summary: Call for papers Xref: biosci bionet.molbio.evolution:1481 bionet.molbio.rapd:448 bionet.population-bio:583 SYMPOSIUM ANNOUNCEMENT RAPDs and Arthropods Entomological Society of America Dallas, TX Dec. 13-17, 1994 A symposium is proposed to discuss the techniques, pitfalls and successes in using the molecular technique known as RAPDs for arthropods. A call for papers for the 1994 program to any researchers using this technique in their work on insects, spiders, mites or related organisms. Deadline: March 15, 1994 If you think you want to participate, please contact: Diana Sammataro Phone: 614 292 9089 Fax: 614 292 2180 Dept. Entomology 103 Botany & Zoology Bldg. 1735 Neil Avenue Columbus, OH 43210-1220 E-mail: dsammata@magnus.acs.ohio-state.edu From owner-rapd@net.bio.net Sun Feb 20 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: rapd news letter Newsgroups: bionet.molbio.rapd Subject: NaOH extraction of DNA for RAPDs Date: 21 Feb 1994 12:22:14 -0000 Lines: 34 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2ka91m$bfu@mserv1.dl.ac.uk> Encoding: 34 TEXT Original-To: postbox I would like to hear whether people have succesfully used the NaOH DNA extraction method of Wang et al (NAR 21; 4153-4154) for RAPD's. We are working on Taraxacum (dandelions) which contain a lot of latex. While CTAB and SDS methods yield DNA that can be used for RFLP studies, PCR amplification is strongly inhibited. The NaOH method is very simple and works well for the amplification of cpDNA. However, with RAPD's there are problems with the reproducibility of the banding patterns, probably because the template DNA concentration is too low when we are using Wang's dilutions (1/100 of the original extract). This seems to be a general problem since at low DNA concentractions the RAPD amplification reaction seems to be chaotic (Williams et al. 1993, Methods Enzymol. 218). Nevertheless, I have heard rumours that some people are using the Wang et al.'s original method for RAPDs. In our hands, increasing the DNA amount per reaction by diluting less gives inhibitor problems again. I am wondering whether people have tried to modify Wang's original method by including other compounds during extraction or by additional precipitations and washes. Another problem with this method is the measurement of the actual DNA concentration, since in crude NaOH extracts the DNA will be single stranded. Hence fluoresence methods will not work. Has anyone tried to measure DNA concentrations? I would greatly appreciate to hear from others! Thanks in advance! Peter van Dijk Dept. of Plant Population Biology Netherlands Institute for Ecological Research pjvandijk@nioo.nl From owner-rapd@net.bio.net Sun Feb 20 22:00:00 1994 Path: biosci!esvax.dnet.dupont.com!rafalski From: rafalski@esvax.dnet.dupont.com Newsgroups: bionet.molbio.rapd Subject: AFLP Date: 21 Feb 1994 12:59:18 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9402211254.AA25779@esds01.es.dupont.com> NNTP-Posting-Host: net.bio.net The best description of AFLP technique is in the European Patent Office publication EP 0 534 858 A1. The AFLP technology is commercialized by Keygene: Keygene n.v. Dr. Marc Zabeau, General Manager Agro Business Park 90 POBox 216, 6700 AE Wageningen The Netherlands Tel 31 8370 24141 fax 31 8370 24939 A very short summary of some AFLP work is in Mazie Genetics Newsletter #67 (1993) page 62 (Smith, Zabeau and Wright). Antoni Rafalski From owner-rapd@net.bio.net Sun Feb 20 22:00:00 1994 Path: biosci!GPU.SRV.UALBERTA.CA!dchong From: dchong@GPU.SRV.UALBERTA.CA (Daniel Chong) Newsgroups: bionet.molbio.rapd Subject: Re: estimation of genome size Date: 21 Feb 1994 22:10:51 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 52 Sender: daemon@net.bio.net Distribution: bionet Message-ID: References: <199402201636.AA12115@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net On 20 Feb 1994, Muhammad A. Lodhi wrote: > Date: 20 Feb 1994 16:35:45 -0000 > From: Muhammad A. Lodhi > To: rapd@net.bio.net > Subject: estimation of genome size > > Hi Friends > Could somebody suggest how can I estimate the total genome size of a > genotype. Or how can I estimate about the %age of the genome size that I > have covered in the genetic maps so far. At present, I have genetic linkage > maps of grape (one for each parent) and analysis of DNA content. Your > responses will be appreciated. Thanks > > Muhammad A. Lodhi > Cornell Univeristy > > > Hi Muhammad; Genome size can be estimated from genetic linkage map using the following equation; G(Z)=[M*(M-1)/2]*2*X(Z)/K(Z) where for a given LOD score, Z, G(Z) is the genome size in cM, M is number of markers analyzed, X(Z) is the maximum cM distance between linked markers, and K(Z) is the number of linkages. The above equation is the method 3 of Chakravarti et al. (1991). You may read the paper for more details. Another good paper is Nelson et al. (1993). They developed a partial linkage map with RAPDs of slash pine using megagametophytes. They used the above formula to estimate genome size and another formula to calculate % coverage of the genome with certain number of RAPDs. Chakravarti A, Lasher LK, Reefer JE (1991) A maximum likelihood method for estimating genome length using genetic linkage data. Genetics 128:175-182. Nelson CD, Nance WL Doudrick RL (1993) A partial genetic linkage map of slash pine (Pinus elliottii Engelm. var.elliottii) based on random amplified polymorphic DNAs. Theor Appl Genet 87:145-151. Hope this will help. Daniel Chong University of Alberta From owner-rapd@net.bio.net Mon Feb 21 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!msuinfo!harbinger.cc.monash.edu.au!uniwa!newsman!vetmac2.csu.murdoch.edu.au!Hopkins From: Richard Hopkins Newsgroups: bionet.molbio.rapd Subject: Scan analysis of RAPD's (Dendron vs Gel Manager) Date: 22 Feb 1994 03:57:10 GMT Organization: Murdoch University Lines: 17 Distribution: world Message-ID: <2kbvqmINNk1q@newsman.csu.murdoch.edu.au> NNTP-Posting-Host: vetmac2.csu.murdoch.edu.au X-UserAgent: Nuntius v1.1.1d17 X-XXMessage-ID: X-XXDate: Tue, 22 Feb 94 12:02:50 GMT Hi netters, We have been looking at software that will allow us to compare scanned images of RFLP and RAPD gels. Two packages that look particulary useful are "Dendron" from Solltech and "Gel Manager" from Biosystematica. They both seem to have very similar functions yet there is a huge difference in price, $10,000 (Australian) and $2,000 respectively. If anyone has had any experience with either of these programmes or others like them we would greatly appreciate any comments. The cost of these packages represents a considerable investment and we really dont want to buy a lemon! Thanks in advance Richard Hopkins Veterinary School Murdoch University From owner-rapd@net.bio.net Wed Feb 23 22:00:00 1994 Path: biosci!PUCCINI.CRL.UMN.EDU!debbys From: debbys@PUCCINI.CRL.UMN.EDU ("Deborah A. Samac") Newsgroups: bionet.molbio.rapd Subject: Thermocycler for RAPDs Date: 24 Feb 1994 21:08:36 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9402242109.AA04035@puccini.crl.umn.edu> NNTP-Posting-Host: net.bio.net Dear Netters, We have been attempting to do RAPD PCR using a Techne PHC-3 thermocycler and had very poor results; we get only a few faint bands or no bands at all. However, duplicate reactions run in a Coy Tempcycler II down the hall give reproducible, bright bands. Both machines were set to run the same profiles of 1 min 94 C, 1 minute at 36 C, 2 minutes 72 C for 40 cycles. We've tried adjusting the ramping of the Techne to match the slower temperature changes of the Coy but this did not improve amplification. The temperture settings on the Techne also had to be adjusted to match the temperatures attained by the Coy. For example, although the Techne was set for 94 C, it only reached 94 degrees for about 5 seconds before starting the next step. We're about ready to pitch the Techne (from a very high window) and purchase a new machine. Any advice on getting the Techne to work will be appreciated. (I have not been able to get any technical support on this problem from Techne.) Thanks, Debby Deborah A. Samac debbys@puccini.crl.umn.edu Department of Plant Pathology, 495 Borlaug Hall University of Minnesota St. Paul, MN 55108 (612) 625-1243 (office) (612) 625-9728 (fax) From owner-rapd@net.bio.net Thu Feb 24 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: nfix@virgo.jcu.cz (Ivo Wiesner) Newsgroups: bionet.molbio.rapd Subject: ??too high RAPD background-ivo w. Date: 25 Feb 1994 16:04:21 -0000 Lines: 29 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2kl7i5$3i0@mserv1.dl.ac.uk> X-Char-Esc: 29 X-Charset: ASCII Original-To: rapd@dl.ac.uk Hello Netters! Could somebody out there share with me his/her experience in RAPD reaction? I work on RAPDs on fescue and pea and have constant problems with too high background in lines on gel which covers more or less the bands themselves. I use200uM dNTPs, 3mMMgCl2, 1U Taq polymerase/25ul, 1uM primer (10 bases), template within 100ng-10pg/25ul, so usual reaction temperature profile 45 cycles with 30deg.annealing orusual 37deg.annealing and originates with denaturation at 95 deg. for 5 min., ending with 10 min.at 72 deg. I tested various concentrations of template, primer (o.2uM-4uM), only 35 cycles with 37 deg.annealing. I constantly receive high background which prevent evaluation of products... Has somebody out there any idea what should be changed or what is going wrong???? Any suggestion highly appreciated!!!!!!!!!!!!!! Greetings Ivo Wiesner nfix@virgo.jcu.cz From owner-rapd@net.bio.net Thu Feb 24 22:00:00 1994 Path: biosci!NET.BIO.NET!kristoff From: kristoff@NET.BIO.NET (David Kristofferson) Newsgroups: bionet.molbio.rapd Subject: IMPORTANT BIOSCI INFORMATION Date: 25 Feb 1994 10:00:09 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 244 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199402251000.CAA24358@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. Sincerely, Dave Kristofferson BIOSCI/bionet Manager kristoff@net.bio.net **** SEARCHING BIOSCI ARCHIVES WITH WAISMAIL **** E-mail users can search the BIOSCI archives by using our waismail e-mail server. For instructions send the message help to waismail@net.bio.net. Leave the Subject: line blank. Other methods of searching the archives via WAIS and gopher are described in the BIOSCI FAQ. **** BIOSCI FREQUENTLY ASKED QUESTIONS (FAQ) SHEET **** New users of BIOSCI/bionet may want to read the "Frequently Asked Questions" or "FAQ" sheet for BIOSCI. The FAQ provides details on how to participate in these forums and is available for anonymous FTP from net.bio.net [134.172.2.69] in pub/BIOSCI/biosci.FAQ or for retrieval by gopher to net.bio.net, port 70. It may also be requested by sending e-mail to biosci@net.bio.net (use plain English for your request). The FAQ is also posted on the first of each month to the newsgroup BIONEWS/bionet.announce immediately following the posting of the BIOSCI information sheet. **** BIOSCI USER ADDRESS DIRECTORY **** Please take this opportunity to add your name and address information to the BIOSCI User Address Database if you have not already done so. Below is the address form that we would like each reader of the BIOSCI/bionet newsgroups to complete and return if you would like to be listed in our database. The database serves as a directory that enables biologists, who are currently using (or even just reading) the BIOSCI newsgroups, to look up e-mail addresses and other information about our users. The address database is reindexed nightly for WAIS and waismail access (waismail is our WAIS e-mail server, more below) and will also be available for access via other gopher sites if they wish to permit it. The raw unindexed data is available for FTP from net.bio.net and is atomized sufficiently to allow import into your local RDBMS should you so desire. Please carefully follow the instructions for completing the form below and return it to either of the following two addresses (whichever is more convenient for you). Thanks in advance for taking the time to complete and return the form. Addresses for returning forms Location Network ----------------------------- -------- ------- biovote@net.bio.net U.S.A. Internet/BITNET biovote@daresbury.ac.uk U.K. JANET MAKING SURE THAT YOUR INFORMATION IS CURRENT This notice will be mailed bimonthly to each newsgroup. You should check our WAIS source or waismail e-mail server from time-to-time to see if your address information is still up-to-date. Send the message help to waismail@net.bio.net for instructions on using waismail. Leave the Subject: line in your message blank. Using Gopher to complete the form --------------------------------- If you don't want to use a text editor, you can also use Dan Jacobson's gopher site to fill out the address database form as follows. Otherwise skip this section on gopher and proceed to the instructions for filling out the form below. > To add yourself to the database just point your > gopher client at merlot.gdb.org and select the following: > > --> 15. Searching For Biologists/ > > --> 9. E-mail Addresses of Biosci-Bionet Users/ > > --> 1. Add (or Correct) Your Address to the BIOSCI User Address > Data.. > > > And fill out the form. or Rob Harper's gopher site in Europe as follows: > Europeans can point their gopher client at gopher.csc.fi and add their > information to the database. All entries will be mailed directly to > Dave for incorporation in a wais source. > > The path to the questionare is as follows. > > ---> 10. Finnish EMBnet BioBox/ > > ---> 8. FAQ Files/ > > FAQ Files > > 1. EMBnet: Information. > 2. EMBnet: Internet resources guide. > 3. A Biologist's Guide to Internet Resources/ > 4. All FAQs (Frequently Asked Questions) Searches and Archives/ > --->5. Bionauts Address Database (questionaire) IMPORTANT INSTRUCTIONS - PLEASE READ CAREFULLY Please enter all responses after the : on each line, leaving one (1) blank space after the : (i.e., before the start of your text). Please do NOT extend your responses past the end of each line (80 characters) or alter any of the field identifiers such as "first name: ". Several lines are provided at the end of the form for comments, but, please adhere to the line length restriction. On the date: line, please enter the date in the DD-MM-YY format, e.g., 05-05-93 for 5 May 1993. This line will tell others when the information was last updated. Please be sure to include the 0's for single digit days or months, e.g., 05-05-93, not 5-5-93. Note that the "e-mail network: " line below is for specifying, e.g., "Internet," "BITNET," "EARN," "JANET," or whatever other network that your computer may be on. If you are uncertain about any field, please feel free to leave it blank, but please DO NOT DELETE the field identifier from the form! In the first field below, "New information or Update ...", please enter "N" if this is the first time that you have registered in the directory or "U" if you are correcting a listing that you sent to us previously. The comment: lines may be used for anything that you like but PLEASE DO NOT DELETE THEM FROM THE FORM OR ALTER THEM. One suggested use is to list the names of the newsgroups in which you participate. Please use the MAILING LIST name (see below - the latest version of the list can be requested from biosci@net.bio.net) instead of the USENET name even if you don't participate by e-mail. WAIS might get confused by the periods in the USENET names. This allows one to retrieve via WAIS or waismail the list of participants in a particular group. For example: comment: ARABIDOPSIS PLANT-BIOLOGY BIONEWS On the comment: lines use these names below ---- NOT the USENET names below MAILING LIST NAME USENET Newsgroup Name ----------------- --------------------- ACEDB-SOFT bionet.software.acedb AGEING bionet.molbio.ageing AGROFORESTRY bionet.agroforestry ARABIDOPSIS bionet.genome.arabidopsis BIOFORUM bionet.general BIO-INFORMATION-THEORY bionet.info-theory BIONAUTS bionet.users.addresses BIONEWS bionet.announce BIO-JOURNALS bionet.journals.contents BIO-MATRIX bionet.molbio.bio-matrix BIO-SOFTWARE bionet.software CHROMOSOMES bionet.genome.chromosomes COMPUTATIONAL-BIOLOGY bionet.biology.computational DROSOPHILA bionet.drosophila EMBL-DATABANK bionet.molbio.embldatabank EMPLOYMENT bionet.jobs GDB bionet.molbio.gdb GENBANK-BB bionet.molbio.genbank GENETIC-LINKAGE bionet.molbio.gene-linkage HIV-MOLECULAR-BIOLOGY bionet.molbio.hiv HUMAN-GENOME-PROGRAM bionet.molbio.genome-program IMMUNOLOGY bionet.immunology INFO-GCG bionet.software.gcg JOURNAL-NOTES bionet.journals.note METHODS-AND-REAGENTS bionet.molbio.methds-reagnts MOLECULAR-EVOLUTION bionet.molbio.evolution NEUROSCIENCE bionet.neuroscience N2-FIXATION bionet.biology.n2-fixation PHOTOSYNTHESIS bionet.photosynthesis PLANT-BIOLOGY bionet.plants POPULATION-BIOLOGY bionet.population-bio PROTEIN-ANALYSIS bionet.molbio.proteins PROTEIN-CRYSTALLOGRAPHY bionet.xtallography RAPD bionet.molbio.rapd SCIENCE-RESOURCES bionet.sci-resources TROPICAL-BIOLOGY bionet.biology.tropical VIROLOGY bionet.virology WOMEN-IN-BIOLOGY bionet.women-in-bio YEAST bionet.molbio.yeast Listing newsgroups on the comment: line is optional, of course. 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 Fri Feb 25 22:00:00 1994 Path: biosci!VTVM1.CC.VT.EDU!RUSSULA From: RUSSULA@VTVM1.CC.VT.EDU ("J. Murphy") Newsgroups: bionet.molbio.rapd Subject: Static electricity Date: 26 Feb 1994 19:26:53 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199402261926.LAA29812@net.bio.net> NNTP-Posting-Host: net.bio.net Hi rapd people; Have any of you noticed a lot of static charge surrounding microfuge tubes when loading them? I first noticed this when adding the mineral oil cap; the charge would literally tear the drop of oil off the pipette tip before the drop was big enough to fall. It strikes me that not only mineral oil would respond to the charge, but possibly also contaminating particles which happen to be around. Not surprisingly, the charge seems to be strongest when cold, dry, high pressure weather prevails. Any ideas/experiences/problems/solutions? -Jack From owner-rapd@net.bio.net Sat Feb 26 22:00:00 1994 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: ??too high RAPD background-ivo w. Date: 27 Feb 1994 00:23:57 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 48 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199402270024.AA26108@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net In response to Ivo Wiesner's following posting; > Could somebody out there share with me his/her >experience in RAPD reaction? >I work on RAPDs on fescue and pea and have constant >problems with too high background in lines on >gel which covers more or less the bands themselves. >I use200uM dNTPs, 3mMMgCl2, 1U Taq polymerase/25ul, >1uM primer (10 bases), template within 100ng-10pg/25ul, >so usual reaction >temperature profile 45 cycles with 30deg.annealing >orusual 37deg.annealing and originates with denaturation >at 95 deg. for 5 min., ending with 10 min.at 72 deg. > >I tested various concentrations of template, primer (o.2uM-4uM), >only 35 cycles with 37 deg.annealing. >I constantly receive high background which prevent >evaluation of products... > >Has somebody out there any idea what should be changed >or what is going wrong???? >Any suggestion highly appreciated!!!!!!!!!!!!!! The following two articles could be useful in finding out the problem that you are facing; 1) Weeden et al. 1992. Inheritance and reliability of RAPD markers. Application of RAPD Tech. to Plant Breeding. pp. 12-17. Joint Plant Breeding Symposia Series of Crop Sci. Soc. of Amer., Amer. Soc. Hort. Sci. and Amer. Genet. Asoc. 2) Williams et al. 1993. Genetic analysis using random amplified polymorphic DNA markers. Methods in Enzymology, 218:705-740. I have observed that the reason for high background is usually higher primer and Taq conc. and lower magnesium conc. These differences are mentioned in the first article. I use 0.5 unit of Taq, 2-3 mM Mg and 120 uM primers. The other possible reasons could be high template DNA conc., over-exposure or over-developing of the film and excessive EtBr staining. You may want to test all the components not only individually but also in combinations. This takes some time but helps in the long run for reliability and repeatability of RAPDs, thats what I found in grapes. Good luck! Muhammad A. Lodhi Cornell University From owner-rapd@net.bio.net Sun Feb 27 22:00:00 1994 Path: biosci!cornell.edu!gy11 From: gy11@cornell.edu (Guangning Ye) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 28 Feb 1994 21:58:05 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199402282158.AA14762@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net subscription