From owner-rapd@net.bio.net Mon Oct 04 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!pipex!uunet!munnari.oz.au!uniwa!newsman!Jim.Cummins From: cummins@possum.murdoch.edu.au (Jim Cummins) Newsgroups: bionet.molbio.rapd Subject: Mitochondrial DNA Message-ID: <28qvisINN3hj@newsman.csu.murdoch.edu.au> Date: 5 Oct 93 05:09:48 GMT Sender: -Not-Authenticated-[6576] Organization: Murdoch University, Western Australia Lines: 8 NNTP-Posting-Host: vetmac3.csu.murdoch.edu.au X-Posted-From: InterNews 1.0@newsman.csu.murdoch.edu.au. Xdisclaimer: No attempt was made to authenticate the sender's name. Forgive me if this is a FAQ, but has anyone applied RAPD to mitochondrial DNA? Jim Cummins School of Veterinary Studies Murdoch University Western Australia 6150 Tel +61-9-360 2668 Fax +61-9-310 4144 For every complex problem there's a simple solution. And it's wrong! From owner-rapd@net.bio.net Tue Oct 05 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: Mitochondrial DNA Message-ID: <6D683610633@uamercury.uark.edu> Date: 6 Oct 93 14:01:31 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 20 >To: rapd@net.bio.net >From: cummins@possum.murdoch.edu.au (Jim Cummins) >Subject: Mitochondrial DNA >Date: 5 Oct 93 05:09:48 GMT >Forgive me if this is a FAQ, but has anyone applied RAPD to >mitochondrial DNA? > >Jim Cummins RAPD would not work very well with mtDNA because of the small genome size. Since so many mtDNAs have been sequenced would it not be better to build consensus degenerate PCR primers ala a Sequence-Tagged-Site. My guess is that in RAPD you would have to worry about amplifying signals from trace contaminanting amounts of genomic DNA. This would be because of more RAPD targetable sites. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Wed Oct 06 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: Chelex Message-ID: <6F64E2F769A@uamercury.uark.edu> Date: 7 Oct 93 21:48:53 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 15 As far as I am concerned the chelex is not necessary unless you have alot of contaminants that interfere with RAPD. With directed-PCR such as STSs you don't need the chelex unless you have a lot of crap staying in the supernate. For an alternative check out the note in the most recent Biotechnique from Steve Lee and North Colorado. He describes a microwave miniprep method that appears to work on just about anything. We had the protocol from a preprint from Steve and it does work as advertised. We tried it on plant, fungi, and bacteria, no problem. How about other people. I figure if you boil to kill the proteases and nucleases it blows open enough cells to get some DNA in the supe to PCR. 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 Oct 06 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Chelex Message-ID: <6EE5F235BCD@uamercury.uark.edu> Date: 7 Oct 93 13:52:58 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 38 > > >>In regards to the question on screening yeast colonies by PCR we have >used >>a procedure for screening bacterial colonies and animal blood that should >>work with just about anything. > >>1) Resuspend a small amount of cells from the colony (tip of a toothpick) >>or 1-2 ul of whole animal blood in 100 ul sterile water. >>2) place in boiling water bath for 10 minutes >>3) spin 5' in the cold >>4) use 2 in a 10ul or 5 ul in a 40ul PCR reaction > >>If you have problems with signal BioRad sells a purified chelex that you >>can add some to the boil that pulls out some inhibitors > >Can you tell me more about the chelex? Actual name, catalog # if you have >it? > >Thanks for your help. > >Michael Lichten >lichten@helix.nih.gov I don't have the Catalog # but the BioRad salesperson was just here with a brochure on the new item. Unfortunately, I passed the notice on to a friend and don't have the new number and can't remember the cute name they gave it. If you call 1-800-4BIORAD and ask them about their new purified chelex for isolation of DNA for PCR they can give you the particulars. We have used the less-pure version of chelex from BioRad for DNA isolations from plant tissue with good success, it does get out some inhibitors. BTW, (standard disclaimer goes here) I don't work for, own stock in, or receive money from the above supplier. 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 Oct 06 23:00:00 1993 Path: biosci!esvax.dnet.dupont.com!rafalski From: rafalski@esvax.dnet.dupont.com Newsgroups: bionet.molbio.rapd Subject: Chelex Message-ID: <9310071437.AA17035@esds01.es.dupont.com> Date: 7 Oct 93 14:37:25 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 8 Chelex-100, biotechnology grade, Bio-Rad catalog p10 #143-2832, 100grams, $85. Other grades also available, see same catalog, p8 (Catalog 1993 "Price list S"). I find it quite interesting that people would rather type a few e-mail messages then reach for a catalog on the shelf. Antoni From owner-rapd@net.bio.net Thu Oct 07 23:00:00 1993 Path: biosci!M.CC.UTAH.EDU!jay.evans From: jay.evans@M.CC.UTAH.EDU Newsgroups: bionet.molbio.rapd Subject: Chelex and DNA extract decay? Message-ID: <9310082112.AA07423@fcom.cc.utah.edu> Date: 8 Oct 93 20:25:00 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 13 Cano and Poinar (Biotechniques, 1993, Vol. 15, p. 432+) have some sobering news regarding DNA extractions using Chelex-100. Apparently both fossilized and modern DNA (extracted via Chelex) may become unamplifiable in fairly short order (two weeks when stored at -20 oC). They do not speculate on causes (DNA degradation vs. some sort of binding process), but it seems to be a potential worry. Have others found similarly short lives for their Chelex-based extracts?? Jay Evans Biology, Univ. of Utah From owner-rapd@net.bio.net Thu Oct 07 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: Chelex Message-ID: <70670411B15@uamercury.uark.edu> Date: 8 Oct 93 13:56:49 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 78 >From: "Huang Bixing" >Date: Fri, 8 Oct 93 11:14:44 EST >Reply-to: >To: DRHOADS@mercury.uark.edu >Subject: Re: Chelex >On 7 Oct 93 15:48:53 CST, Doug Rhoads wrote: > >>As far as I am concerned the chelex is not necessary unless you have alot >>of contaminants that interfere with RAPD. With directed-PCR such as STSs >>you don't need the chelex unless you have a lot of crap staying in the >>supernate. For an alternative check out the note in the most recent >>Biotechnique from Steve Lee and North Colorado. He describes a microwave >>miniprep method that appears to work on just about anything. We had the >>protocol from a preprint from Steve and it does work as advertised. We >>tried it on plant, fungi, and bacteria, no problem. > > >Dear Dr. Rhoads, > >I am doing my PhD project which is ivloved in phenol/choroform and chelex >extraction of DNA. I would like to have a copy of the protocol you >mentioned above, which is extremely easy and quick. Could you mail or fax >me a copy, please. > >My postal address is : > Bixing Huang > Department of Biology > Deakin University > Geelong, Vic 3217 > Australia. >Fax number is 61 52 272022. > >Thank you very much for your time. > >With regards. > >Your respectfully > >Bixing Huang >----- > >School of Biological and Chemical Sciences, Deakin University, Geelong >----- I don't send out other peoples reprints or preprints. I would suggest you consult the original article for the details. Here is my condensation of the procedure into steps. Microwave Isolation of Total DNA for PCR/RAPD Analysis This procedure was developed by D. Goodwin and S. Lee (slee@goldng8.univnorthco.edu) at U of North Colorado. Reference: Biotechniques (1993) 15:438-444. Purportedly works with everything: bacteria, plants, fungi and protists. Solutions: Lysis Buffer- 50 mM TrisCl pH 7.2, 50 mM Na2EDTA, 3% SDS, 1% - Mercaptoethanol Phenol-Hcl3- 50% Phenol, 48% Hcl3, 2% Isoamyl alcohol 1. Place 0.5 cm2 of mycelium (fungi: 0.1-2 g wet weight or equivalent weight of other sample) in 1.5 ml microfuge tube containing 50-100 l of lysis buffer. 2. With tube open and loosely covered with plastic wrap, microwave on high (400 W check your microwave output) for 15", then 10", and then 5". Total of 30" but NO BOILING. 3. Add 300-350 l lysis buffer (vol=400 l) and incubate 10' at 80oC 4. Add 400 l Phenol-CHCl3, vortex and centrifuge 10' room temp 10k x g. 5. Remove aqueous to new tube, add NaOAc to 0.3 M and isopropanol to 54% (about 1.1 volumes), spin 2' at 15 krpm or 4' at 10k x g at room temp., decant and rinse pellet with cold 80% EtOH. 6. Resuspend in 50 to 150 l Te (depending on experience) YIELD: 0.1 to 10 g/ l depending on source and starting quantity of tissue Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Sun Oct 10 23:00:00 1993 Path: biosci!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!europa.eng.gtefsd.com!uunet!biosci!M.CC.UTAH.EDU!jay.evans From: jay.evans@M.CC.UTAH.EDU Newsgroups: bionet.molbio.rapd Subject: Chelex and DNA extract decay? Message-ID: <9310082112.AA07423@fcom.cc.utah.edu> Date: 8 Oct 93 20:25:00 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 11 Cano and Poinar (Biotechniques, 1993, Vol. 15, p. 432+) have some sobering news regarding DNA extractions using Chelex-100. Apparently both fossilized and modern DNA (extracted via Chelex) may become unamplifiable in fairly short order (two weeks when stored at -20 oC). They do not speculate on causes (DNA degradation vs. some sort of binding process), but it seems to be a potential worry. Have others found similarly short lives for their Chelex-based extracts?? Jay Evans Biology, Univ. of Utah From owner-rapd@net.bio.net Mon Oct 11 23:00:00 1993 Path: biosci!NMSUVM1.NMSU.EDU!G5930716 From: G5930716@NMSUVM1.NMSU.EDU ("Tito P. Alcantara") Newsgroups: bionet.molbio.rapd Subject: subscription Message-ID: <9310121729.AA20252@net.bio.net> Date: 12 Oct 93 15:29:41 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 3 Please include me in your list of subscribers. My e-mail address is: Thanks a lot. From owner-rapd@net.bio.net Mon Oct 11 23:00:00 1993 Path: biosci!NMSU.EDU!talcanta From: talcanta@NMSU.EDU Newsgroups: bionet.molbio.rapd Subject: subscription Message-ID: <9310122053.AA14216@NMSU.Edu> Date: 12 Oct 93 20:53:02 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 9 Please include me in your list of subscribers. My e-mail address is: Thanks for your consideration. Tito P. Alcantara Plant Breeding and Genetics Department of Agronomy and Horticulture New Mexico State University Las Cruces, NM 88003-0003 From owner-rapd@net.bio.net Tue Oct 12 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: aqprc1@uk.ac.stir (Rita Castilho) Newsgroups: bionet.molbio.rapd Subject: microwave pcr Message-ID: <1993Oct13.121409.16544@gserv1.dl.ac.uk> Date: 13 Oct 93 12:09:24 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 17 Precedence: first-class Original-To: rapd@uk.ac.daresbury (rapd) X-Mailer: ELM [version 2.3.S2 PL3] > Microwave Isolation of Total DNA for PCR/RAPD Analysis > This procedure was developed by D. Goodwin and S. Lee > (slee@goldng8.univnorthco.edu) at U of North Colorado. > Reference: Biotechniques (1993) 15:438-444. Purportedly works with > everything: bacteria, plants, fungi and protists. Does anyone have any idea if it will also works for fish muscle? Thanks! Rita Castilho Fax: +44.(0)786.472133 Institute of Aquaculture E-mail: aqprc1@forth.stir.ac.uk University of Stirling Stirling FK9 4LA U.K. From owner-rapd@net.bio.net Tue Oct 12 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: microwave pcr on Fish Muscle Message-ID: <7803FF85253@uamercury.uark.edu> Date: 13 Oct 93 15:44:36 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 30 >To: rapd@net.bio.net >From: aqprc1@uk.ac.stir (Rita Castilho) >Subject: microwave pcr >Date: 13 Oct 93 12:09:24 GMT > > >> Microwave Isolation of Total DNA for PCR/RAPD Analysis >> This procedure was developed by D. Goodwin and S. Lee >> (slee@goldng8.univnorthco.edu) at U of North Colorado. >> Reference: Biotechniques (1993) 15:438-444. Purportedly works with >> everything: bacteria, plants, fungi and protists. > >Does anyone have any idea if it will also works for fish muscle? > >Thanks! > >Rita Castilho I think the procedure would work fine if you homogenized before microwaving. However, if you homogenize then you don't need to microwave. We routinely isolate from fish parts and mammal muscle by homogenizing with a tissuemizer (modified Dremel tool) in TE. Then add SDS, EDTA and pronase for a while. Extract with Phenol:CHCl3. EtOH ppt and you got DNA. It isn't fast but it works. 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 Oct 13 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: DIBAGEF1@IT.CNR.FI.IDG.VM (Priscilla Bettini) Newsgroups: bionet.molbio.rapd Subject: Request Message-ID: <1993Oct14.132443.5326@gserv1.dl.ac.uk> Date: 14 Oct 93 14:20:20 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Organization: Dipartimento di Biologia Animale e Genetica Lines: 17 Precedence: first-class from VM.IDG.FI.CNR.IT by UKACRL.BITNET (Mailer R2.07) with BSMTP id 3525; Thu, 14 Oct 93 14: 21:47 BST Original-To: rapd@uk.ac.daresbury I would like to be included in the RAPD network. Priscilla Bettini Department of animal Biology and Genetics, Florence (Italy). E-mail address: DIBAGEF1@VM.IDG.FI.CNR.IT ------------------------------------------- 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@idg.fi.cnr.it ------------------------------------------- From owner-rapd@net.bio.net Wed Oct 13 23:00:00 1993 Path: biosci!bloom-beacon.mit.edu!news.kei.com!sol.ctr.columbia.edu!math.ohio-state.edu!howland.reston.ans.net!agate!doc.ic.ac.uk!daresbury!daresbury!news From: aqprc1@uk.ac.stir (Rita Castilho) Newsgroups: bionet.molbio.rapd Subject: microwave pcr Message-ID: <1993Oct13.121409.16544@gserv1.dl.ac.uk> Date: 13 Oct 93 12:09:24 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 17 Precedence: first-class Original-To: rapd@uk.ac.daresbury (rapd) X-Mailer: ELM [version 2.3.S2 PL3] > Microwave Isolation of Total DNA for PCR/RAPD Analysis > This procedure was developed by D. Goodwin and S. Lee > (slee@goldng8.univnorthco.edu) at U of North Colorado. > Reference: Biotechniques (1993) 15:438-444. Purportedly works with > everything: bacteria, plants, fungi and protists. Does anyone have any idea if it will also works for fish muscle? Thanks! Rita Castilho Fax: +44.(0)786.472133 Institute of Aquaculture E-mail: aqprc1@forth.stir.ac.uk University of Stirling Stirling FK9 4LA U.K. From owner-rapd@net.bio.net Thu Oct 14 23:00:00 1993 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN TERSE MURRAY) Newsgroups: bionet.molbio.rapd Subject: Re Microwave minniprep extract of DNA Message-ID: <9310151534.AA21443@phibred.phibred.com> Date: 15 Oct 93 15:34:58 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 11 Hi Folks, I rapidly tried the method and tried several different treatments. I know that a second microwave treatment shears the DNA and a treatment of 15 sec, 15 and then 10 secs using 5% SDS yields some high molecular weight DNA and some sheared DNA as well. Oh yeah, that 10 min lysis step at 80 C is essential. If anyone else has obtained good DNA for RAPD, please inform me. I used plant cotyledons for DNA extraction. Murayian@phibred.com From owner-rapd@net.bio.net Thu Oct 14 23:00:00 1993 Path: biosci!bloom-beacon.mit.edu!usc!howland.reston.ans.net!pipex!sunic!corax.udac.uu.se!zeta.bmc.uu.se!daresbury!daresbury!news From: DIBAGEF1@IT.CNR.FI.IDG.VM (Priscilla Bettini) Newsgroups: bionet.molbio.rapd Subject: Request Message-ID: <1993Oct14.132443.5326@gserv1.dl.ac.uk> Date: 14 Oct 93 14:20:20 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Organization: Dipartimento di Biologia Animale e Genetica Lines: 17 Precedence: first-class from VM.IDG.FI.CNR.IT by UKACRL.BITNET (Mailer R2.07) with BSMTP id 3525; Thu, 14 Oct 93 14: 21:47 BST Original-To: rapd@uk.ac.daresbury I would like to be included in the RAPD network. Priscilla Bettini Department of animal Biology and Genetics, Florence (Italy). E-mail address: DIBAGEF1@VM.IDG.FI.CNR.IT ------------------------------------------- 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@idg.fi.cnr.it ------------------------------------------- From owner-rapd@net.bio.net Fri Oct 15 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: degenerate PCR Message-ID: <7C7BB8F233F@uamercury.uark.edu> Date: 16 Oct 93 15:13:05 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 33 >To: yeast@net.bio.net >From: djdlab@sun.com (Dean Danner Lab) >Subject: degenerate PCR >Date: 15 Oct 93 15:33:05 GMT >Reply-to: djdlab@sun.com > >To anyone who is a PCR god or goddess, I am about to embark on a project >involving PCR amplification of a not yet found yeast gene. I am going to >design the primers from human sequence. Is it better to produce >degenerate primers or primers using those codons preferred most by yeast? >Does anyone know of a reference that has the preferred codon usage for >yeast? > > Margaret M Lanterman Emory University We have used degenerate primers based on ribosomal protein genes using conserved protein domains that were then reverse translated. We have found that primers with as many as 128 degeneracies work fine in PCR if you lower the anneal about 3-4 C and add 3x more of each primer. The problem is, from your statement, that you may not know any conserved domains. How will you know the yeast gene has the same amino acids. As far as codon bias, you can generate your own codon bias table for your favorite organism by downloading 4-6 mRNA sequences for the organism from GenBank and then get a copy of SEQAID II (available from several BioGophers) for use on a DOS machine. There is a module that builds codon bias tables for use in identification of exons. 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 Fri Oct 15 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: degenerate PCR Message-ID: <7C7D1DB1BD8@uamercury.uark.edu> Date: 16 Oct 93 15:18:24 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 33 >To: yeast@net.bio.net >From: djdlab@sun.com (Dean Danner Lab) >Subject: degenerate PCR >Date: 15 Oct 93 15:33:05 GMT >Reply-to: djdlab@sun.com > >To anyone who is a PCR god or goddess, I am about to embark on a project >involving PCR amplification of a not yet found yeast gene. I am going to >design the primers from human sequence. Is it better to produce >degenerate primers or primers using those codons preferred most by yeast? >Does anyone know of a reference that has the preferred codon usage for >yeast? > > Margaret M Lanterman Emory University We have used degenerate primers based on ribosomal protein genes using conserved protein domains that were then reverse translated. We have found that primers with as many as 128 degeneracies work fine in PCR if you lower the anneal about 3-4 C and add 3x more of each primer. The problem is, from your statement, that you may not know any conserved domains. How will you know the yeast gene has the same amino acids. As far as codon bias, you can generate your own codon bias table for your favorite organism by downloading 4-6 mRNA sequences for the organism from GenBank and then get a copy of SEQAID II (available from several BioGophers) for use on a DOS machine. There is a module that builds codon bias tables for use in identification of exons. 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 Sat Oct 16 23:00:00 1993 Path: biosci!bcm!cs.utexas.edu!uunet!biosci!NMSU.EDU!talcanta From: talcanta@NMSU.EDU Newsgroups: bionet.molbio.rapd Subject: subscription Message-ID: <9310122053.AA14216@NMSU.Edu> Date: 12 Oct 93 20:53:02 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 9 Please include me in your list of subscribers. My e-mail address is: Thanks for your consideration. Tito P. Alcantara Plant Breeding and Genetics Department of Agronomy and Horticulture New Mexico State University Las Cruces, NM 88003-0003 From owner-rapd@net.bio.net Sat Oct 16 23:00:00 1993 Path: biosci!bcm!cs.utexas.edu!uunet!biosci!NMSUVM1.NMSU.EDU!G5930716 From: G5930716@NMSUVM1.NMSU.EDU ("Tito P. Alcantara") Newsgroups: bionet.molbio.rapd Subject: subscription Message-ID: <9310121729.AA20252@net.bio.net> Date: 12 Oct 93 15:29:41 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 3 Please include me in your list of subscribers. My e-mail address is: Thanks a lot. From owner-rapd@net.bio.net Sat Oct 16 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!decwrl!decwrl!usenet.coe.montana.edu!news.uoregon.edu!netnews.nwnet.net!ns1.nodak.edu!plains!borovkov From: borovkov@plains.NoDak.edu (Alex Borovkov) Newsgroups: bionet.molbio.rapd Subject: Re: degenerate PCR Message-ID: Date: 17 Oct 93 00:26:32 GMT References: <7C7D1DB1BD8@uamercury.uark.edu> Sender: usenet@ns1.nodak.edu (Usenet login) Distribution: bionet Organization: North Dakota Higher Education Computing Network Lines: 43 Nntp-Posting-Host: plains.nodak.edu X-Newsreader: TIN [version 1.2 PL1] Doug Rhoads (DRHOADS@MERCURY.UARK.EDU) wrote: : >To: yeast@net.bio.net : >From: djdlab@sun.com (Dean Danner Lab) : >Subject: degenerate PCR : >Date: 15 Oct 93 15:33:05 GMT : >Reply-to: djdlab@sun.com : > : >To anyone who is a PCR god or goddess, I am about to embark on a project : >involving PCR amplification of a not yet found yeast gene. I am going to : >design the primers from human sequence. Is it better to produce : >degenerate primers or primers using those codons preferred most by yeast? : >Does anyone know of a reference that has the preferred codon usage for : >yeast? : > : > Margaret M Lanterman : Emory University : We have used degenerate primers based on ribosomal protein genes using : conserved protein domains that were then reverse translated. We have : found that primers with as many as 128 degeneracies work fine in PCR if : you lower the anneal about 3-4 C and add 3x more of each primer. The : problem is, from your statement, that you may not know any conserved : domains. How will you know the yeast gene has the same amino acids. : As far as codon bias, you can generate your own codon bias table for your : favorite organism by downloading 4-6 mRNA sequences for the organism from : GenBank and then get a copy of SEQAID II (available from several : BioGophers) for use on a DOS machine. There is a module that builds : codon bias tables for use in identification of exons. : 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 Degenerated primers didn't work for me but I got the product when used an inosine in position of degeneration instead. Try it. -- Dr. Alex Borovkov, | Please excuse me for my English. Plant Pathology Dept., | My Russian is getting worse too North Dakota State University | and soon I will know nothing Fargo, ND 58105 | but few dirty words tel: 701 237 8340 ; FAX: 701 237 7851 ; Internet: borovkov@plains.NoDak.edu. From owner-rapd@net.bio.net Sat Oct 16 23:00:00 1993 Path: biosci!bcm!cs.utexas.edu!uunet!biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: microwave pcr on Fish Muscle Message-ID: <7803FF85253@uamercury.uark.edu> Date: 13 Oct 93 15:44:36 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 30 >To: rapd@net.bio.net >From: aqprc1@uk.ac.stir (Rita Castilho) >Subject: microwave pcr >Date: 13 Oct 93 12:09:24 GMT > > >> Microwave Isolation of Total DNA for PCR/RAPD Analysis >> This procedure was developed by D. Goodwin and S. Lee >> (slee@goldng8.univnorthco.edu) at U of North Colorado. >> Reference: Biotechniques (1993) 15:438-444. Purportedly works with >> everything: bacteria, plants, fungi and protists. > >Does anyone have any idea if it will also works for fish muscle? > >Thanks! > >Rita Castilho I think the procedure would work fine if you homogenized before microwaving. However, if you homogenize then you don't need to microwave. We routinely isolate from fish parts and mammal muscle by homogenizing with a tissuemizer (modified Dremel tool) in TE. Then add SDS, EDTA and pronase for a while. Extract with Phenol:CHCl3. EtOH ppt and you got DNA. It isn't fast but it works. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Sun Oct 17 23:00:00 1993 Path: biosci!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!pipex!sunic!corax.udac.uu.se!zeta.bmc.uu.se!daresbury!daresbury!news From: DIBAGEF1@IT.CNR.FI.IDG.VM (Priscilla Bettini) Newsgroups: bionet.molbio.rapd Subject: Request Message-ID: <1993Oct14.132443.5326@gserv1.dl.ac.uk> Date: 14 Oct 93 14:20:20 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Organization: Dipartimento di Biologia Animale e Genetica Lines: 17 Precedence: first-class from VM.IDG.FI.CNR.IT by UKACRL.BITNET (Mailer R2.07) with BSMTP id 3525; Thu, 14 Oct 93 14: 21:47 BST Original-To: rapd@uk.ac.daresbury I would like to be included in the RAPD network. Priscilla Bettini Department of animal Biology and Genetics, Florence (Italy). E-mail address: DIBAGEF1@VM.IDG.FI.CNR.IT ------------------------------------------- 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@idg.fi.cnr.it ------------------------------------------- From owner-rapd@net.bio.net Mon Oct 18 23:00:00 1993 Path: biosci!bcm!avdms8.msfc.nasa.gov!europa.eng.gtefsd.com!uunet!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd Subject: Re: Re Microwave minniprep extract of DNA Message-ID: <2a0m54$olg@oj.rsmas.miami.edu> Date: 19 Oct 93 12:21:56 GMT References: <9310151534.AA21443@phibred.phibred.com> Distribution: bionet Organization: R.S.M.A.S./University of Miami Lines: 10 NNTP-Posting-Host: oj.rsmas.miami.edu In <9310151534.AA21443@phibred.phibred.com> IAN TERSE MURRAY wrote: >I rapidly tried the method and tried several different treatments. I know that >a second microwave treatment shears the DNA and a treatment of 15 sec, 15 and >then 10 secs using 5% SDS yields some high molecular weight DNA and some sheared >DNA as well. Oh yeah, that 10 min lysis step at 80 C is essential. And is probably all that is required. I skip the microwave step and just boil my samples in TRIS buffer at 85 -95C for 15 minutes then aliquot to PCR buffer as 50% of total buffer in tubes. I get the same results as boiling without the shearing. From owner-rapd@net.bio.net Mon Oct 18 23:00:00 1993 Path: biosci!agate!ames!decwrl!decwrl!usenet.coe.montana.edu!news.uoregon.edu!netnews.nwnet.net!ns1.nodak.edu!plains!borovkov From: borovkov@plains.NoDak.edu (Alex Borovkov) Newsgroups: bionet.molbio.rapd Subject: Re: degenerate PCR Message-ID: Date: 17 Oct 93 00:26:32 GMT References: <7C7D1DB1BD8@uamercury.uark.edu> Sender: usenet@ns1.nodak.edu (Usenet login) Distribution: bionet Organization: North Dakota Higher Education Computing Network Lines: 43 Nntp-Posting-Host: plains.nodak.edu X-Newsreader: TIN [version 1.2 PL1] Doug Rhoads (DRHOADS@MERCURY.UARK.EDU) wrote: : >To: yeast@net.bio.net : >From: djdlab@sun.com (Dean Danner Lab) : >Subject: degenerate PCR : >Date: 15 Oct 93 15:33:05 GMT : >Reply-to: djdlab@sun.com : > : >To anyone who is a PCR god or goddess, I am about to embark on a project : >involving PCR amplification of a not yet found yeast gene. I am going to : >design the primers from human sequence. Is it better to produce : >degenerate primers or primers using those codons preferred most by yeast? : >Does anyone know of a reference that has the preferred codon usage for : >yeast? : > : > Margaret M Lanterman : Emory University : We have used degenerate primers based on ribosomal protein genes using : conserved protein domains that were then reverse translated. We have : found that primers with as many as 128 degeneracies work fine in PCR if : you lower the anneal about 3-4 C and add 3x more of each primer. The : problem is, from your statement, that you may not know any conserved : domains. How will you know the yeast gene has the same amino acids. : As far as codon bias, you can generate your own codon bias table for your : favorite organism by downloading 4-6 mRNA sequences for the organism from : GenBank and then get a copy of SEQAID II (available from several : BioGophers) for use on a DOS machine. There is a module that builds : codon bias tables for use in identification of exons. : 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 Degenerated primers didn't work for me but I got the product when used an inosine in position of degeneration instead. Try it. -- Dr. Alex Borovkov, | Please excuse me for my English. Plant Pathology Dept., | My Russian is getting worse too North Dakota State University | and soon I will know nothing Fargo, ND 58105 | but few dirty words tel: 701 237 8340 ; FAX: 701 237 7851 ; Internet: borovkov@plains.NoDak.edu. From owner-rapd@net.bio.net Mon Oct 18 23:00:00 1993 Path: biosci!bcm!cs.utexas.edu!uunet!spool.mu.edu!umn.edu!pathobiology-206.med.umn.edu!user From: fredboyd@bmec.micro.umn.edu (Fred Boyd, Ph.D.) Newsgroups: bionet.molbio.rapd Subject: Re: degenerate PCR Message-ID: Date: 19 Oct 93 16:52:27 GMT References: <7C7D1DB1BD8@uamercury.uark.edu> Sender: news@news.cis.umn.edu (Usenet News Administration) Followup-To: bionet.molbio.rapd Distribution: bionet Organization: University of Minnesota Lines: 48 Nntp-Posting-Host: pathobiology-206.med.umn.edu In article <7C7D1DB1BD8@uamercury.uark.edu>, DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") wrote: > >To: yeast@net.bio.net > >From: djdlab@sun.com (Dean Danner Lab) > >Subject: degenerate PCR > >Date: 15 Oct 93 15:33:05 GMT > >Reply-to: djdlab@sun.com > > > >To anyone who is a PCR god or goddess, I am about to embark on a project > >involving PCR amplification of a not yet found yeast gene. I am going to > >design the primers from human sequence. Is it better to produce > >degenerate primers or primers using those codons preferred most by yeast? > >Does anyone know of a reference that has the preferred codon usage for > >yeast? > > > > Margaret M Lanterman > Emory University > > We have used degenerate primers based on ribosomal protein genes using > conserved protein domains that were then reverse translated. We have > found that primers with as many as 128 degeneracies work fine in PCR if > you lower the anneal about 3-4 C and add 3x more of each primer. The > problem is, from your statement, that you may not know any conserved > domains. How will you know the yeast gene has the same amino acids. > As far as codon bias, you can generate your own codon bias table for your > favorite organism by downloading 4-6 mRNA sequences for the organism from > GenBank and then get a copy of SEQAID II (available from several > BioGophers) for use on a DOS machine. There is a module that builds > codon bias tables for use in identification of exons. > > > 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 I have "librarie" which have been selected for growth inhibitory activities which I would like to screen by PCR for "interesting" classes of genes like kinases, cyclins, transcription factors, etc. Does anyone have some primers we could use for this fishing expedition? Thanks. Please reply by E-mail. -- Fred Boyd, Ph.D. Voice- 612-624-8150 Lab. Med. Path. Fax- 612-616-2444 Cell Biol. Neuro. Lab- 612-624-8154 Box 609, UMHC University of Minnesota E-Mail fredboyd@bmec.micro.umn.edu Minneapolis, MN 55455 From owner-rapd@net.bio.net Fri Oct 22 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: RAPD primers Message-ID: <8725289607B@uamercury.uark.edu> Date: 23 Oct 93 17:47:16 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 65 Moira has asked me the following question and I think I will post the answer to the group as I think everyone should want to hear my opinions, ha-ha. Actually, maybe we can try this thread for discussion for a while. I have considered putting a small note together but maybe people don't think it is of general enough interest. Therefore, take a shot at the following, please!! >Dear Dr Rhoads: > >As you have used the UBC primers on a variety of species, John Hobbs >suggested I contact you to see whether certain sets have been better >than others for particular taxa. Initially I would like to use RAPD on >a primitive grasshopper. Any pointers as to which set might be best? >Many thanks, > >Moira van Staaden The simplest answer is I don't have any pointers to the `best' set. What I can tell you is that band yield is genus/species specific. That the major factor is in the 3' most 2 bases and CG content should be 5 or more out of 10. What I could guarantee is that if you picked a set of 100 there would be at least one primer that would give you more than 10 bands. The average yield of scorable bands for an insect genome should hit around 2 bands/primer. Somewhere around 40-60% of the primers will give at least one band. Here is our best data for tomato and chicken (larger genomes) distilled down into their briefest essence. Tomato Chicken Average # of bands per primer 3.37+-3.46 2.57+-2.42 Percent of primers giving >0 bands 64.2 67.2 Best 3' dinucleotide AA CT/GT Average # of bands for best 3' dinuc 5.7+-3.9 3.4+-2.4 Percent >0 bands for best 3' dinuc 90.0 82.8 Avg. Band production based on #GCs in a 10 mer 3 4 5 6 7 8 9 tomato 0 0 1.5 3.2 4.4 5.4 3.4 chicken 0 0 1.5 2.5 3.1 3.8 5.4 Frequency of Band production based on #GCs in a 10mer 3 4 5 6 7 8 9 tomato 0 0 40 64 79 86 80 chicken 0 0 47 68 82 90 100 That's pretty much it in a nutshell. I have tried to look at other sequence affects but there are no `magic bullet' primers. Of course the 2base primers do a better job in these complex genomes. For tomato using a set of 24 2base primers the average number of bands per primer is 9.8+-5.4 and 96% of the primers give at least one band. I just wish I had a few 2base primers with AA at the 3' end. Those that remember when I got on the 2 base kick I specifically excluded primers that end in A or T. Don't I look stupid now. In fact, my biggest point is that all along I (we??) have believed that a C or G at the 3' end makes for the best primer. Is this REALLY true??? The data above certainly say NO. 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 Fri Oct 22 23:00:00 1993 Path: biosci!esvax.dnet.dupont.com!rafalski From: rafalski@esvax.dnet.dupont.com Newsgroups: bionet.molbio.rapd Subject: YoPro - DNA conc. Message-ID: <9310222327.AA25988@esds01.es.dupont.com> Date: 22 Oct 93 23:27:57 GMT Sender: shibumi@net.bio.net Distribution: bionet Lines: 40 YoPro assay for the determination of DNA concentration: Developed for use with RAPDs by John G.K. Williams, with contributions from JoAnne Lynch and Karla Butler. Mix 10 ul DNA (0.2-30 ng DNA) with 190 ul of YoPro / RNase mix. We use a microtiter dish (96-well) Incubate at R.T. for 30 min. Measure fluorescence using a plate reading fluorimeter (we use a Millipore instrument). Make a calibration curve by using genomic DNA of known concentration - a series of dilutions at 0.15-0.35 ug/ml. We re-do the standard curve each time we do measurements - by putting the standard DNA in one of rows/or columns of the 96 well dish. YoPro-RNase mix: 10 mM Tris-HCl pH7.5 1 mM EDTA 50 mM NaCl 1 uM YoPro 10 ug/ml RNAse A (DNAse free) YoPro: YO-PRO-1 iodide (exc max 491/em.max 509nm , 1 mM in DMSO cat.#Y3603, 1 ml $ 95, 5x1ml $380 Molecular Probes, Inc POB 22010 Eugene OR 97402-0414 503- 465-8300 techn ass 503-465-8353 cust serv 503-465-8338 fax 503-344-6504 Of course, the method can also be used w a standard fluorimeter. YoPro is a dimeric intercalating dye, binding to DNA much stronger then EtBr. Fluorsecence enhancement upon binding to DNA is the principle of the assay. RNA (esp. with secondary structure) may interfere. If you have questions, you'll have to wait - a am going away and will be back Nov. 3rd. Antoni Rafalski From owner-rapd@net.bio.net Fri Oct 22 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: YoPro - DNA conc. Message-ID: <86ECC672953@uamercury.uark.edu> Date: 23 Oct 93 14:15:56 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 22 On Fri, 22 Oct 93 19:27:57 -0400 Antoni Rafalski said: >YoPro assay for the determination of DNA concentration: >Developed for use with RAPDs by John G.K. Williams, with contributions from >JoAnne Lynch and Karla Butler. [rest deleted] We have been using our Hoefer fluorimeter and Hoechst dye to quantitate RAPD reactions directly. The primers don't interfere significantly as the Hoechst only fluoresces on binding double stranded nucleic acids. Response is nearly linear over a nearly 1000 fold range. One standard adjustment is needed then you can read between 5 and 2000 ng/ul. It only takes 1 or 2 ul for a reading. What is the sensitivity of the YoPro method? 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 Sat Oct 23 23:00:00 1993 Path: biosci!bloom-beacon.mit.edu!xlink.net!news.dfn.de!mailgzrz.TU-Berlin.DE!lise!steveb From: steveb@lise.physik.tu-berlin.de (Steve B.) Newsgroups: bionet.molbio.rapd Subject: VA mycorrhizae Keywords: VA mycorrhizae Message-ID: <2ae8cr$dpv@mailgzrz.TU-Berlin.DE> Date: 24 Oct 93 15:52:59 GMT Organization: TUBerlin/ZRZ Lines: 21 NNTP-Posting-Host: lise.physik.tu-berlin.de I am concerned with a project dealing with the identification of VA mycorrhizae by different PCR strategies. I am currently working with rDNA and RAPD primers but I would prefer primers that are specific to VA fungi genera DOES ANYBODY HAVE SUCH PRIMERS ? Thanks a lot in advance, Christine P.S.: Respond via e-mail to steveb@marie.physik.tu-berlin.de (reply will do) -- Stefan Berreth steveb@marie.physik.tu-berlin.de From owner-rapd@net.bio.net Sat Oct 23 23:00:00 1993 Path: biosci!VTVM1.CC.VT.EDU!TURNER From: TURNER@VTVM1.CC.VT.EDU (Bruce Turner) Newsgroups: bionet.molbio.rapd Subject: new subscriber for rapd network Message-ID: <9310242145.AA24505@net.bio.net> Date: 24 Oct 93 21:43:53 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 6 lease enroll the following individual in the rapd bionet: Rdawley @ Ursinus (bitnet address) Thanks From owner-rapd@net.bio.net Sat Oct 23 23:00:00 1993 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!agate!iat.holonet.net!t_duda From: t_duda@orac.holonet.net (Tom Duda) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD primers Message-ID: Date: 24 Oct 93 02:13:00 GMT References: <8725289607B@uamercury.uark.edu> Sender: usenet@iat.holonet.net (USENET News System) Distribution: bionet Organization: HoloNet National Internet Access System: 510-704-1058/modem Lines: 26 Nntp-Posting-Host: orac.holonet.net Clark and Lanigan (1993, Mol Biol Evol 10: 1096-1111) present a variety of equations (theoretical of course) that predict the number of bands that should be observed. Summary: V = expected number of fragments between length s1 and s2 a = 0.25^n n = number of bases in the primer V = (1 - 2a)^(s1-1) - (1 - 2a)^(s1) N = size of the genome Exp # of bands = N/(2/a) Exp # of bands between length s1 and s2 = V * Exp # of bands In their report, they also use these equations to formulate an estimate of the expected # of bands between length 500 and 3500 bp using a 10-nt primer for a species whose genome size is about 3 * 10^9. This value is equal to 8.16. Actual RAPD analysis with this species has yielded an average of 11 bands/individual based on 22 primers. They predict that the non-randomness of the genome may be responsible for the difference. Another factor could be mis-priming of recognition sites. From owner-rapd@net.bio.net Sun Oct 24 22:00:00 1993 Path: biosci!NET.BIO.NET!kristoff From: kristoff@NET.BIO.NET (David Kristofferson) Newsgroups: bionet.molbio.rapd Subject: IMPORTANT BIOSCI INFORMATION Message-ID: <9310250900.AA09287@net.bio.net> Date: 25 Oct 93 09:00:07 GMT Sender: news@net.bio.net Distribution: bionet Lines: 243 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. 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Thanks again for your cooperation! --------------- please cut here and return portion below --------------- New information or Update to old record (enter N or U): date (DD-MM-YY): first name: middle initial: family name: job title: e-mail address: e-mail network: phone number: FAX number: institution: address1: address2: address3: city: state/province: country: postal code: research interest: research interest: comment: comment: comment: comment: comment: From owner-rapd@net.bio.net Sun Oct 24 22:00:00 1993 Path: biosci!daresbury!zeta.bmc.uu.se!corax.udac.uu.se!sunic!pipex!uunet!decwrl!decwrl!usenet.coe.montana.edu!news.uoregon.edu!netnews.nwnet.net!news.u.washington.edu!stein2.u.washington.edu!toby From: toby@stein2.u.washington.edu (Toby Bradshaw) Newsgroups: bionet.molbio.rapd Subject: Re: Linkage analysis with pseudotest cross population Message-ID: <2ago39$fip@news.u.washington.edu> Date: 25 Oct 93 14:33:13 GMT References: <199310250043.AA23874@postoffice.mail.cornell.edu> Distribution: bionet Organization: University of Washington, Seattle Lines: 57 NNTP-Posting-Host: stein.u.washington.edu In article <199310250043.AA23874@postoffice.mail.cornell.edu>, Muhammad A. Lodhi wrote: > Does anyone have better idea about linkage analysis of test cross >populations where phase is missing (pseudotest cross). The situation >becomes something in between test cross and F2. Currently I have been >using MAPMAKER 3.0 but it is not easy and reliable to use this software for >such situations. I tried JOINMAP and LINKAGE-I but both of these are based >on only pairwise analysis, as far as I understood. With mapmaker I am able >to identify the linkage groups but it can't handle dominant markers >(segregating 1:1) and segregating 3:1 simultaneously. Moreover, it gives >high distances between markers. The 1:1 pseudo-testcross markers must be coded as "f2 backcross", then MAPMAKER will interpret the data correctly. You'll end up with two maps of cis-linked markers if you're using diploid tissue. >I am working on grapes and I have RAPD, >isozyme and RFLP markers from one population. I am wondering if there is >any reliable software for such populations. As you may have already discovered, MAPMAKER will use all the codominant data and the 3:1 RAPD data IF you can code the data as a cross between inbred lines. Otherwise... Packages to try (no personal experience with these, as our tree maps are made from interspecific F2s, where MAPMAKER works fine for most experiments) are GMENDEL from Steve Knapp (Oregon State U.) or Ben Liu (NC State) and, perhaps, a package being worked on by Tom Mitchell-Olds at U. Montana (tmo@selway.umt.edu may be the right email address). >I am aware of work done by a >group in NC on forest trees Ron Sederoff's group, although the use RAPDs almost excelusively, and with their pine work have been able to avoid the dominance problem by using haploid megagametophyte tissue. Dario Grattapaglia has made good RAPD maps in Eucalyptus in Ron's lab, using only markers segregating in a pseudo-testcross configuration (last I heard). >and Norman Weeden on apple. I believe Norm has written some simple linkage analysis for Excel spreadsheets. >There is an >increasing demand by the geneticists working on woody plants to find >something reliable for data analysis. No doubt about that. Dave Neale has had to deal with this problem as much as anyone, at the USFS PSW station now in Albany, CA. Write me if you need his email address. -Toby Bradshaw toby@u.washington.edu From owner-rapd@net.bio.net Sun Oct 24 22:00:00 1993 Path: biosci!UXA.CSO.UIUC.EDU!dlohnes From: dlohnes@UXA.CSO.UIUC.EDU (lohnes david glenn) Newsgroups: bionet.molbio.rapd Subject: Re: IMPORTANT BIOSCI INFORMATION Message-ID: <199310251524.AA12970@uxa.cso.uiuc.edu> Date: 25 Oct 93 15:24:13 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 3 From owner-rapd@net.bio.net Sun Oct 24 22:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: VA mycorrhizae Message-ID: <8A083265954@uamercury.uark.edu> Date: 25 Oct 93 15:58:22 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 27 I tried to reply to steveb@lise.physik.tu-berlin.de but the connection timed out so I am posting this back to rapd in hopes that the origin sees my reply. >I am concerned with a project dealing with the identification of VA >mycorrhizae by different PCR strategies. I am currently working with rDNA >and RAPD primers but I would prefer primers that are specific to VA fungi >genera > > DOES ANYBODY HAVE SUCH PRIMERS ? > >Thanks a lot in advance, > Christine >P.S.: Respond via e-mail to steveb@marie.physik.tu-berlin.de (reply will >do) > Stefan Berreth > steveb@marie.physik.tu-berlin.de Why not create your own?? Just buy a set of Operon primers. Find one or two that work with your genus. Identify a band or bands that discriminate the genotypes. Cut it out, clone it, sequence it and develop longer, specific primers. For that matter just clone any 2 kbp fragment, sequence it and develop the ends as PCR primers. These are known as SCARs. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Sun Oct 24 22:00:00 1993 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Linkage analysis with pseudotest cross population Message-ID: <199310250043.AA23874@postoffice.mail.cornell.edu> Date: 24 Oct 93 21:48:24 GMT Sender: news@net.bio.net Distribution: bionet Lines: 27 Dear Friends Does anyone have better idea about linkage analysis of test cross populations where phase is missing (pseudotest cross). The situation becomes something in between test cross and F2. Currently I have been using MAPMAKER 3.0 but it is not easy and reliable to use this software for such situations. I tried JOINMAP and LINKAGE-I but both of these are based on only pairwise analysis, as far as I understood. With mapmaker I am able to identify the linkage groups but it can't handle dominant markers (segregating 1:1) and segregating 3:1 simultaneously. Moreover, it gives high distances between markers. I am working on grapes and I have RAPD, isozyme and RFLP markers from one population. I am wondering if there is any reliable software for such populations. I am aware of work done by a group in NC on forest trees and Norman Weeden on apple. There is an increasing demand by the geneticists working on woody plants to find something reliable for data analysis. I will appreciate your help and comments. Thanks in advance ====================================== Muhammad A. Lodhi Cornell Univ. Geneva Campus NYSAES, 123 Hedrick Hall Geneva, NY 14456 Ph :315-787-2239 Fax :315-787-2216 e-mail:mal5@cornell.edu From owner-rapd@net.bio.net Mon Oct 25 22:00:00 1993 Path: biosci!daresbury!zeta.bmc.uu.se!corax.udac.uu.se!sunic!mcsun!uunet!decwrl!decwrl!usenet.coe.montana.edu!netnews.nwnet.net!ns1.nodak.edu!plains!mcclean From: mcclean@plains.NoDak.edu (Phillip McClean) Newsgroups: bionet.molbio.rapd Subject: AFLP Message-ID: Date: 26 Oct 93 14:59:08 GMT Sender: usenet@ns1.nodak.edu (Usenet login) Organization: North Dakota Higher Education Computing Network Lines: 7 Nntp-Posting-Host: plains.nodak.edu X-Newsreader: TIN [version 1.2 PL1] I am posting this for Alex Krupkin who does not have access to the Net. He is interested in the AFLP procedure. Could you send any information regarding the company, pubications and procedures to me and I will forward them to him. Thanks in advance. Phil McClean mcclean@plains.nodak.edu From owner-rapd@net.bio.net Mon Oct 25 22:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!psinntp!barilvm!kineret.huji.ac.il!wisipc.weizmann.ac.il!weizmann.weizmann.ac.il!BCRUBIN From: BCRUBIN@weizmann.weizmann.ac.il (Eitan Rubin) Newsgroups: bionet.molbio.rapd Subject: PCR degenerated primers: can one predict specifity? Message-ID: <16C72D5B4.BCRUBIN@weizmann.weizmann.ac.il> Date: 25 Oct 93 15:11:48 GMT Sender: news@wisipc.weizmann.ac.il (News User) Organization: Weizmann Institute of Science Lines: 7 X-Newsreader: NNR/VM S_1.3.2 Hi. I'm working on a program that might help predict the specifity of a particu lar pair of primers. I am looking for examples of degenrated primers that worke d (and gave 1-2) bands but mostly for those that gave many bands. If you have an example or know of one, please send me more info. I will also be more then happy to run my program on anyone's primers, give hi m my prediction, and when he runs the PCR, compare them. I hope my idea would be explained in more details in some paper soon.... From owner-rapd@net.bio.net Tue Oct 26 22:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!uunet!yeshua.marcam.com!news.kei.com!sol.ctr.columbia.edu!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!network.ucsd.edu!usenet From: andy@pooh.ucsd.edu Newsgroups: bionet.population-bio,bionet.molbio.rapd,bionet.molbio.evolution,bionet.jobs Subject: PostDoc Available Message-ID: <2ammadINNuu@network.ucsd.edu> Date: 27 Oct 93 22:40:24 GMT Organization: The Avant-Garde of the Now, Ltd. Lines: 55 Xref: biosci bionet.population-bio:505 bionet.molbio.rapd:304 bionet.molbio.evolution:1219 bionet.jobs:2544 NNTP-Posting-Host: adizon.ucsd.edu MIME-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII POSTDOCTORAL RESEARCH FELLOW POSITION AVAILABLE Southwest Fisheries Science Center Marine Mammal Division A one-year National Research Council Associateship (renewable to three years) is available in the Marine Mammal Molecular Genetics Laboratory of the Southwest Fishery Science Center located in La Jolla, California. Ph.D. with experience and/or interest in population genetics or modelling is sought to formulate quantitative means for using genetic data to define biologically meaningful stock units on which to base conservation and management efforts for marine mammals. Stocks, not species, are the units on which conservation and management efforts, by law, must be focussed, yet how to do this remains one of the most important questions in conservation biology today. In spite of rapid progress in the molecular arena, development of quantitative means for using the information gained in the laboratory to define biologically meaningful stock units has not been fully realized. Methodologies using molecular data to estimate population-genetic parameters, molecular phylogenies, gene flow and dispersal distance, etc., are available, but there has been growing realization that this area is much more complex and pitfall-ridden than had been thought, especially when the methods are applied to incompletely isolated populations. Nevertheless, the continuing rapid advances in molecular methods, the reality of laboratory automation, and the ability to measure genetic diversity directly from pooled samples will provide data of heretofore unavailable breadth and scope. A concentrated research effort focusing directly on how to use these data to define stock units in potentially politically controversial situations is needed. Envisioned, for example, is the development of an individual-based model of evolving and interchanging local populations, which could be stochastically sampled in order to test the utility of various analyses of population genetic structure for defining management stocks. Such a model would also serve as a means of designing practical sampling programs for such purposes and, in the course of which, measures of the statistical power of the quantitative indices should be developed. NRC Associates receive a stipend from the National Research Council while carrying out his or her proposed research. The current annual stipend for a Regular Associate (< 5yr from PhD) is $30,000 per year. Suitable relocation reimbursement will be determined for an awardee, and funds are available for professional travel during tenure. Applicants should direct initial inquires to Dr. Andrew Dizon (andy@pooh.ucsd.edu) or Dr. William Perrin, PO Box 271, SWFSC, La Jolla, CA, 92038. From owner-rapd@net.bio.net Wed Oct 27 22:00:00 1993 Path: biosci!MUSKWA.UCS.UALBERTA.CA!dchong From: dchong@MUSKWA.UCS.UALBERTA.CA (Chong Daniel) Newsgroups: bionet.molbio.rapd Subject: (none) Message-ID: <9310280328.AA20022@muskwa.ucs.ualberta.ca> Date: 28 Oct 93 03:28:36 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 12 Similarity index for RAPDS ----------------------------------- Several reports have used a straitford statistic, F = 2Nxy/(Nx + Ny), as the measure of similarity between two individuals, and between two populations for RAPD data. F does not count situations that both have no bands. If we compare samples between two populations, should both no band situations be taken into consideration for estimating similarity? Daniel. From owner-rapd@net.bio.net Wed Oct 27 22:00:00 1993 Path: biosci!UNR.EDU!hoelzer From: hoelzer@UNR.EDU (Guy A Hoelzer) Newsgroups: bionet.molbio.rapd Subject: Re: your mail Message-ID: Date: 28 Oct 93 16:26:46 GMT References: <9310280328.AA20022@muskwa.ucs.ualberta.ca> Sender: daemon@net.bio.net Reply-To: Guy A Hoelzer Distribution: bionet Lines: 32 On Wed, 27 Oct 1993, Chong Daniel wrote: > > Similarity index for RAPDS > ----------------------------------- > > Several reports have used a straitford statistic, F = 2Nxy/(Nx + Ny), > as the measure of similarity between two individuals, and between two > populations for RAPD data. F does not count situations that both have > no bands. If we compare samples between two populations, should both no > band situations be taken into consideration for estimating similarity? > > Daniel. Unlike Doug Rhodes, I agree with this point. If you are interested in the distribution of genetic variation across populations you may underestimate the relative similarity of individuals within a population by ignoring shared absences of a band present in individuals of another population. The only problem I see with this is that many different kinds of mutations could cause a band to disappear; thus, a shared absence may not reflect genetic similarity. Of course, this is also true of another kind of presence/absence genetic data, namely restriction sites. Why not study RAPD polymorphism in the same way as restriction site data? Any thoughts? Guy From owner-rapd@net.bio.net Wed Oct 27 22:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: Message-ID: <8E68B8807BB@uamercury.uark.edu> Date: 28 Oct 93 13:59:58 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 22 >Similarity index for RAPDS >----------------------------------- > > Several reports have used a straitford statistic, F = 2Nxy/(Nx + Ny), >as the measure of similarity between two individuals, and between two >populations for RAPD data. F does not count situations that both have >no bands. If we compare samples between two populations, should both no >band situations be taken into consideration for estimating similarity? > >Daniel. If you want to count no band situations then there would be a large number of `no bands', far greater than the shared bands. This could be taken to mean how many genomic sites failed to give an RAPD band. In the case of comparisons of 14 individuals vs 25 individuals you would come up with a different score (i.e., does the intra relationship change by the inclusion of an unrelated individual). Pair wise comparisons seem to be the only rational way of approaching this. 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 Thu Oct 28 22:00:00 1993 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Linkage analysis with pseudotest cross population Message-ID: <199310291903.AA08601@postoffice.mail.cornell.edu> Date: 29 Oct 93 16:08:19 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 28 Dear Friends Does anyone have better idea about linkage analysis of test cross populations where phase is missing (pseudotest cross). The situation becomes something in between test cross and F2. Currently I have been using MAPMAKER 3.0 but it is not easy and reliable to use this software for such situations. I tried JOINMAP and LINKAGE-I but both of these are based on only pairwise analysis, as far as I understood. With mapmaker I am able to identify the linkage groups but it can't handle dominant markers (segregating 1:1) and segregating 3:1 simultaneously. Moreover, it gives high distances between markers. I am working on grapes and I have RAPD, isozyme and RFLP markers from one population. I am wondering if there is any reliable software for such populations. I am aware of work done by a group in NC on forest trees and Norman Weeden on apple. There is an increasing demand by the geneticists working on woody plants to find something reliable for data analysis. I will appreciate your help and comments. Thanks in advance ====================================== Muhammad A. Lodhi Cornell Univ. Geneva Campus NYSAES, 123 Hedrick Hall Geneva, NY 14456 Ph :315-787-2239 Fax :315-787-2216 e-mail:mal5@cornell.edu From owner-rapd@net.bio.net Thu Oct 28 22:00:00 1993 Path: biosci!news.cs.umb.edu!hsdndev!howland.reston.ans.net!pipex!sunic!corax.udac.uu.se!zeta.bmc.uu.se!daresbury!daresbury!news From: ZANDELPW@nl.rug.biol (Louis van de Zande) Newsgroups: bionet.molbio.rapd Subject: Re: Similarity RAPDs Message-ID: <1993Oct29.091926.12780@gserv1.dl.ac.uk> Date: 29 Oct 93 08:59:27 GMT Sender: list-admin@daresbury.ac.uk Reply-To: ZANDELPW@nl.RUG.BIOL Distribution: bionet Organization: Department of Biology, RUGroningen Lines: 47 X-Pmrqc: 1 X-Envelope-To: rapd@dl.AC.UK Content-Identifier: Re: Similarit... Precedence: first-class Original-To: rapd@uk.ac.daresbury Content-Transfer-Encoding: 7BIT X-Mailer: Pegasus Mail v2.3 (R4). > > > Similarity index for RAPDS > ----------------------------------- > > Several reports have used a straitford statistic, F = 2Nxy/(Nx + Ny), > as the measure of similarity between two individuals, and between two > populations for RAPD data. F does not count situations that both have > no bands. If we compare samples between two populations, should both no > band situations be taken into consideration for estimating similarity? > > Daniel. > > > There is more here than one might think at first sight. Compare three individuals, both displaying two RAPD bands. Something like: 1 2 3 --- --- --- --- --- --- Evidently(?), comparing 1 and 2 you will not take into account the unique fragment amplified from 3. However, comparing both 1 and 2 with 3 you suddenly will, thus changing similarities between 1 and 2. And there is more. Comparing haploid genomes will not give the same results as comparing diploids (or worse). But then again, is it fair to count the absence of a fragment as a similarity if the cause for the absence can be different in two organisms? To my opinion, it would be safe to use RAPDs as genetic MARKERS and as "identifyers" but not for quantitative measures. Just have a look at the assumptions you have to make before doing some statistics with RAPDs (Clark and Lanigan in MBE 10; 1096-1111; 1993). Good luck L.van de Zande Dept. of Genetics University of Groningen P.O. Box 14 9750 AA Haren The Netherlands Tel. +31 50 632126 Fax. +31 50 632348 From owner-rapd@net.bio.net Fri Oct 29 22:00:00 1993 Path: biosci!agate!howland.reston.ans.net!europa.eng.gtefsd.com!uunet!news.claremont.edu!paris.ics.uci.edu!news.service.uci.edu!miledi1.bio.uci.edu!oocyte From: oocyte@darwin.bio.uci.edu (Miledi Lab) Newsgroups: bionet.molbio.rapd Subject: looking for chromosome 6 cosmids Keywords: chromosome 6, cosmids Message-ID: Date: 21 Oct 93 21:57:41 GMT Distribution: methods Organization: UCI Lines: 16 Nntp-Posting-Host: miledi1.bio.uci.edu X-Newsreader: Trumpet for Windows [Version 1.0 Rev Final Beta #10] Hi netters! Does anybody know where can I get chromosome specific cosmids? or on the other hand who offers screening of cosmid libraries? I will appreciate any information! Ata Ataulfo Martinez-Torres LAboratory of Molecular an Cell. Neurobiology Department of Psychobiology University of California, Irvine California 92717 email: mataulfo@darwin.bio.uci.edu