From owner-rapd@net.bio.net Wed Dec 01 22:00:00 1993 Path: biosci!news.cs.umb.edu!hsdndev!purdue!lerc.nasa.gov!magnus.acs.ohio-state.edu!dsammata From: dsammata@magnus.acs.ohio-state.edu (Diana Sammataro) Newsgroups: bionet.molbio.rapd Subject: Rapds neg. control bands etc. Message-ID: <2dlk7q$lcd@charm.magnus.acs.ohio-state.edu> Date: 2 Dec 93 20:46:50 GMT Distribution: bionet Organization: The Ohio State University Lines: 72 NNTP-Posting-Host: bottom.magnus.acs.ohio-state.edu P. Parker Protocol, November 1993 OSU A. Making Up tubes: For 24 tubes for a comb of 26 (+ 2 ladders), make enough for 25 Reactant ul per sample needed for 25 tubes H20 14.5 337.5ul MgCl2 3.0 75.0 dNTP's 3.5 87.5 10x Buffer 2.5 62.5 Primer 1.0 25.0 Taq 0.2 to 0.5 5.0 to 12.5 DNA 1.0 25.0 TOTAL 25.0 625/25 = 25 tubes B. Procedure 1. Place all open Eppy tubes, pipette tips, pipettors and autoclaved, ddH20 under a still UV light hood for 24 hrs. Cover with foil or saran wrap that has been under the UV light too. 2. PUT ON GLOVES. Take reactants out of freezer and cool on ice. Take out DNA samples and keep on ice. Label small PCR tubes. Keep covered. 3. Clean table and pipettors with bleach. 4. Mix first 4 ingredients into clean, UV'd Eppy tube; Note: varying amounts of MgCl2 can change banding patterns. Change or rinse gloves down with bleach, frequently. 5. Add together Taq first vortexing it gently then spinning it down. Once added, vortex and spin down reaction mix to make sure the heavier Taq is well distributed. 6. Aliquot 24 5l Rxn mix into each labeled PCR tube. 7. Add 1 5l DNA template to each tube except the negative control tube. 8. Add 1 drop oil to top of each tube, spin down in centrifuge equipped with larger tube with tops cut off to keep mini tubes from falling thru. 9. Place in PCR machine that has been warming up for 15 m 10. Run machine. I have had good luck with 45 cycles at 94!C 1m, 37! 1 m, 72! 1min. This runs for about 5 or 6 hours. Annealing temps can also change banding patterns, so try 94!C 1 min, 35! 1 min ramp to 72!, 72! for 1 min. 11. When run is complete, file #99 to cool (4!C) and store tubes in frig. 12. To run out gel, add 4 ul Blue Juice to each tube, spin thru oil 3 sec and heat for 5 min at 65!C with ladder. I use the 123 and 100 kb ladders, 2 ul LDR, 2ul dH20, 1ul BJ. 13. Make a 1.2% agarose gel, (2.4g agarose in 200 ml 1x TBE) store in frig ON covered if not ready to run. 14. Run large get 4V/cm gel length, dye in ethidium bromide 30-45m, rinse 10 m and visualize on UV. C. Determining Stock Sample concentrations 1. Once you've read your DNA and get the concentration, plug it into the following formula to get a 100 ng/ul solution: a. if [0.8]DNA then x ul x 0.8 ug/ul = 20ul x 100 ng/ul then 800 ng/ul = 20 ul x 100 ng/ul x = 2000 ul 800 = 2.5 ul DNA is needed less 20 ul dH20 = 17.5 water added to make 20 ul of sample at 100 ng. b. if 5 ng needed, use 100 instead of 2000. I am doing Mites in bees and spiroplasmas (bacteria) in mites and bees. Everything was going fine unitl they changed the buffer to buffer + MgCl and I am beginning to think I should try all new stuff again. I have been using Gibco Taq and things have been OK up until about a week ago. Any suggestions? I have finally gotten rid of my negative control bands using UV hood. Diana Sammataro "Go to the microbe, thou scientist, consider its ways Dept. Entomology and be wise" 1735 Neil Ave -ANON???????? Columbus, OH 43210-1220 Ohio State University email: dsammata@magnus.acs.ohio-state.edu Phone: 614 292 9089 From owner-rapd@net.bio.net Wed Dec 01 22:00:00 1993 Path: biosci!CS.Arizona.EDU!organpipe.uug.arizona.edu!uunet!europa.eng.gtefsd.com!library.ucla.edu!agate!usenet.ins.cwru.edu!news.ysu.edu!psuvm!cunyvm!yvax.byu.edu!farmerj From: farmerj@yvax.byu.edu Newsgroups: bionet.molbio.rapd Subject: Program for primer design? Message-ID: <1993Nov30.150459.3514@yvax.byu.edu> Date: 30 Nov 93 22:04:54 GMT Organization: Brigham Young University Lines: 26 I have just begun to recover from a series of minor computer disasters associated with switching all my files to a new computer. In the process, I lost an e-mail announcement that included a description of a (free?) program that designs PCR primers. From the description, the program seemed to have all the features I need. If you know where I may obtain this program, I would appreciate it very much if you would send me the information. Thank you. James L. Farmer Department of Zoology 571 WIDB Brigham Young University Provo, Utah 84602, USA (801) 378-2153 (OFFICE) (801) 378-7499 (FAX) FARMERJ@YVAX.BYU.EDU FARMERJ@BYUVAX.BITNET From owner-rapd@net.bio.net Wed Dec 01 22:00:00 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: HELP Ive Lost It!!! Message-ID: Date: 26 Nov 93 17:28:07 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 32 >To: rapd@net.bio.net >From: smw@waikato.ac.nz >Subject: HELP Ive Lost It!!! >Date: 25 Nov 93 05:28:25 GMT >hello > >a while ago someone posted a reference for a paper on RAPD analysis >unfortunately I have lost the ref so if anyone out there has it or could >point me in the right direction I would be most grateful > > >Shawn Walsh >Biology Dept >University of Waikato >New Zealand > >SMW@Waikato.ac.nz > Shwan, you will have to be more specific. There are hundreds of papers on RAPD analysis. If you just want a paper describing the technique check out: Williams,JGK; Hanafey,MK; Rafalski,JA; Tingey,SV (1993): Genetic analysis using random amplified polymorphic DNA markers. Meth. Enzym. 218, 704-740. Good luck. 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 Dec 01 22:00:00 1993 Path: biosci!news.cs.umb.edu!hsdndev!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd Subject: Re: AmpliTaq polymerase LD Message-ID: <2dku50$2pd@oj.rsmas.miami.edu> Date: 2 Dec 93 14:29:52 GMT References: <199311302308.AA12764@postoffice.mail.cornell.edu> Distribution: bionet Organization: R.S.M.A.S./University of Miami Lines: 25 NNTP-Posting-Host: oj.rsmas.miami.edu In <199311302308.AA12764@postoffice.mail.cornell.edu> muhammad a. lodhi wrote: >Huang Bixing wrote that; >>I am trying every percaution to avoid non-specific bands in my RAPD >>negative control. > >continued. After a while I stopped worrying about them as they seemed >'friendly' to me. I could not find a single factor responsible for this >amplification but some time it helped to work under laminar flow hood as >mostly I worked on the bench-top without any further protection. I ran >reactions in 2-3 replications and found very consistent results. Its >probably one of those mysteries that come with RAPD. > I have been told that the random bands in negative controls are normal primer artifacts. They have never shown up in any reactions when template is added. I now use them as an indicator that everything else in my reaction mix is working properly and worry if I don't see them which seems to indicate that I did something wrong OR the conditions are not optimal for that particular primer. If I optimize to see RANDOM artifacts in negative controls I get good consistant patterns with that primer with template. The key seems to be that the artifactual bands are truely random, e.g. different each reaction with the same primer, and not consistant primer dimers or other artifacts which persist even when template is present. 2 negative controls are required to verify this for each primer. From owner-rapd@net.bio.net Thu Dec 02 22:00:00 1993 Path: biosci!lhc!darwin.sura.net!europa.eng.gtefsd.com!uunet!decwrl!decwrl!usenet.coe.montana.edu!news.uoregon.edu!netnews.nwnet.net!news.u.washington.edu!stein1.u.washington.edu!toby From: toby@stein1.u.washington.edu (Toby Bradshaw) Newsgroups: bionet.molbio.rapd Subject: Re: Grinding of tissue for DNA or protein extraction Message-ID: <2dl3ac$d48@news.u.washington.edu> Date: 2 Dec 93 15:58:04 GMT References: <9312012056.AA03108@phibred.phibred.com> Distribution: bionet Organization: University of Washington, Seattle Lines: 12 NNTP-Posting-Host: stein.u.washington.edu In article <9312012056.AA03108@phibred.phibred.com>, IAN MURRAY wrote: >I am looking for disposable/ resuable pestles to fit into the wells of >a 96 well microtitre plate or a 1 ml tube, the ones that I have are supplied >by Bio Rad. Reusable pestles are pertty easy to make using your tube or plate as a mold and a good epoxy as the pestle material. Put a shaft in the epoxy before it hardens and, voila, a pestle. -Toby Bradshaw toby@u.washington.edu From owner-rapd@net.bio.net Fri Dec 03 22:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!sunic!news.funet.fi!klaava!cc.helsinki.fi!louhelainen From: louhelainen@cc.helsinki.fi Newsgroups: bionet.molbio.rapd Subject: Artifical bands in RAPD Message-ID: <1993Dec4.014551.1@cc.helsinki.fi> Date: 4 Dec 93 01:45:51 GMT References: <199311302308.AA12764@postoffice.mail.cornell.edu> <2dku50$2pd@oj.rsmas.miami.edu> Distribution: bionet Organization: University of Helsinki Lines: 50 NNTP-Posting-Host: hylkb.helsinki.fi In article <2dku50$2pd@oj.rsmas.miami.edu>, kramer@oj.rsmas.miami.edu (Jack Kramer) writes: > In <199311302308.AA12764@postoffice.mail.cornell.edu> muhammad a. lodhi wrote: >>Huang Bixing wrote that; >>>I am trying every percaution to avoid non-specific bands in my RAPD >>>negative control. >> >>continued. After a while I stopped worrying about them as they seemed >>'friendly' to me. I could not find a single factor responsible for this >>amplification but some time it helped to work under laminar flow hood as >>mostly I worked on the bench-top without any further protection. I ran >>reactions in 2-3 replications and found very consistent results. Its >>probably one of those mysteries that come with RAPD. >> > > I have been told that the random bands in negative controls are normal > primer artifacts. They have never shown up in any reactions when > template is added. I now use them as an indicator that everything else > in my reaction mix is working properly and worry if I don't see them > which seems to indicate that I did something wrong OR the conditions are > not optimal for that particular primer. If I optimize to see RANDOM > artifacts in negative controls I get good consistant patterns with that > primer with template. The key seems to be that the artifactual bands > are truely random, e.g. different each reaction with the same primer, > and not consistant primer dimers or other artifacts which persist even > when template is present. 2 negative controls are required to verify > this for each primer. > > As I see it, those "normal" artifacts are formed because of contaminating DNA from autoclave and other sources. Use radiation sterilation instead , maybe those bands disappear. This could explain the fact that those bands always form a different pattern. In your sample tubes the real template DNA overrules the contaminants by number so that they are not formed. If you can, you could use a true positive control in addition to the true negative. This setup would tell you that your labware is free of contaminants and your reaction mixture is working optimally. I have seen the same situation when I have used low annealing temperatures typical to RAPD in "normal" PCR reaction. One of my collegues have emphasised the importancy of the water quality in RAPD - he lost his artifical bands after starting to use prebottled, molbio-grade water. -- ( Jari Pekka Louhelainen ( ( University of Helsinki, Dept. of General Microbiology ( ( Pl 41 (Mannerheimintie 172), SF-00014 HELSINGIN YLIOPISTO, FINLAND ( ( Phone: +358-0-4735425 or +358-40-5002405 Fax: +358-0-4735426 ( From owner-rapd@net.bio.net Fri Dec 03 22:00:00 1993 Path: biosci!daresbury!not-for-mail From: abrpanch@reading.ac.uk (Naresh C. Pancholi) Newsgroups: bionet.molbio.rapd Subject: DNA extraction from potatoes leaf Message-ID: <2do4c5$1sk@mserv1.dl.ac.uk> Date: 3 Dec 93 19:34:29 GMT Sender: daemon@mserv1.dl.ac.uk Reply-To: "Naresh C. Pancholi" Distribution: bionet Lines: 109 Original-To: rapd@dl.ac.uk Original-Sender: "Naresh C. Pancholi" --1358963620-1314005280-754947178:#19479 Content-Type: TEXT/PLAIN; CHARSET=US-ASCII Dear Raul, The potato extraction protocol as follows: /// take approx. 6 to 8 leaves from in vitro grown plant. /// crush and grind in liquid nitrogen. /// transfer powder immediately into a sterile eppendorf tube. /// add 0.5 ml CTAB buffer. It should be pre-warmed in a water bath at 60oC for 10 mins. /// under fume cupboard add 200 fl chloroform (i.e., 24:1; chloroform:isoamyl alcohol). Also add 5 fl a-mercaptoethanol. Be extremely careful. /// mix the content thoroughly. Tight lids properly. /// incubate tubes in a waterbath at 60oC for 15 mins. /// spin in a mini centrifuge for 5 mins. - approx. 13000 rpm. /// use large bore tips to remove supernant to a fresh tube. /// add 1.2 volume of chilled isopropanol and mix gently for approx. 30 seconds. DNA will precipitate. /// store at -20oC for 30 mins. /// spin for 1 minute at 13000 rpm to pallet DNA. /// pour off isopropanol and wash the pallet with 0.5 ml chilled absolute ethanol for 30 seconds. /// spin for 1 minute at 13000 rpm to pallet DNA. /// carefully pour off ethanol. /// suck up any remaining ethanol by using pipette. /// leave tube open to dry for 20 mins. or more. /// dissolve DNA pallet in freshly prepared TE buffer (50 fl). /// leave for 1 hour before PCR/RAPD or alternatively store at 4oC overnight or at -20oC for long term storage. Note: always wear gloves and safety glasses where necessary particularly when handling a- mercaptoethanol. My main area of work is banana tissue culture. However, I am looking into somaclonal variation. A friend of mine used this protocol for potatoes and now I am following it for bananas. RAPD can detect the variation, if there is any, at the early stage of development. I hope, RAUL, this will serve the purpose. Good luck. Naresh Pancholi abrpanch@reading.ac.uk Agricultural Botany University of Reading Dear Raul, --1358963620-1314005280-754947178:#19479 Content-Type: APPLICATION/octet-stream; name=cast Content-ID: Content-Description: The potato extraction protocol as follows: /// take approx. 6 to 8 leaves from in vitro grown plant. /// crush and grind in liquid nitrogen. /// transfer powder immediately into a sterile eppendorf tube. /// add 0.5 ml CTAB buffer. It should be pre-warmed in a water bath at 60oC for 10 mins. /// under fume cupboard add 200 fl chloroform (i.e., 24:1; chloroform:isoamyl alcohol). Also add 5 fl a-mercaptoethanol. Be extremely careful. /// mix the content thoroughly. Tight lids properly. /// incubate tubes in a waterbath at 60oC for 15 mins. /// spin in a mini centrifuge for 5 mins. - approx. 13000 rpm. /// use large bore tips to remove supernant to a fresh tube. /// add 1.2 volume of chilled isopropanol and mix gently for approx. 30 seconds. DNA will precipitate. /// store at -20oC for 30 mins. /// spin for 1 minute at 13000 rpm to pallet DNA. /// pour off isopropanol and wash the pallet with 0.5 ml chilled absolute ethanol for 30 seconds. /// spin for 1 minute at 13000 rpm to pallet DNA. /// carefully pour off ethanol. /// suck up any remaining ethanol by using pipette. /// leave tube open to dry for 20 mins. or more. /// dissolve DNA pallet in freshly prepared TE buffer (50 fl). /// leave for 1 hour before PCR/RAPD or alternatively store at 4oC overnight or at -20oC for long term storage. Note: always wear gloves and safety glasses where necessary particularly when handling a- mercaptoethanol. My main area of work is banana tissue culture. However, I am looking into somaclonal variation. A friend of mine used this protocol for potatoes and now I am following it for bananas. RAPD can detect the variation, if there is any, at the early stage of development. I hope, RAUL, this will serve the purpose. Good luck. Naresh Pancholi abrpanch@reading.ac.uk Agricultural Botany University of Reading UK --1358963620-1314005280-754947178:#19479-- From owner-rapd@net.bio.net Fri Dec 03 22:00:00 1993 Path: biosci!daresbury!not-for-mail From: abrpanch@reading.ac.uk (Naresh C. Pancholi) Newsgroups: bionet.molbio.rapd Subject: Re: DNA extraction from potatoes leaf Message-ID: <2do5fn$2k1@mserv1.dl.ac.uk> Date: 3 Dec 93 19:53:27 GMT Sender: daemon@mserv1.dl.ac.uk Reply-To: "Naresh C. Pancholi" Distribution: bionet Lines: 12 Original-To: rapd@dl.ac.uk Original-Sender: "Naresh C. Pancholi" Dear Netters, I am sorry for some mistakes in my message which was posted a while ago. It reads fl but it should be micro litre (ul). Similarly, a-mercaptoethanol should be read beta-mercaptoethanol. This happened because of file transfer from DOS to UNIX system! Please also tolerate the entire text repetition. Naresh Pancholi From owner-rapd@net.bio.net Mon Dec 06 22:00:00 1993 Path: biosci!JUDY.ENG.UCI.EDU!liang From: liang@JUDY.ENG.UCI.EDU (liang) Newsgroups: bionet.molbio.rapd Subject: CHINESES_BIOTECH_NET_FOUNDED Message-ID: <9312061946.AA20558@judy.eng.uci.edu> Date: 6 Dec 93 19:46:14 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 12 CBNet (Chinese Biotechnology Network) is a non-profit organization composed of professionals in biological, chemical, medical sciences, engineering and related fields. The CBNet sponsors the Chinese Biotechnology Internet Forum (CBIF) newsletter. To subscribe CBIF, please send an email to Listserv@UCSD.Edu with the message body: Add CB-Net. From owner-rapd@net.bio.net Mon Dec 06 22:00:00 1993 Path: biosci!JUDY.ENG.UCI.EDU!liang From: liang@JUDY.ENG.UCI.EDU (liang) Newsgroups: bionet.molbio.rapd Subject: CHINESES_BIOTECH_NET_FOUNDED Message-ID: <9312060501.AA19457@judy.eng.uci.edu> Date: 6 Dec 93 05:01:19 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 12 CBNet (Chinese Biotechnology Network) is a non-profit organization composed of professionals in biological, chemical, medical sciences, engineering and related fields. The CBNet sponsors the Chinese Biotechnology Internet Forum (CBIF) newsletter. To subscribe CBIF, please send an email to Listserv@UCSD.Edu with the message body: Add CB-Net. From owner-rapd@net.bio.net Mon Dec 06 22:00:00 1993 Path: biosci!ZYGOTE.HSC.USC.EDU!jbell From: jbell@ZYGOTE.HSC.USC.EDU (Jeff Bell) Newsgroups: bionet.molbio.rapd Subject: (none) Message-ID: Date: 4 Dec 93 19:18:18 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 1 unsubscribe Jeff Bell From owner-rapd@net.bio.net Wed Dec 08 22:00:00 1993 Path: biosci!VTVM1.CC.VT.EDU!TURNER From: TURNER@VTVM1.CC.VT.EDU (Bruce Turner) Newsgroups: bionet.molbio.rapd Subject: (none) Message-ID: <9312091755.AA08798@net.bio.net> Date: 9 Dec 93 17:53:31 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 5 Please enroll following on rapd net: Russula at vtvm1.cc.vt.edu Thanks Turner From owner-rapd@net.bio.net Wed Dec 08 22:00:00 1993 Path: biosci!PUCCINI.CRL.UMN.EDU!martinez From: martinez@PUCCINI.CRL.UMN.EDU ("") Newsgroups: bionet.molbio.rapd Subject: Re: Program for primer design? Message-ID: <9312092339.AA08186@puccini.crl.umn.edu> Date: 9 Dec 93 23:39:17 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 17 In message <9312092152.AA23378@net.bio.net> writes: > > > If you know where I may obtain this program, I would appreciate > it very much if you would send me the information. Well I know of one program called Oligo Selection Program (OSP) from the univ of washington med school. It's available in SUN, MAC, PC, or VAX format. It was featured in PCR Methods and Applications 1:124-128 (1991). Best of all it's free- my kind of program. Hope this helps. I also know of a few other free Mac oligo programs, if your interested, but they may not be as good as OSP. Pat M. From owner-rapd@net.bio.net Wed Dec 08 22:00:00 1993 Path: biosci!bcm!TAMUTS.TAMU.EDU!cs.utexas.edu!swrinde!emory!europa.eng.gtefsd.com!library.ucla.edu!agate!usenet.ins.cwru.edu!news.ysu.edu!psuvm!cunyvm!yvax.byu.edu!farmerj From: farmerj@yvax.byu.edu Newsgroups: bionet.molbio.rapd Subject: Program for primer design? Message-ID: <1993Nov30.150459.3514@yvax.byu.edu> Date: 30 Nov 93 22:04:54 GMT Organization: Brigham Young University Lines: 26 I have just begun to recover from a series of minor computer disasters associated with switching all my files to a new computer. In the process, I lost an e-mail announcement that included a description of a (free?) program that designs PCR primers. From the description, the program seemed to have all the features I need. If you know where I may obtain this program, I would appreciate it very much if you would send me the information. Thank you. James L. Farmer Department of Zoology 571 WIDB Brigham Young University Provo, Utah 84602, USA (801) 378-2153 (OFFICE) (801) 378-7499 (FAX) FARMERJ@YVAX.BYU.EDU FARMERJ@BYUVAX.BITNET From owner-rapd@net.bio.net Mon Dec 13 22:00:00 1993 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!uunet!news.moneng.mei.com!uwm.edu!vixen.cso.uiuc.edu!newsrelay.iastate.edu!news.iastate.edu!mgkaiser From: mgkaiser@iastate.edu (Michael G Kaiser) Subject: lack of product from RAPD PCR Message-ID: Originator: mgkaiser@pv7b88.vincent.iastate.edu Sender: news@news.iastate.edu (USENET News System) Organization: Iowa State University, Ames, Iowa (USA) Date: Tue, 14 Dec 1993 22:40:08 GMT Lines: 10 I am a new user of this newsgroup but have been doing RAPD for two years. Using Operon primers and chicken DNA the RAPD PCR have worked very well. Lately, none of the reactions have had any product. I have replaced all reagents, taq poly, used new lots of primer and various template DNA and still nothing works. I would like to hear any sugestions as to what might be wrong or what I might try. -- Michael G Kaiser mgkaiser@iastate.edu From owner-rapd@net.bio.net Tue Dec 14 22:00:00 1993 Path: biosci!POMONA.CLAREMONT.EDU!JNUSSER From: JNUSSER@POMONA.CLAREMONT.EDU (John Nusser) Newsgroups: bionet.molbio.rapd Subject: percentage of polymorphic loci Date: 14 Dec 1993 17:40:20 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01H6H6N04YIQ8ZE4RA@POMONA.CLAREMONT.EDU> NNTP-Posting-Host: net.bio.net Hello! I am interested in comparing the results of my RAPD survey on a wild population of clapper rails with RAPD surveys on other bird species. Specifically, I would like to know in other species, what percentage of loci are polymorphic using 4-base or 2-base 10-mer primers. I believe I have found low levels of variability in these birds, but I have very little data to compare mine to. For 4-base primers, I have found less than .5% of loci are polymorphic, and for 2-base primers, I have found less than .7% of loci are polymorphic. I would appriciate any estimates of what percentage of RAPD loci are polymorphic. Although most of my analysis is based upon results generated with Nei's diversity statistics, I am still interested in getting a general idea of what other people are finding. Thanks in advance. John Nusser Jnusser@Pomona.claremont.edu From owner-rapd@net.bio.net Tue Dec 14 22:00:00 1993 Path: biosci!agate!library.ucla.edu!europa.eng.gtefsd.com!paladin.american.edu!gatech!usenet.ufl.edu!schema.fiu.edu!serss0.fiu.edu!solix!harkinsd From: harkinsd@solix.fiu.edu (Derek M. Harkins) Newsgroups: bionet.molbio.rapd Subject: Re: lack of product from RAPD PCR Date: 15 Dec 1993 21:28:51 GMT Organization: Florida International University Lines: 32 Message-ID: <2envij$9b0@serss0.fiu.edu> References: NNTP-Posting-Host: solix-gw.fiu.edu X-Newsreader: TIN [version 1.2 PL0] Michael G Kaiser (mgkaiser@iastate.edu) wrote: : I am a new user of this newsgroup but have been doing RAPD for two : years. Using Operon primers and chicken DNA the RAPD PCR have worked : very well. Lately, none of the reactions have had any product. I have : replaced all reagents, taq poly, used new lots of primer and various : template DNA and still nothing works. I would like to hear any : sugestions as to what might be wrong or what I might try. : -- : Michael G Kaiser : mgkaiser@iastate.edu A colleague of mine has a personal experience to recount concerning this issue.: --- I recently lost 2 months work due to the same problem. This may sound dumb, but it seemed to be all in the pipetting. Be very careful that every bit of reagent gets into the master mix, when aliquoting, etc. That is, you have to actually look at the pipet tip every time . Yes, this is very tedious, but now all my reactions work. Also, when you aliquot the master mix, sometimes a small amount will enter the tip before you actually take up sample. Can't let that happen. By the way, I tried all the things you did, and they didn't work for me either. Apparently, volume is very critical. Good luck. Cathy Ronning, USDA-ARS, Miami FL. -- * Derek M. Harkins | All opinions here stated, if * * United States Department of Agriculture | correct and lucid, are solely * * Agricultural Research Service NCGR-MIA | my own. All others you can * * Miami, FL 33158 USA (305) 238-9321 | blame on my evil twin. * From owner-rapd@net.bio.net Tue Dec 14 22:00:00 1993 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!xlink.net!scsing.switch.ch!rzusuntk.unizh.ch!newsadm From: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Subject: re: percentage of polymorphic loci Message-ID: <1993Dec15.141943.12179@rzu-news.unizh.ch> Organization: University of Zurich, Switzerland References: <01H6H6N04YIQ8ZE4RA@POMONA.CLAREMONT.EDU> Date: Wed, 15 Dec 1993 14:19:43 GMT Lines: 21 In article <01H6H6N04YIQ8ZE4RA@POMONA.CLAREMONT.EDU> JNUSSER@POMONA.CLAREMONT.EDU (John Nusser) writes: >Hello! > > I am interested in comparing the results of my RAPD survey on a wild >population of clapper rails with RAPD surveys on other bird species. >Specifically, I would like to know in other species, what percentage of loci >are polymorphic using 4-base or 2-base 10-mer primers. > > I believe I have found low levels of variability in these birds, but I >have very little data to compare mine to. For 4-base primers, I have found >less than .5% of loci are polymorphic, and for 2-base primers, I have found >less than .7% of loci are polymorphic. I would appriciate any estimates of >what percentage of RAPD loci are polymorphic. Although most of my analysis >is based upon results generated with Nei's diversity statistics, I am still >interested in getting a general idea of what other people are finding. > > Thanks in advance. > > John Nusser > Jnusser@Pomona.claremont.edu From owner-rapd@net.bio.net Wed Dec 15 22:00:00 1993 Path: biosci!bcm!cs.utexas.edu!swrinde!sgiblab!sgigate.sgi.com!olivea!decwrl!decwrl!usenet.coe.montana.edu!netnews.nwnet.net!news.u.washington.edu!stein1.u.washington.edu!johnreed From: johnreed@stein1.u.washington.edu (John Reed) Newsgroups: bionet.molbio.rapd Subject: population genetics with RAPDs, again Date: 16 Dec 1993 19:20:10 GMT Organization: University of Washington, Seattle Lines: 42 Message-ID: <2eqcda$gql@news.u.washington.edu> NNTP-Posting-Host: stein.u.washington.edu Hi all. I am trying to find some useful methods of analyzing RAPD data from a population genetic perspective. I have written a simple program to find the number of differences between individuals and create a "distance" matrix. This seems like useful information, but how does one analyze it? I have used the analysis of molecular variance (AMOVA) program of Excoffier et al. (Genetics, 131: 479-491, 1992), but in communicating with L. Excoffier he said that this anlysis was not valid for RAPD data (though, it mught be an approximation of the correct analysis?). This was for reasons I really do not understand and I was hoping someone could enlighten me. Some background first.. AMOVA takes distance matrices calculated from HAPLOTYPE (not RAPD) data and generates a probability distribution from the matrix. It then partitions variance into the 3 groups, among individuals within pops, among pops within groups and among groups. It also calculates some F-stat analogues, which the authors call phi-stats (if I remember correctly). This is very useful. Now, the reason that Mr. Excoffier gave for the invalidity of using this method on RAPD data was that RAPD data are dominant from diploid organisms. This I knew, but WHY does this matter (Mr. Excoffier, are you out there)? There is surely some simple and logical reason, but I haven't yet thought of it. I have also thought of using regular ANOVA to analyze these distance matrices (my distances seem to be normally distributed), but how do I structure the analysis? A single data point represents the number of differences between two individuals, not a property of one individual. Another question. How would one go about testing for differences at a single locus among populations or groups? The method I have used is a simple chi-square analogue, the log-likelihood ratio (G statistic). Is this appropriate? Any help will be highly appreciated. Thanks, John -------------------------------------------------------------------------- John Reed College of Forest Resources, AR-10 University of Washington Seattle, WA 98195 Internet: johnreed@u.washington.edu -------------------------------------------------------------------------- From owner-rapd@net.bio.net Wed Dec 15 22:00:00 1993 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Latest version of PAUP? Date: 15 Dec 1993 19:56:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199312160356.AA21936@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net Dear Friends Does anyone there know which is the latest version of PAUP. I have PAUP 3.0r. If there is one after 3.0, where can I get it. I will appreciate any help in this regard. Thanks in advance Muhammad A. Lodhi Cornell University From owner-rapd@net.bio.net Thu Dec 16 22:00:00 1993 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!xlink.net!scsing.switch.ch!rzusuntk.unizh.ch!newsadm From: newsadm@rzu-news.unizh.ch (CNEWS ADMINISTRATION) Subject: RAPD with NaOH-extraction Message-ID: <1993Dec17.083427.13429@rzu-news.unizh.ch> Organization: University of Zurich, Switzerland Date: Fri, 17 Dec 1993 08:34:27 GMT Lines: 12 RAPD with NaOH-extraction We try to establish a fast PCR DNA extraction method (NAR 1993 21(17):4153-4154) together with RAPD-10mer primers for amplification. We work with Phaseolus vulgaris vulgaris (bean) varieties. Is there anybody who got some results using the NaOH extraction and 10mer primers for the same or other species? It seems to be quite difficult to find the suitable reaction conditions and the corresponding cycler program. Can anybody give some hints? Our expieriences so far show less banding than expected (about 3 per primer). Is this to be expected using this fast extraction? Thanks for any information, Rolf Meile From owner-rapd@net.bio.net Thu Dec 16 22:00:00 1993 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!usenet.ins.cwru.edu!magnus.acs.ohio-state.edu!dlohnes From: dlohnes@magnus.acs.ohio-state.edu (David G Lohnes) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD with NaOH-extraction Date: 17 Dec 1993 14:04:26 GMT Organization: The Ohio State University Lines: 29 Sender: dlohnes@magnus.acs.ohio-state.edu Message-ID: <2ese9a$kge@charm.magnus.acs.ohio-state.edu> References: <1993Dec17.083427.13429@rzu-news.unizh.ch> NNTP-Posting-Host: bottom.magnus.acs.ohio-state.edu I have just tried the NaOH-extraction method for use with RAPDs and have very good results. Instead of the 1 microliter of the diluted DNA, I add 4 microliters. The cycling temperatures are as follows: 94 C for 2 min 3 cycles of 94 C for 15 sec 35 C for 15 sec ramp to 72 C in 60 sec 72 C for 75 sec 40 cycles of 94 C for 15 sec 40 C for 15 sec ramp to 72 C in 60 sec 72 C for 75 sec 72 C for 7 min 4 C indefinitely This is with soybean DNA. Hope it helps. David Lohnes OARDC/OSU Wooster, Ohio From owner-rapd@net.bio.net Thu Dec 16 22:00:00 1993 Newsgroups: bionet.molbio.rapd Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!howland.reston.ans.net!xlink.net!scsing.switch.ch!swidir.switch.ch!news.unige.ch!divsun!laurent From: laurent@divsun.unige.ch (EXCOFFIER Laurent) Subject: Re: population genetics with RAPDs Message-ID: <1993Dec17.082855.12967@news.unige.ch> Sender: usenet@news.unige.ch Organization: University of Geneva, Switzerland Date: Fri, 17 Dec 1993 08:28:55 GMT Lines: 54 In article gql@news.u.washington.edu, johnreed@stein1.u.washington.edu (John Reed) writes: > Hi all. I am trying to find some useful methods of analyzing RAPD data > from a population genetic perspective .... > I have used the analysis of molecular variance (AMOVA) > program of Excoffier et al. (Genetics, 131: 479-491, 1992), but in > communicating with L. Excoffier he said that this anlysis was not valid > for RAPD data (though, it mught be an approximation of the correct > analysis?). This was for reasons I really do not understand and I was > hoping someone could enlighten me. > John, I did not say that AMOVA could not be used with RAPDs, but that it was not specifically designed to deal with these markers. In essence AMOVA is just a technique of Analysis of Variance applied to molecular data. In the right context, ANOVA allows to estimate the degree of population structure with analogues of F-statitics as shown by Cockerham in the sixties. AMOVA extracts also these analogues of F-statitics, that we call Phi-statistics. A useful property of these statistics is that they reflect a population structure independently of which locus was used in the analysis. They can thus be estimated for different markers, and averaged to give better estimates. When using AMOVA on RAPDs, my concern is that the inferred Phi-statistics are qualitatively different from those obtained with other markers where genotypes are known. AMOVA applied to RAPDs partitions phenotypic variance instead of genotypic variance as is done for other molecular markers. These estimates thus cannot be compared. However, AMOVA can be used to partition total phenotypic variance into different components (individuals, populations, and group of populations), whose significance can be tested. Thus, population structure can be tested using this approach, as was done by Huff, Peakall and Smouse (1993, Theor. Appl. Genet. 86:927-934). We are now in the (slow) process of adapting AMOVA to specifically deal with RAPDs, in order to extract estimates of population structure comparable with those of other molecular markers. Two distinct problems emerge: the dominance of the bands and the (often unknown) amount of inbreeding in plant populations studied with RAPDs. Another approach has been explored by Lynch and Milligan (submitted) to deal with this issue. I hope this makes it a little bit clearer. ---- Laurent Excoffier, Genetics & Biometry Lab, Dept of Anthropology, U. of Geneva, 12, G. Revilliod, 1227 Geneva, Switzerland. Tel: +41 22 702 6965 Fax: 300 0351 From owner-rapd@net.bio.net Fri Dec 17 22:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!howland.reston.ans.net!usenet.ins.cwru.edu!news.ysu.edu!psuvm!cunyvm!yvax.byu.edu!farmerj From: farmerj@yvax.byu.edu Newsgroups: bionet.molbio.rapd Subject: RE: population genetics with RAPDS Message-ID: <1993Dec17.151321.3734@yvax.byu.edu> Date: 17 Dec 93 15:13:21 -0700 Organization: Brigham Young University Lines: 31 The continuing interest in how to use RAPD data in population genetics suggests to me that we may wish to open a dialogue with the people who subscribe to CLASS-L, a classification listserv group that seems to be interested in problems of this sort. I am a casual, amateur observer of that group, and I do not believe that I have seen RAPD data discussed before. Perhaps those of you who are more knowledgeable than I about population genetics would like to join that group and begin a discussion in front of the experts. On the assumption that some of you might be interested, here is the address: Subscribing: Send the message SUB CLASS-L yourname where "yourname" is your name to LISTSERV@SBCCVM. The address to which to post messages is CLASS-L@SBCCVM. Be prepared for very technical criticism of your ideas. It helps to be very knowledgeable about statistics. Good luck. Jim Farmer farmerj@yvax.byu.edu P.S. Please pardon the gibberish which follows. How do you erase when using the "POST"command? " CVMLIwh2 From owner-rapd@net.bio.net Sun Dec 19 22:00:00 1993 Newsgroups: bionet.molbio.rapd Path: biosci!daresbury!doc.ic.ac.uk!uknet!pipex!sunic!news.funet.fi!convex!ridell From: ridell@convex.csc.FI (Jouko Ridell) Subject: reproducibility of bacterial RAPD Message-ID: <1993Dec20.153712.4931@funet.fi> Keywords: rapd, staphylococcus aureus, reproducibility Sender: usenet@funet.fi (#Kotilo NEWS system ) Nntp-Posting-Host: convex.csc.fi Organization: Finnish University and Research Network FUNET Distribution: bionet Date: Mon, 20 Dec 1993 15:37:12 GMT Lines: 26 I have just started to use RAPD to study the molecular epidemiology of Staphylococcus aureus. Some general questions: 1) Is the reproducibility of RAPD dependent on the size of the genome? Eg. with bigger genomes (plants, fungi, mammals) random primers have bigger chance to find spesific priming sites than with smaller genomes (bacteria). According to theoretical predictions bacterial RAPD should not produce so many products as is seen. Suggested explanation is mispriming (Clark and Lanigan 1993, Mol. Biol. Evol. 10:1096-1111). If so, does this mean that bacterial RAPD is more vulnerable to artifactual variations caused by the slight differences in the PCR conditions ? 2) What is known at present about the effect of the quality/type/purity of template DNA on bacterial RAPD ? According to some articles purification of DNA is not needed and boiled whole cell lysates can be used as well. I'm eager to hear your opinions and experiences. Jouko Ridell Email: Ridell@vetmed.fi University of Veterinary Medicine Department of Food and Environmental Hygiene Telefax: int-358-0-3931718 FINLAND -- From owner-rapd@net.bio.net Mon Dec 20 22:00:00 1993 Newsgroups: bionet.molbio.rapd Path: biosci!daresbury!doc.ic.ac.uk!warwick!pipex!uunet!europa.eng.gtefsd.com!library.ucla.edu!news.ucdavis.edu!pbmac-22.ucdavis.edu!user From: tdlong@ucdavis.edu (Tony Long) Subject: Re: percentage of polymorphic loci Message-ID: Followup-To: bionet.molbio.rapd Sender: usenet@ucdavis.edu (News Administrator) Organization: Center for Population Biology References: <01H6H6N04YIQ8ZE4RA@POMONA.CLAREMONT.EDU> Distribution: bionet Date: Tue, 21 Dec 1993 20:11:03 GMT Lines: 33 In article <01H6H6N04YIQ8ZE4RA@POMONA.CLAREMONT.EDU>, JNUSSER@POMONA.CLAREMONT.EDU (John Nusser) wrote: > > Hello! > > I am interested in comparing the results of my RAPD survey on a wild > population of clapper rails with RAPD surveys on other bird species. > Specifically, I would like to know in other species, what percentage of loci > are polymorphic using 4-base or 2-base 10-mer primers. > > I believe I have found low levels of variability in these birds, but I > have very little data to compare mine to. For 4-base primers, I have found > less than .5% of loci are polymorphic, and for 2-base primers, I have found > less than .7% of loci are polymorphic. I would appriciate any estimates of > what percentage of RAPD loci are polymorphic. Although most of my analysis > is based upon results generated with Nei's diversity statistics, I am still > interested in getting a general idea of what other people are finding. > > Thanks in advance. > > John Nusser > Jnusser@Pomona.claremont.edu Be Careful!! The percentage of loci polymorphic is a function of sample size and can not therefore be compared with such estimates from an unequal sample size. You are likely better off obtaining some estimate of theta (see Nei) Tony Long Center for Population Biology U. C. Davis Davis, CA tdlong@ucdavis.edu From owner-rapd@net.bio.net Wed Dec 22 22:00:00 1993 Path: biosci!lhc!darwin.sura.net!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: "Naresh C. Pancholi" Newsgroups: bionet.molbio.rapd Subject: Help needed Date: 23 Dec 1993 11:54:30 -0000 Lines: 25 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2fc0tm$4ft@mserv1.dl.ac.uk> Reply-To: "Naresh C. Pancholi" Original-Sender: "Naresh C. Pancholi" Original-To: RAPD , Plant Biology , Methods-and-Reagents , Bionauts Dear Netters, I want to know the e-mail address of Dr. David Jones (Parc Scientifiqe Institute, Montpellier, France). I have e-mail address of that institute (cgi150) but that is not enough to contact. Can someone over there help me? I am going to post this message to more than one groups so I am sorry if you have come across this message once again. Thanks. +-----------------------------------------------+ | Naresh Pancholi abrpanch@reading.ac.uk | | Agricultural Botany Phone: +44 734 318092 | | Reading University Fax: +44 734 316577 | | Reading RG6 2AS | | UK | +-----------------------------------------------+ From owner-rapd@net.bio.net Wed Dec 22 22:00:00 1993 Path: biosci!cornell.edu!mal5 From: mal5@cornell.edu (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Phylogenetic analysis with DNA content data Date: 16 Dec 1993 17:52:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199312170152.AA03982@postoffice.mail.cornell.edu> Dear Friends Can someone guide me how to do phylogenetic analysis on the data that I describe in the following, if possible; I have analyzed nuclear DNA content from 24 species (almost all diploid except one) of Vitis (grape), 2 other genera (other than Vitis, one genotype in each case) and several hybrids and cultivars belonging to 24 Vitis species. Vitis has ~60 species in all, the DNA content values are very close to each other and probably non significantly different. I collected that data for two years so in a sense I have two replications. I am wondering if there is a way of establishing any phylogenetic relationship (or partial relationship) among the species and genera. I am open to any suggestions and collaboration. Thanks in advance =============================================================================== Muhammad A. Lodhi Cornell University Grape Breeding and Genetics Program Geneva, NY 14456 From owner-rapd@net.bio.net Thu Dec 23 22:00:00 1993 Path: biosci!hubcap!darwin.sura.net!paladin.american.edu!howland.reston.ans.net!nctuccca.edu.tw!TWNMOE10.Edu.TW!YMMCS023 From: YMMCS023@TWNMOE10.Edu.TW Newsgroups: bionet.molbio.rapd Subject: CAG repeat in human brain cDNA Date: Sat, 25 Dec 93 04:26:35 GMT Organization: MOE Computer Center, Taiwan Lines: 7 Message-ID: <16CAF3E7B.YMMCS023@TWNMOE10.Edu.TW> NNTP-Posting-Host: twnmoe10.edu.tw X-Newsreader: NNR/VM S_1.3.2 Do anybody think there must be further neuropsychiatric diseases caused by CAG or other trinucleotide repeat expansion? Is it possible that schizophrenia or b ipolar disorder is related to trinucleotide repeat mutation? Is there any lab w orking on the relating topic? Any information posted will be highly appreciated . Horng e-mail: ymmcs023@twnmoe10.edu.tw