From owner-rapd@net.bio.net Tue May 03 23:00:00 1994 Path: biosci!UKCC.UKY.EDU!EQUIGENE From: EQUIGENE@UKCC.UKY.EDU Newsgroups: bionet.molbio.rapd Subject: program Date: 4 May 1994 08:11:02 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199405041510.IAA26183@net.bio.net> NNTP-Posting-Host: net.bio.net I would also like information on a computer program for systematics of RAPDs. Thanks. Teri L. Lear University of Kentucky Dept. of Veterinary Science Gluck Equine Research Center Lexington, KY 40546-0099 From owner-rapd@net.bio.net Tue May 03 23:00:00 1994 Path: biosci!TAUNIVM.TAU.AC.IL!VPMBJ%VOLCANI.bitnet From: VPMBJ%VOLCANI.bitnet@TAUNIVM.TAU.AC.IL Newsgroups: bionet.molbio.rapd Subject: Computer program Date: 4 May 1994 06:30:42 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199405041330.GAA21485@net.bio.net> NNTP-Posting-Host: net.bio.net Dose anybody knows on a computer program (for mac or PC) specific for analysis of population or systematics with RAPD. If so, where can this program be obtain. Amos Goaz The Volcani Center, ISRAEL. From owner-rapd@net.bio.net Tue May 03 23:00:00 1994 Path: biosci!ABRSLE.AGR.CA!DEMEKE From: DEMEKE@ABRSLE.AGR.CA Newsgroups: bionet.molbio.rapd Subject: computer program for RAPD Date: 4 May 1994 10:29:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01HBXY0W06QA000HDV@GW.AGR.CA> NNTP-Posting-Host: net.bio.net Hi: The best way is to refer to the recent publications on RAPD analysis and classifications. Some of the programs used so far are: 1. UPGMA- Applied Biostatistics, Inc., 2. NTSYS-pc- Exeter Software, Setauket, N.Y., 3. PCO- one source- Dr. R.P. Adams, Baylor University, Plant Biotechnology Center, Waco, Texas, 76798-7372, U.S.A. Good luck! Tigst Demeke From owner-rapd@net.bio.net Wed May 04 23:00:00 1994 Path: biosci!CORNELL.EDU!MAL5 From: MAL5@CORNELL.EDU (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: QTL analysis on woody plants Date: 5 May 1994 06:17:25 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199405051313.JAA20339@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net Dear Friends Has there been any report published or submitted on QTL analysis in woody plants and/or pseudotestcross populations. Any information would be highly appreciated. Thanks in advance ========================================== Muhammad A. Lodhi Program in Grape Breeding and Genetics Cornell University From owner-rapd@net.bio.net Wed May 04 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!wupost!news.miami.edu!not-for-mail From: kramer@oj.rsmas.miami.edu (Jack Kramer) Newsgroups: bionet.molbio.rapd Subject: Re: QTL analysis on woody plants Date: 5 May 1994 10:14:08 -0400 Organization: R.S.M.A.S. Lines: 10 Distribution: bionet Message-ID: <2qauvg$gnh@oj.rsmas.miami.edu> References: <199405051313.JAA20339@postoffice.mail.cornell.edu> NNTP-Posting-Host: oj.rsmas.miami.edu In article <199405051313.JAA20339@postoffice.mail.cornell.edu>, Muhammad A. Lodhi wrote: >Dear Friends >Has there been any report published or submitted on QTL analysis in woody >plants and/or pseudotestcross populations. Any information would be highly >appreciated. Try searching citations by Ray Schnell. He has been doing QTL analyses with RAPDS on tropical fruit crop trees for several years. From owner-rapd@net.bio.net Thu May 05 23:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!EU.net!howland.reston.ans.net!noc.near.net!transfer.stratus.com!bigboote.WPI.EDU!wpi.WPI.EDU!jbagshaw From: jbagshaw@wpi.WPI.EDU (Joseph C. Bagshaw) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.rapd Subject: A grinding problem Date: 6 May 1994 19:48:20 GMT Organization: Worcester Polytechnic Institute Lines: 1 Message-ID: <2qe6u4$976@bigboote.WPI.EDU> NNTP-Posting-Host: wpi.wpi.edu Xref: biosci bionet.molbio.methds-reagnts:14024 bionet.molbio.rapd:552 From owner-rapd@net.bio.net Sun May 08 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: Use of PCR for chromosome walking? Date: 9 May 1994 11:35:42 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 26 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9405091836.AA21543@gw1.phibred.com> NNTP-Posting-Host: net.bio.net Hello Netters, I have a large primer, about 1-2kb and wish t use it in PCR reactions to amplify large regions of DNA (about 10kb or more). In this way we could chromosome walk. Here is the crunch> 1) How can I increase the stringency of binding of such a large piece of DNA??? 2) Are there any thermocyclers, programmes, or reagent concentrations (or polymerase enzyme) that would enable this to be done? 3) With such large pieces of synthetic DNA, is the decrease in efficiency greater than in normal PCR? 4) Does the final product become too viscous, and decrease mixing ? If anyone has suggestions, references or helping hints, I would be greatly appreciative. It would save my coleagues and I a lot of time. Yours Sincerely, Ian Murray Murrayian@phibred.com From owner-rapd@net.bio.net Sun May 08 23:00:00 1994 Path: biosci!UNAMVM1.DGSCA.UNAM.MX!carvalho From: carvalho@UNAMVM1.DGSCA.UNAM.MX (Carvalho Torres Alexandro cassio-UACPYP) Newsgroups: bionet.molbio.rapd Subject: RAPD CLONING Date: 9 May 1994 14:13:24 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Hi, At the moment I am intending to clone a rapd fragment of 1.6Kb, we have some difficult. What is the most pratical tip to cloning this band. What is the strategy used by the people that cloned RAP fragments. Thanks. ###################################################### Alexandro C.T. Carvalho Instituto Nacional de la Nutricion Salvador Zubiran Mexico, DF FAX 655 9675 ####################################################### From owner-rapd@net.bio.net Sun May 08 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: RAPD CLONING Date: 9 May 1994 14:37:02 -0700 Organization: University of Arkansas Lines: 27 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: carvalho@unamvm1.dgsca.unam.mx (Carvalho Torres Alexandro cassio-UACPYP) >Subject: RAPD CLONING >Date sent: 9 May 1994 14:13:24 -0700 > >Hi, >At the moment I am intending to clone a rapd fragment of 1.6Kb, we have >some difficult. What is the most pratical tip to cloning this band. What >is the strategy used by the people that cloned RAP fragments. >Thanks. >###################################################### >Alexandro C.T. Carvalho >Instituto Nacional de la Nutricion Salvador Zubiran >Mexico, DF >FAX 655 9675 >####################################################### The method we prefer to use is resolution on an agarose gel, excise the band, rinse for 5-10 minutes in dH2O, soak for several hours in 100ul of H2O or low EDTA buffer, reamplify using the same primer on 2ul of eluate, and then ligate into a PCR cloning vector such as pGEMT from Promega. 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 May 08 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: Use of PCR for chromosome walking? Date: 9 May 1994 12:09:36 -0700 Organization: University of Arkansas Lines: 34 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: murrayian@phibred.com (IAN MURRAY) >Subject: Use of PCR for chromosome walking? >Date sent: 9 May 1994 11:35:42 -0700 >Hello Netters, >I have a large primer, about 1-2kb and wish t use it in PCR reactions >to amplify large regions of DNA (about 10kb or more). In this way we >could chromosome walk. Here is the crunch> >1) How can I increase the stringency of binding of such a large piece of > DNA??? >2) Are there any thermocyclers, programmes, or >reagent concentrations (or >polymerase enzyme) that would enable this to be done? >3) With such large pieces of synthetic DNA, is the decrease in efficiency >greater than in normal PCR? >4) Does the final product become too viscous, and decrease mixing ? >If anyone has suggestions, references or helping hints, I would be greatly >appreciative. It would save my coleagues and I a lot of time. > >Yours Sincerely, >Ian Murray Murrayian@phibred.com > I am not sure why anyone would want to try and use 1kb primers?? My guess is that you actually have 1kbp dsDNA fragments and you want to amplify the region between them?? Just about the maximum size primer you should want is around 25-30 bases depending on the CG content. At 50%CG most 30mers have a Tm of >70C. 90C melts anything but you have to go down to 70-72 for the Taq enzyme to function maximally. 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 Mon May 09 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: RAPD CLONING Date: 10 May 1994 07:47:58 -0700 Organization: University of Arkansas Lines: 56 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net >On 9 May 1994, Doug Rhoads wrote: > >> > >> >Hi, >> >At the moment I am intending to clone a rapd fragment of 1.6Kb, we have >> >some difficult. What is the most pratical tip to cloning this band. What >> >is the strategy used by the people that cloned RAP fragments. >> >Thanks. >> >###################################################### >> >Alexandro C.T. Carvalho >> >> The method we prefer to use is resolution on an agarose gel, excise the >> band, rinse for 5-10 minutes in dH2O, soak for several hours in 100ul of >> H2O or low EDTA buffer, reamplify using the same primer on 2ul of eluate, >> and then ligate into a PCR cloning vector such as pGEMT from Promega. >> >> Doug Rhoads || Dept. of Biological Sciences >> >Dear Dr. Hoads, >I try to dp this but I can't success, because I didn't amplify the >fragment of 1.6 Kb. Exist some special program to amplify this fragment. >I use the follow program. >94C...........10 sec X 35c >36C...........30 sec >72C...........1 min >You don't fill in your band or make a specila treatment the plasmid, like >alcaline phosphatase reaction. >Please if you have some tips, send to me. I will very happy with more >information. >Thanks a lot. Alexandro Carvalho > I would guess that when you re-amplified you got several of the other bands as well. One thing you can do is go to anneals at 42-45C since by now you have a perfect match on the termini. Also you shouldn't need to run more than 20-25 cycles. You may also want to consider going to an acrylamide gel for better resolution of you band. A 4% gel from 39:1 acryl:MBA should work even for a 1.6 kbp band. As far as the plasmid, you need something with a EcoRV site in the multi-cloning site in a LacZ gene. We have used pGEM5Zf from Promega. Digest with EcoRV and then incubate with several units of Taq Pol and ONLY dTTP to add a single T to the 3' ends (not efficient but can work). The plasmid is then ligated to the PCR product which naturally is A tailed by the Taq Pol. Transfect into XL1 blue or other suitable host and select with Amp. Color determination with Xgal IPTG should identify colonies with inserts (white). Generally we expect about 1% white colonies. If we use the pGEMT vector from Promega we get more like 10% whites. I guess they do a better job than we do. Standard disclaimer that I am not associated with or remunerated by any company mentioned above. 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 Mon May 09 23:00:00 1994 Path: biosci!UNAMVM1.DGSCA.UNAM.MX!carvalho From: carvalho@UNAMVM1.DGSCA.UNAM.MX (Carvalho Torres Alexandro cassio-UACPYP) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD CLONING Date: 9 May 1994 19:45:59 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 46 Sender: daemon@net.bio.net Distribution: bionet Message-ID: References: NNTP-Posting-Host: net.bio.net On 9 May 1994, Doug Rhoads wrote: > >To: rapd@net.bio.net > >From: carvalho@unamvm1.dgsca.unam.mx (Carvalho Torres Alexandro cassio-UACPYP) > >Subject: RAPD CLONING > >Date sent: 9 May 1994 14:13:24 -0700 > > > > >Hi, > >At the moment I am intending to clone a rapd fragment of 1.6Kb, we have > >some difficult. What is the most pratical tip to cloning this band. What > >is the strategy used by the people that cloned RAP fragments. > >Thanks. > >###################################################### > >Alexandro C.T. Carvalho > >Instituto Nacional de la Nutricion Salvador Zubiran > >Mexico, DF > >FAX 655 9675 > >####################################################### > > The method we prefer to use is resolution on an agarose gel, excise the > band, rinse for 5-10 minutes in dH2O, soak for several hours in 100ul of > H2O or low EDTA buffer, reamplify using the same primer on 2ul of eluate, > and then ligate into a PCR cloning vector such as pGEMT from Promega. > > 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 > > Dear Dr. Hoads, I try to dp this but I can't success, because I didn't amplify the fragment of 1.6 Kb. Exist some special program to amplify this fragment. I use the follow program. 94C...........10 sec X 35c 36C...........30 sec 72C...........1 min You don't fill in your band or make a specila treatment the plasmid, like alcaline phosphatase reaction. Please if you have some tips, send to me. I will very happy with more information. Thanks a lot. Alexandro Carvalho From owner-rapd@net.bio.net Mon May 09 23:00:00 1994 Path: biosci!PHIBRED.COM!huangb From: huangb@PHIBRED.COM (Bin Huang (905)8464446) Newsgroups: bionet.molbio.rapd Subject: please subscribe me Date: 10 May 1994 07:40:57 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9405101447.AA16774@gw1.phibred.com> NNTP-Posting-Host: net.bio.net Please subscribe. HUANGB@PHIBRED.COM From owner-rapd@net.bio.net Mon May 09 23:00:00 1994 Path: biosci!sfu.ca!corley From: corley@sfu.ca ("Graham Corley-Smith") Newsgroups: bionet.molbio.rapd Subject: RE: Use of PCR for chromosome walking? Date: 9 May 1994 21:54:11 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 45 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <75015.corley@sfu.ca> Reply-To: corley@sfu.ca NNTP-Posting-Host: net.bio.net In Message 9 May 1994 11:35:42 -0700, murrayian@phibred.com (IAN MURRAY) writes: >Hello Netters, > >I have a large primer, about 1-2kb and wish t use it in PCR reactions >to amplify large regions of DNA (about 10kb or more). In this way we >could chromosome walk. Here is the crunch> > >1) How can I increase the stringency of binding of such a large piece of > DNA??? >2) Are there any thermocyclers, programmes, or >reagent concentrations (or >polymerase enzyme) that would enable this to be done? > >3) With such large pieces of synthetic DNA, is the decrease in efficiency >greater than in normal PCR? > >4) Does the final product become too viscous, and decrease mixing ? > > >If anyone has suggestions, references or helping hints, I would be greatly >appreciative. It would save my coleagues and I a lot of time. > >Yours Sincerely, > >Ian Murray > >Murrayian@phibred.com Have you seen the article entitled 'Long PCR' Leaps Into Larger DNA Sequences, that was published in Science 263:1564-1565 (March 18, 1994)? Graham ________________ Graham E. Corley-Smith Institute of Molecular Biology and Biochemistry Biological Sciences Department Simon Fraser University Burnaby, B.C., V5A 1S6 Canada Phone: (604) 291-3021 Fax: (604) 291-5583 From owner-rapd@net.bio.net Mon May 09 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!EU.net!uunet!panix!zip.eecs.umich.edu!newsxfer.itd.umich.edu!jobone!lynx.unm.edu!news.cs.indiana.edu!babbage.ece.uc.edu!ucbeh!rogstad From: rogstad@ucbeh.san.uc.edu Newsgroups: bionet.molbio.rapd Subject: JOB: EPA POSTDOC: PLANT POPULATION GENETICS Message-ID: <1994May9.231550.7302@ucbeh> Date: 9 May 94 23:15:50 EST Distribution: world Organization: University of Cincinnati Lines: 29 POSTDOCTORAL POSITION: PLANT MOLECULAR POPULATION GENETICIST Oak Ridge Institute of Science and Education Research Fellowship tenable at the Department of Biological Sciences, University of Cincinnati, and sponsored by the U.S. Environmental Protection Agency Environmental Monitoring Systems Laboratory - Cincinnati. The position is available immediately and is funded for two years, although application for extension will be expected. The research involves all or part of the following: * Investigations, utilizing DNA variation, of effects of anthropogenic disturbance on population genetics of selected plant species. * Learning, and developing further, methods to investigate genetic diversity of selected plant species. This may involve the use of microsatellite, minisatellite, RFLPS, or RAPD analyses. * Working with statistical analyses to optimize methods for data analysis. * Collecting and analyzing DNA variation of field samples from impacted and control field sites. QUALIFICATIONS: Applicants must have a Ph.D. degree by the time of appointment. Individuals with extensive experience in molecular population genetics including field botany skills, plant DNA extraction, RFLP Southern hybridization techniques, PCR methodology, and data analysis will be preferred. APPLICATION: Send curriculum vitae, brief statement of research experience and goals, and list of three references (with phone numbers) to S. Rogstad, Biological Sciences ML6, University of Cincinnati, Cincinnati, OH 45229-0006. From owner-rapd@net.bio.net Tue May 10 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!EU.net!Austria.EU.net!newsfeed.ACO.net!info.univie.ac.at!lab8486.abc.univie.ac.at!ma From: ma@ABC.univie.ac.at (Manuel Simon) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD CLONING Date: Wed, 11 May 1994 18:28:30 Organization: Inst. Biochemistry and Molecular Cell Biology Lines: 39 Distribution: bionet Message-ID: References: NNTP-Posting-Host: lab8486.abc.univie.ac.at X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] In article DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") writes: >From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") >Subject: Re: RAPD CLONING >Date: 10 May 1994 07:47:58 -0700 High, some remarks: >I would guess that when you re-amplified you got several of the other >bands as well. One thing you can do is go to anneals at 42-45C since by >now you have a perfect match on the termini. Also you shouldn't need to >run more than 20-25 cycles. If you do so, of course you could get unspecific internal priming and therefore shorter fragments. >As far as the plasmid, you need something with a EcoRV site in the >multi-cloning site in a LacZ gene. We have used pGEM5Zf from Promega. >Digest with EcoRV and then incubate with several units of Taq Pol and ONLY >dTTP to add a single T to the 3' ends (not efficient but can work). The >plasmid is then ligated to the PCR product which naturally is A tailed by >the Taq Pol. Transfect into XL1 blue or other suitable host and select >with Amp. Color determination with Xgal IPTG should identify colonies >with inserts (white). Generally we expect about 1% white colonies. If we >use the pGEMT vector from Promega we get more like 10% whites. I guess >they do a better job than we do. There are also other sites available which cut blunt ended (e.g. SmaI) which are usually in all commercial vectors. The main problem is, that Taq polymerase attaches a 3´ protruding nucleotide to the synthesized strand and therefore these fragments are not so easy to ligate. If you are not successful with the protocol above try to polish the PCR fragment with T4-DNA polymerase in the presence of 100 uM dNTP. Then degradation shall stop at the beginning of double strand. Good luck Manuel From owner-rapd@net.bio.net Tue May 10 23:00:00 1994 Path: biosci!BRADLEY.BRADLEY.EDU!rrs From: rrs@BRADLEY.BRADLEY.EDU (Robert Stephens) Newsgroups: bionet.molbio.rapd Subject: computer program for systematics Date: 11 May 1994 08:42:36 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9405111543.AA20416@bradley.bradley.edu> NNTP-Posting-Host: net.bio.net For the mac try getting a copy of PAUP 3.1.1 from swofford@onyx.si.edu and for the pc try getting a copy of HENNIG86 1.5 from Steve Farris at State Unmiversity of New York Stony Brook . Don't know Farris' e-mail or complete address. Also, R. Page has a pc program called Component (?). Last I read he was affiliated with the British Museum. Hope this helps. From owner-rapd@net.bio.net Tue May 10 23:00:00 1994 Path: biosci!AGRADM.Lan.McGill.CA!MATHER-1 From: MATHER-1@AGRADM.Lan.McGill.CA ("DM-Lab-1") Newsgroups: bionet.molbio.rapd Subject: Help Date: 11 May 1994 14:35:59 -0700 Organization: McGill University, Macdonald Campus Lines: 2 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199405112136.RAA06935@sifon.CC.McGill.CA> NNTP-Posting-Host: net.bio.net Subscribe Help From owner-rapd@net.bio.net Tue May 10 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: Failing RAPD reactions Date: 11 May 1994 11:07:19 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9405111809.AA24930@gw1.phibred.com> NNTP-Posting-Host: net.bio.net >From: murrayian@phibred.com (IAN MURRAY) >Subject: RAPD primer eqaution >Date sent: 22 Apr 1994 12:23:58 -0700 >Hello, > > > >On another note............ > >I have used the 96 well plate MJ PTC100 thermocycler for a long time now, >constantly ((6mo). It now does not work and the reair service said that >there was nothing wrong with it. The same reagents work on another >thermocycler that we use (Ericomp using 0.5 ul tubes) to yield RAPD >products. Simultaneous experiments showed that the MJ just did not work for >RAPD and PCR. Does anyone have an idea of what is wrong?? Our Idaho did this to us and we solved the problem by lowering the denature temp to 90C. We attributed it to overshoot when the room was warmer. 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 --------------------------------------------------------------------- Also Mike@mj.com suggested that the 96 well round bottom plates were at fault. A suggestion was to wash them with BSA, so that the Polymerase would not be bound to the plate. I have not yet tried th above, but if anyone has faile RAPD reactions, PCR as well and has ruled out the reagents (and mineral oil) as the cause..... The plates may be the culprit. Murrayian@phibred.com Ian Murray Pioneer Hi-Bred From owner-rapd@net.bio.net Tue May 10 23:00:00 1994 Path: biosci!VM.CC.FAMU.EDU!XQU From: XQU@VM.CC.FAMU.EDU Newsgroups: bionet.molbio.rapd Subject: (none) Date: 11 May 1994 08:37:09 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199405111537.IAA12879@net.bio.net> NNTP-Posting-Host: net.bio.net Please subscribe me. From owner-rapd@net.bio.net Tue May 10 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Julian Paul Robinson Newsgroups: bionet.molbio.rapd Subject: Tm and Operon Technologies Date: 11 May 1994 15:24:54 +0100 Lines: 14 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2qqprm$dc4@mserv1.dl.ac.uk> Reply-To: Julian Paul Robinson Original-Sender: Julian Paul Robinson Original-To: rapd@dl.ac.uk Does any one out-there know how to calculate Tm for primers of 10 bp?? Also, does anyone know the address/fax no. of Operon Technologies, Inc. (or distributor for the UK) :-) Julian From owner-rapd@net.bio.net Wed May 11 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: 'Long PCR' : leapds into larger DNA sequences Date: 12 May 1994 12:48:42 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9405121951.AA22481@gw1.phibred.com> NNTP-Posting-Host: net.bio.net Hello, Has everyone read the articl 'long PCR' Leaps into larger DNA sequences in Science Vol 263 pp 1564-1565 (18 March 1994) ? In summary, long PCR products were produced (20 - 45 Kb) by altering either the polymerases or the PCR reagents. However, I heard through the grapevine that it has not been reproduced. Has anyone tried it ? If anyone has comments or further references, they will be appreciated. Murrayian@phibred.com Ian Murray From owner-rapd@net.bio.net Wed May 11 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!spool.mu.edu!torn!utnut!utzoo!mes From: mes@zoo.toronto.edu (Mark Siddall) Subject: Re: computer program for systematics Message-ID: Date: Thu, 12 May 1994 05:31:53 GMT Distribution: bionet References: <9405111543.AA20416@bradley.bradley.edu> Organization: U of Toronto Zoology Lines: 45 In article <9405111543.AA20416@bradley.bradley.edu> rrs@BRADLEY.BRADLEY.EDU (Robert Stephens) writes: >For the mac try getting a copy of PAUP 3.1.1 from >swofford@onyx.si.edu and for the pc try getting a copy of HENNIG86 >1.5 from Steve Farris at State Unmiversity of New York Stony Brook No go!!! Farris hasn't been at Stoney Brook for about a year or so. He's in Sweden somewhere. Stoney Brook may still distribute Hennig86 though I don't know. >. Don't know Farris' e-mail or complete address. Also, R. Page >has a pc program called Component (?). Last I read he was affiliated >with the British Museum. Hope this helps. Rod's email address is rod.page@zoology.oxford.ac.uk however COMPONENT is not so much for phylogeny reconstruction as it is for biogeographic and host-parasite questions. To continue: there's Kevin Nixon's ClaDos for examining character optimization. Also, Felsenstein's Phylip. And the latest is MEGA. Most of these packages cost $ but not much. MEGA is $15, PAUP is pretty cheap given what it does. (There's also my own software Random Cladistics, which does not calculate phylogenies but does allow PC users who like the speed and reliability of Hennig86 to do bootstrapping, Faith and Cranston's PTP, random-tree histograms and a couple of other things. Requires you have Hennig86 independently. It's available (free) by anonymous ftp to zoo.toronto.edu whereupon one must "cd pub" (no quotes) and get both random.exe and random.doc in binary mode. Read random.doc for instructions on intalling the package to your DOS pc. Let me know if you get it as there's a couple of helpful hints and limitations one should know about). Sorry for banging my own drum, but it is free after all. Mark > -- Mark E. Siddall "In saying what is obvious, never choose cunning. mes@vims.edu Yelling works better." - C. Ozick Virginia Inst. Marine Sci. Gloucester Point, VA, 23062 From owner-rapd@net.bio.net Wed May 11 23:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!EU.net!howland.reston.ans.net!cs.utexas.edu!usc!nic-nac.CSU.net!charnel.ecst.csuchico.edu!psgrain!nntp.cs.ubc.ca!unixg.ubc.ca!quartz.ucs.ualberta.ca!gpu!asimmond From: asimmond@gpu.srv.ualberta.ca (Andrew Simmonds) Newsgroups: bionet.molbio.rapd Subject: RAPD on Confier Needles Date: 12 May 1994 21:58:54 GMT Organization: University of Alberta Lines: 17 Message-ID: <2qu8qu$n6u@quartz.ucs.ualberta.ca> NNTP-Posting-Host: gpu.srv.ualberta.ca X-Newsreader: TIN [version 1.2 PL2] Might anyone have any suggestions on how to prepare DNA from confier needles for RAPD analysis. We are looking for something quick and dirty, other than the usual grinding in liquid nitrogen. We need clean DNA, and CsCl gradients are not an option. Thanks in advance.. Mark Hicks Andrew Simmonds ______________________________________________________________________________ Department of Genetics - University of Alberta CANADA ASIMMOND@GPU.SRV.UALBERTA.CA G216 Biological Sciences Centre Edmonton, Alberta, CANADA T6G 2E9 ****************************************************************************** "Does steel wool come from magnetic sheep?" ****************************************************************************** From owner-rapd@net.bio.net Thu May 12 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!moe.ksu.ksu.edu!pst.pp.ksu.edu!user From: pst@ksu.ksu.edu (Paul St. Amand) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD on Confier Needles Followup-To: bionet.molbio.rapd Date: Fri, 13 May 1994 13:04:55 -0500 Organization: USDA/ARS & KSU Agronomy dept. Lines: 23 Message-ID: References: <2qu8qu$n6u@quartz.ucs.ualberta.ca> NNTP-Posting-Host: pst.pp.ksu.edu In article <2qu8qu$n6u@quartz.ucs.ualberta.ca>, asimmond@gpu.srv.ualberta.ca (Andrew Simmonds) wrote: > Might anyone have any suggestions on how to prepare DNA from confier > needles for RAPD analysis. We are looking for something quick and dirty, > other than the usual grinding in liquid nitrogen. We need clean DNA, and > CsCl gradients are not an option. Thanks in advance.. Try NaOH extraction. I can't remember the citation, but I think it was in NAR 1993. It's easy, cheap, and very fast. I get very good PCR results using it on single alfalfa leaflets. You simply grind fresh tissue in 0.5 Normal NaOH (10ul per mg of fresh tissue) and pipette 5ul of the liquid phase into 495ul of Tris (100mM, pH = 8.0). Shake well and use or freeze immediately. Use 1 to 10ul of the Tris solution per RAPD reaction. Let me know if it works. Paul C. St. Amand Research Geneticist ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ USDA/ARS Agronomy Dept. 122 Throckmorton Hall (913) 532-6168 Kansas State University Fax (913) 532-5692 Manhattan KS 66506-5501 PST@KSU.KSU.Edu From owner-rapd@net.bio.net Thu May 12 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!swrinde!ihnp4.ucsd.edu!munnari.oz.au!newsroom.utas.edu.au!ml.csiro.au!mac_1303.ml.csiro.au!chris.bolch From: Christopher J. S. Bolch Subject: subscribe Bolch Message-ID: <1994May12.225118.336@ml.csiro.au> X-Xxmessage-Id: X-Xxdate: Thu, 12 May 94 21: 53:27 GMT Sender: news@ml.csiro.au Organization: CSIRO Division of Fisheries, Hobart, Tasmania X-Useragent: Version 1.1.3 Date: Thu, 12 May 1994 22:51:18 GMT Lines: 1 subscribe chris.bolch@ml.csiro.au From owner-rapd@net.bio.net Thu May 12 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!swrinde!ihnp4.ucsd.edu!munnari.oz.au!newsroom.utas.edu.au!ml.csiro.au!mac_1303.ml.csiro.au!chris.bolch From: Christopher J. S. Bolch Subject: RAPD program Message-ID: <1994May12.225448.466@ml.csiro.au> X-Xxmessage-Id: X-Xxdate: Thu, 12 May 94 21: 56:58 GMT Sender: news@ml.csiro.au Organization: CSIRO Division of Fisheries, Hobart, Tasmania X-Useragent: Version 1.1.3 References: <199405041510.IAA26183@net.bio.net> Date: Thu, 12 May 1994 22:54:48 GMT Lines: 7 In article <199405041510.IAA26183@net.bio.net> , EQUIGENE@UKCC.UKY.EDU writes: >I would also like information on a computer program for systematics of >RAPDs. Thanks. So would I. I have seen analyses of data with other programs (e.g. PAUP) but would like to know if there are other approaches available. From owner-rapd@net.bio.net Thu May 12 23:00:00 1994 Path: biosci!agate!dog.ee.lbl.gov!overload.lbl.gov!bks From: bks@s27w007.pswfs.gov (Bradley K. Sherman) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD on Confier Needles Date: 13 May 1994 20:45:15 GMT Organization: Dendrome, A Genome Database for Forest Trees Lines: 27 Message-ID: <2r0osr$h7m@overload.lbl.gov> References: <2qu8qu$n6u@quartz.ucs.ualberta.ca> NNTP-Posting-Host: s27w007.pswfs.gov asimmond@gpu.srv.ualberta.ca (Andrew Simmonds) wrote: > Might anyone have any suggestions on how to prepare DNA from confier > needles for RAPD analysis. We are looking for something quick and dirty, > other than the usual grinding in liquid nitrogen. We need clean DNA, and > CsCl gradients are not an option. Thanks in advance.. We are attempting to compile protocols used in studying the molecular biology of forest trees. If you have not already looked there, there is a section on the Dendrome gopher (s27w007.pswfs.gov port 70): ... 6. Forest tree genome resources (including TreeGenes)/ .... 4. Protocols/ where you might find something useful. This section of our server is maintained by Kim Marshall (kam@s27w007.pswfs.gov). Suggestions, comments and contributed protocols are welcome. --bks -- Bradley K. Sherman P.O. Box 245 Computer Scientist Berkeley, CA, 94701 Dendrome Project 510-559-6437 FAX: 510-559-6440 Institute of Forest Genetics Internet: bks@s27w007.pswfs.gov From owner-rapd@net.bio.net Sat May 14 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bloom-beacon.mit.edu!grapevine.lcs.mit.edu!olivea!koriel!cs.utexas.edu!howland.reston.ans.net!agate!doc.ic.ac.uk!uknet!EU.net!uunet!utcsri!utzoo!mes From: mes@zoo.toronto.edu (Mark Siddall) Subject: Re: RAPD program Message-ID: Date: Sun, 15 May 1994 16:08:49 GMT References: <199405041510.IAA26183@net.bio.net> <1994May12.225448.466@ml.csiro.au> Organization: U of Toronto Zoology Lines: 35 In article <1994May12.225448.466@ml.csiro.au> Christopher J. S. Bolch writes: >In article <199405041510.IAA26183@net.bio.net> , EQUIGENE@UKCC.UKY.EDU >writes: >>I would also like information on a computer program for systematics of >>RAPDs. Thanks. > >So would I. I have seen analyses of data with other programs (e.g. PAUP) >but would like to know if there are other approaches available. I (think) I have responded to this once already regarding the software available out there for phylogenetic work. Upon re-reading the query it appears as though the poster might be under the impression that there is RAPD-systematics oriented software. There is not. Nor is there any need as such. Phylogenetic software (e.g., PAUP, Hennig86, PHYLIP etc) are character-based. It doesn't matter what your characters are. There is some software designed for analyzing character information in long DNA sequences (e.g., MEGA) so that the experimentor doesn't have to go cross-eyed looking at the sequences. This is not a problem for people doing RAPDs or RFLPs or what have you. This brings me to my next point. Though RAPDs may be suitable for population-level phenomena, they are not (in my opinion) at all suitable for phylogenetic studies! For the same reasons that RFLPs are not either. Oh, sure, people are doign it, but when you start asking questions of what is heritable, what is homology, and what constitutes a character state transformation, it falls apart. I'm probably going to be unpopular around here for having said this. Mark -- Mark E. Siddall "In saying what is obvious, never choose cunning. mes@vims.edu Yelling works better." - C. Ozick Virginia Inst. Marine Sci. Gloucester Point, VA, 23062 From owner-rapd@net.bio.net Sun May 15 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!edwards From: edwards@nature.Berkeley.EDU (Owain Edwards) Newsgroups: bionet.molbio.rapd Subject: Re: band detection programs Date: 16 May 1994 14:16:59 GMT Organization: U.C. College of Natural Resources Lines: 28 Message-ID: <2r7v8r$48i@agate.berkeley.edu> References: NNTP-Posting-Host: nature.berkeley.edu KARI KAMMIOVIRTA writes: >Hi all sisters and brothers in PCR, > >I've been working with RAPD for two years now and noticed that worst part of >the work is comparing bands in pictures. Especially when I have lot of >samples (about 250) there is 13 different fotographs I have to compare. >Has anybody used any programs for this. I have found only one that seems >suitable, Pharmacia's RFLPrint, does somebody have any comments about this >program? Price is nice (14000 $) and it would be good to have some >experienced opinions. >Thanks in advance, > >Kari I am also interested in comparing among patterns. I have been searching for a while for a program that will do this, and I think I may have found one, though I have not tried it yet. I am sending for a demo for a program called WHOLE BAND MATCHER which is part of the BioImage Gel Scanner package from Millipore. The documentation that I have claims that it lets you compare bands within and between gels and the program creates dendrograms and similarity indeces directly from gels. Has anyone had any experience with this program, or has anyone heard anything about it? Thanks, Owain Edwards (edwards@nature.berkeley.edu) From owner-rapd@net.bio.net Sun May 15 23:00:00 1994 Path: biosci!parc!decwrl!decwrl!spool.mu.edu!agate!doc.ic.ac.uk!lyra.csx.cam.ac.uk!warwick!pipex!sunic!news.funet.fi!news.csc.fi!news.helsinki.fi!MMKAB-01208.pc.Helsinki.FI!kkammiov From: kkammiov@viikki21.helsinki.fi (KARI KAMMIOVIRTA (MMKAB)) Newsgroups: bionet.molbio.rapd Subject: band detection programs Date: Mon, 16 May 1994 07:43:01 GMT Organization: University of Helsinki Lines: 12 Message-ID: NNTP-Posting-Host: mmkab-01208.pc.helsinki.fi Hi all sisters and brothers in PCR, I've been working with RAPD for two years now and noticed that worst part of the work is comparing bands in pictures. Especially when I have lot of samples (about 250) there is 13 different fotographs I have to compare. Has anybody used any programs for this. I have found only one that seems suitable, Pharmacia's RFLPrint, does somebody have any comments about this program? Price is nice (14000 $) and it would be good to have some experienced opinions. Thanks in advance, Kari From owner-rapd@net.bio.net Sun May 15 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!netnews.nwnet.net!ns1.nodak.edu!badlands!dahleen From: dahleen@badlands.NoDak.edu (Lynn S Dahleen) Subject: Re: RAPD CLONING Sender: usenet@ns1.nodak.edu Message-ID: Date: Mon, 16 May 1994 19:01:19 GMT Distribution: bionet References: Nntp-Posting-Host: badlands.nodak.edu Organization: North Dakota Higher Education Computing Network X-Newsreader: TIN [version 1.2 PL2] Lines: 39 Doug Rhoads (DRHOADS@MERCURY.UARK.EDU) wrote: : >On 9 May 1994, Doug Rhoads wrote: : > : >> > : >> >Hi, : >> >At the moment I am intending to clone a rapd fragment of 1.6Kb, we have : >> >some difficult. What is the most pratical tip to cloning this band. What : >> >is the strategy used by the people that cloned RAP fragments. : >> >Thanks. : >> >###################################################### : >> >Alexandro C.T. Carvalho : >> : >> The method we prefer to use is resolution on an agarose gel, excise the : >> band, rinse for 5-10 minutes in dH2O, soak for several hours in 100ul of : >> H2O or low EDTA buffer, reamplify using the same primer on 2ul of eluate, : >> and then ligate into a PCR cloning vector such as pGEMT from Promega. : >> : >> Doug Rhoads || Dept. of Biological Sciences : >> I too have had great success with this method using eluates from both agarose and acrylamide gels. However, after re-amplification of the eluate, I typically add 5units of klenow to the reaction and incubate 30 min at 37 degrees C to blunt end the DNA. I then add 50-200ng of SmaI or EcoRV cut plasmid (bluescript in my case) to the mix. I then Phenol extract once, Chloroform extract once and then ethanol precipitate in order to get rid of excess ATPs that might (?) inhibit blunt ended ligations. After air drying the resulting DNA pellet, I resuspend in 17ul water and then add 2ul of 10X ligation buffer (NEB) and 1ul of NEBs concentrated T4 DNA ligase. I allow the ligation reaction to go overnight at room temperature (about 22 degrees C). I typically use half of the reaction to transform 100ul of competent DH5a cells using Richard Swartzes increadible competency proceedure. (Does anyone have a reference for this? I could use one as I have only an old beatup protocol from god knows where, and I would like to reference it in a paper I am preparing.) I hope this is of some additional help. Dave Horvath USDA/ARS/NCSL (701)239-1335 From owner-rapd@net.bio.net Mon May 16 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!agate!lsa3mac34.berkeley.edu!user From: dobson@mendel.berkeley.edu (Steve Dobson) Newsgroups: bionet.molbio.rapd Subject: Server for bionet.molbio.rapd Followup-To: bionet.molbio.rapd Date: 17 May 1994 17:23:47 GMT Organization: U C Berkeley Lines: 12 Message-ID: NNTP-Posting-Host: lsa3mac34.berkeley.edu I have a friend who wants to link up to bionet.molbio.rapd. I am linked up via NewsWatcher for the MacIntosh. He is unable to use this program. Can someone please e-mail him info on how to link up to bionet.molbio.rapd via his e-mail account. I assume this can be done, though I don't know how. His address is : DGMILL00@UKCC.uky.edu (the "0's" are zeros). Thanks, Steve From owner-rapd@net.bio.net Mon May 16 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: RAPD CLONING Date: 17 May 1994 06:09:55 -0700 Organization: University of Arkansas Lines: 53 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <34124E674D@uamercury.uark.edu> NNTP-Posting-Host: net.bio.net >Doug Rhoads (DRHOADS@MERCURY.UARK.EDU) wrote: >: >On 9 May 1994, Doug Rhoads wrote: >: > >: >> > >: >> >Hi, >: >> >At the moment I am intending to clone a rapd fragment of 1.6Kb, we have >: >> >some difficult. What is the most pratical tip to cloning this band. What >: >> >is the strategy used by the people that cloned RAP fragments. >: >> >Thanks. >: >> >###################################################### >: >> >Alexandro C.T. Carvalho >: >> >: >> The method we prefer to use is resolution on an agarose gel, excise the >: >> band, rinse for 5-10 minutes in dH2O, soak for several hours in 100ul of >: >> H2O or low EDTA buffer, reamplify using the same primer on 2ul of eluate, >: >> and then ligate into a PCR cloning vector such as pGEMT from Promega. >: >> >: >> Doug Rhoads || Dept. of Biological Sciences >: >> > I too have had great success with this method using eluates from both >agarose and acrylamide gels. However, after re-amplification of the >eluate, I typically add 5units of klenow to the reaction and incubate 30 >min at 37 degrees C to blunt end the DNA. I then add 50-200ng of SmaI >or EcoRV cut plasmid (bluescript in my case) to the mix. I then Phenol >extract once, Chloroform extract once and then ethanol precipitate in >order to get rid of excess ATPs that might (?) inhibit blunt ended >ligations. After air drying the resulting DNA pellet, I resuspend in >17ul water and then add 2ul of 10X ligation buffer (NEB) and 1ul of NEBs >concentrated T4 DNA ligase. I allow the ligation reaction to go >overnight at room temperature (about 22 degrees C). I typically use half >of the reaction to transform 100ul of competent DH5a cells using Richard >Swartzes increadible competency proceedure. (Does anyone have a reference >for this? I could use one as I have only an old beatup protocol from god >knows where, and I would like to reference it in a paper I am preparing.) >I hope this is of some additional help. > > Dave Horvath > USDA/ARS/NCSL > (701)239-1335 Michael Weiner of Stratagene has pointed out that Klenow is not the best enzyme to `polish' ends of PCR products. This is mostly true for products whose 3' nucleotide is NOT-A. From the info he sent me it looks like if the products already terminates in A no further transferase activity is exhibited by the Taq Pol. If you try and `blunt' products with Klenow the those with A,C or G at the 3' end (5' of primer NOT T) the Klenow treatment doesn't do as well for cloning as does T4 Pol or Pfu Pol. This really means that a lot of us have been very lucky in the past. 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 Mon May 16 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: Summary of Long PCR responses *thanks for the contributions" Date: 17 May 1994 09:58:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 190 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9405171703.AA19167@gw1.phibred.com> NNTP-Posting-Host: net.bio.net Hello Netters, Sorry for the out of order sequence of some of the posts. The postings have led to my greater interest in long PCR. I was wondering if fragment extension was limited by: 1) those factors discussed in Science, March 1994. (263:15654-1565) 2) the length of primer. I propose that long primers can cause looping of the DNA, or at least increase the amount of unwound DNA. On the down side, longer primers require longer annealing times. 3) lack of helicases and gyrases to release the torsional strain on the DNA strands _ I assume that the Polymerase extension causes strand separation. Any comments??? Furthermore, Could anyone provide me with protocol for long PCR. Thanks in advance. Murrayian@phibred.com Ian Murray Pioneer Hi-Bred --------------++++++++++++++++++++++++_______________________ Hi: I am surprised that you have a synthetic DNA of this length. You could definitely help everyone if you can say how you managed to get it. I think you may probably have a ds kb length DNA. In this case the whole story changes because of its tendency to come together. One main thing you need to be concerned about in using long primers is their relative ease at which they degrade at high temperature.s This is a combination of both temperature and the acidity of the reaction mix resulting from temperature association of Tris' pH. The change in viscosity is one of the last things to be worried about. I would be very much surprised if you can get the PCR going using such large primers in the first place. Do let me know if you are successful. Raj Shankarappa D.V.M., Ph.D bsh@med.pitt.edu 745 Scaife Hall Department of Pathology University of Pittsburgh School of Medicine Pittsburgh PA 15261 Tel: (412) 648-9763 or 648-9573 or 648-9026 (Hey, I can't help it!) Fax: (412) 648-1916 ----------------------------------------------------- Subj: Use of PCR for chromosome walking? Hello Netters, I have a large primer, about 1-2kb and wish t use it in PCR reactions to amplify large regions of DNA (about 10kb or more). In this way we could chromosome walk. Here is the crunch> 1) How can I increase the stringency of binding of such a large piece of DNA??? 2) Are there any thermocyclers, programmes, or reagent concentrations (or polymerase enzyme) that would enable this to be done? 3) With such large pieces of synthetic DNA, is the decrease in efficiency greater than in normal PCR? 4) Does the final product become too viscous, and decrease mixing ? If anyone has suggestions, references or helping hints, I would be greatly appreciative. It would save my coleagues and I a lot of time. Yours Sincerely, Ian Murray Murrayian@phibred.com ---------------------------------------------- Subj: Amplifying large DNAs Interesting questions...why does the primer need to be so long? Otherwise, yes, there are polymerase/thermal profile considerations that could be of use to you. We have obtained larger than 10 kb amplifications, but the primers were of "normal" length. I cannot see why your primers would need to be so large. I can give some suggestions on the rest, though. Give me a message if you are interested. Sincerely, Bruno WS Sobral Staff Scientist California Institute of Biological Research (CIBR) 11099 N. Torrey Pines Road, Suite 300 La Jolla CA USA 92037 (619)535-5483 (office) (619)535-5491 (lab) E-mail: sobral@lifsci.sdsu.edu or brunosfly@aol.com (619)535-5472, 535-5490 or 535-5481 (faxes) -------------------------------------- > I am not sure why anyone would want to try and use 1kb primers?? My guess is that you actually have 1kbp dsDNA fragments and you want to amplify the region between them?? Just about the maximum size primer you should want is around 25-30 bases depending on the CG content. At 50%CG most 30mers have a Tm of >70C. 90C melts anything but you have to go down to 70-72 for the Taq enzyme to function maximally. 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 -------------------------------------- Ian, I read your question on the RAPDs network and agree with Doug Rhoades about the primer size. A very expensive primer I bet!! I do know that Boehringer- Mannheim has been able to amplify very large DNA fragments using their Taq DNA polymerase (up to 10kb) using special techniques. You might want to contact their technical sevices department for the methods. I have always gotten good advice .... Hope this helps. Teri -------------------------------------------- If you know the sequence of your 1 kb "primer", then ... use a portion of it (up to about 30 bases). At larger lengths, it is true that you will have a higher melting temperature, but Taq polymerase will need you to "cool" to 72 C for extension anyway, so anything above that is useless for increasing PCR specificity per se. As to production of larger amplification fragments, you need to use other polymerases (than just Taq) for best results. Sincerely, Bruno WS Sobral E-mail: sobral@lifsci.sdsu.edu or brunosfly@aol.com -------------------------------------------------------- Have you seen the article entitled 'Long PCR' Leaps Into Larger DNA Sequences, that was published in Science 263:1564-1565 (March 18, 1994)? Graham Graham E. Corley-Smith Institute of Molecular Biology and Biochemistry Biological Sciences Department Simon Fraser University Burnaby, B.C., V5A 1S6 Canada Phone: (604) 291-3021 Fax: (604) 291-5583 ---------------------------------- Ian, ..... I still doubt large primers will work the way you expect. The article in Science makes sense to read, but I heard that no one else could reproduce the results (grapevine). I have not tried such a large amplification here, but 15 kb or so is pretty standard. For walking, you may wish to look at BioTechniques 16(5):792-798. Never tried it, but it sounds as easy as it's going to get for what I believe you want to do. I also saw a posting of yours about the PTC100 failures. The BSA is critical. We keep 200 micrograms/ml in all our reactions in the round- bottom 96-well plates. Or you can use the conical bottom tubes, which do not require BSA. Good luck! Sincerely, Bruno WS Sobral E-mail: sobral@lifsci.sdsu.edu or brunosfly@aol.com ------------------------------------------------ Ian, I use it all the time and it does work. Wayne barnes' article was published in the March PNAS. michael_weiner@stratagene (please be advised that Stratagene sells one of the ingredients necessary for the reaction) From owner-rapd@net.bio.net Tue May 17 23:00:00 1994 Path: biosci!daresbury!trane.uninett.no!eunet.no!nuug!EU.net!howland.reston.ans.net!europa.eng.gtefsd.com!emory!news.cc.emory.edu!news.cc.emory.edu!not-for-mail From: vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) Newsgroups: bionet.molbio.rapd Subject: Cloning with TA Date: 18 May 1994 17:39:10 -0400 Organization: Emory University Lines: 9 Message-ID: <2re1tu$5ms@emoryu1.cc.emory.edu> NNTP-Posting-Host: emoryu1.cc.emory.edu X-Newsreader: TIN [version 1.2 PL2] I saw the postings about cloning rapd fragments and wondering why TA cloning kits are not used more often. The protocols are simpler. Is it the expense? We tried it once (from Novagen, I think) and appeared to work fine. I would like to hear others' experience with the TA kits before we embark on more cloning. Thanks From owner-rapd@net.bio.net Tue May 17 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!EU.net!uknet!daresbury!not-for-mail From: nfix@virgo.jcu.cz (Ivo Wiesner) Newsgroups: bionet.molbio.rapd Subject: ?RAPD sampling for outcross popul.?? Date: 18 May 1994 13:05:55 +0100 Lines: 25 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2rd0b3$nsr@mserv1.dl.ac.uk> X-Char-Esc: 29 X-Charset: ASCII Original-To: rapd@dl.ac.uk Hello Netters outthere !!! could anybody share with me his/her experience with: what is the best and fastest strategy for estimation of the representative sample size (number of individuals) when I have to discriminate among outcrossing populations, I mean populations of outcrossing plants (mutually isolated) using RAPD technology.??? (My problem are populations of fescue and ryegrass). ThnaX in advance for any info or reference !!!!!!!!!!! Greetings Ivo Wiesner nfix@virgo.jcu.cz Inst.Plant Mol.Biol. Branisovska 31 Ceske Budejovice CZ-370 05 Czech Republic From owner-rapd@net.bio.net Wed May 18 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!usenet.ufl.edu!gnv.ifas.ufl.edu!afc From: afc@gnv.ifas.ufl.edu (Andrew Cockburn) Newsgroups: bionet.molbio.rapd Subject: Re: band detection programs Message-ID: <1994May19.092946.4438@tower> Date: 19 May 94 09:29:46 -0500 References: <2r7v8r$48i@agate.berkeley.edu> Organization: USDA/ARS Lines: 49 In article <2r7v8r$48i@agate.berkeley.edu>, edwards@nature.Berkeley.EDU (Owain Edwards) writes: > KARI KAMMIOVIRTA writes: >>Hi all sisters and brothers in PCR, >> >>I've been working with RAPD for two years now and noticed that worst part of >>the work is comparing bands in pictures. Especially when I have lot of >>samples (about 250) there is 13 different fotographs I have to compare. >>Has anybody used any programs for this. I have found only one that seems >>suitable, Pharmacia's RFLPrint, does somebody have any comments about this >>program? Price is nice (14000 $) and it would be good to have some >>experienced opinions. >>Thanks in advance, >> >>Kari > > I am also interested in comparing among patterns. I have been > searching for a while for a program that will do this, and I think I may > have found one, though I have not tried it yet. I am sending for a demo > for a program called WHOLE BAND MATCHER which is part of the BioImage > Gel Scanner package from Millipore. The documentation that I have > claims that it lets you compare bands within and between gels and the > program creates dendrograms and similarity indeces directly from gels. > > Has anyone had any experience with this program, or has anyone > heard anything about it? > > Thanks, > > Owain Edwards (edwards@nature.berkeley.edu) We have a Millipore BioImage system. It does some nice stuff, such as finding bands automatically (not very reliable, though), calculating fragment sizes (much better than doing it yourself), matching patterns between gels, and exporting results for printing or storage. The BioImage system was originally designed for plant breeders, who have to follow lots of anonymous markers in lots of crosses. It does this very well. However, it runs only on a Sun SparcStation. If you don't happen to have one sitting in your lab, it will probably cost you $30k to $40k to get the system set up, counting software, computer, and scanner. Of course, you really want to add a $50k phosphorimager: think of all the money you will save on darkroom supplies! Our main problem has been user resistance. Most of my people have enough trouble using their PCs, and the Sun terrifies them. Anyone who uses it on a routine basis thinks that it is great. Andrew Cockburn USDA From owner-rapd@net.bio.net Wed May 18 23:00:00 1994 Path: biosci!BORCIM.WUSTL.EDU!woods From: woods@BORCIM.WUSTL.EDU (Jon Woods) Newsgroups: bionet.molbio.rapd Subject: Re: Cloning with TA Date: 18 May 1994 17:02:48 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: <199405190003.AA08001@wugate.wustl.edu> NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) >Subject: Cloning with TA >Date: 18 May 1994 17:39:10 -0400 > >I saw the postings about cloning rapd fragments and wondering why TA >cloning kits are not used more often. The protocols are simpler. Is it the >expense? > We tried it once (from Novagen, I think) and appeared to work fine. >I would like to hear others' experience with the TA kits before we embark >on more cloning. > >Thanks We've found TA cloning of PCR fragments to work reasonably well, without a kit or buying vector already containing 3' T overhangs. Insert: PCR fragment, most molecules having 3' single A overhangs. Vector: Digest cloning vector of choice with enzyme leaving blunt ends. Incubate DNA with thermostable polymerase, e.g., Taq, at 72C in the presence of dTTP as the sole nucleotide. Thermostable polymerases prefer to use A to extend a blunt end by 1 nucleotide, but will use other nucleotides if these are provided without A. Thus, this procedure creates vector molecules with 3' single T overhangs, compatible with the PCR fragment. Then ligate. Jon Jon Woods Department of Molecular Microbiology Box 8230 Washington University School of Medicine 660 S. Euclid Av. St. Louis, MO 63110-1093 phone (314) 362-2741 FAX (314) 362-1232 e-mail: woods@borcim.wustl.edu From owner-rapd@net.bio.net Wed May 18 23:00:00 1994 Path: biosci!BORCIM.WUSTL.EDU!woods From: woods@BORCIM.WUSTL.EDU (Jon Woods) Newsgroups: bionet.molbio.rapd Subject: Re: TA cloning Date: 19 May 1994 11:43:57 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 60 Sender: daemon@net.bio.net Distribution: world Message-ID: <199405191844.AA04799@wugate.wustl.edu> NNTP-Posting-Host: net.bio.net >> >> Vector: Digest cloning vector of choice with enzyme leaving blunt ends. >> Incubate DNA with thermostable polymerase, e.g., Taq, at 72C in the >> presence of dTTP as the sole nucleotide. Thermostable polymerases prefer >> to use A to extend a blunt end by 1 nucleotide, but will use other >> nucleotides if these are provided without A. Thus, this procedure creates >> vector molecules with 3' single T overhangs, compatible with the PCR >> fragment. >> >> Then ligate. >> >> Jon >> > >Hi Jon, this protocol seems good to me, I wonder if you treat the vector >with CIAP before cloning. What is the vector amount (1-2 micrograms?) and >the dTTP concentration (.1 mM?) that you use for the reaction ? >Thank you very much for any answer. >Francesco Argenton, Dipartimento di Biologia >Universita' di Padova, Via Trieste 75 >35121 Padova Italy > > Francesco, For adding a 3' T to blunt-ended vectors for cloning PCR fragments with overhanging A termini: 1. We do not dephosphorylate the vector. If adding the Ts works, vector circularization without insert is very inefficient. We have been doing this procedure with polylinkers using lacZ color detection of inserts, and if the Ts are NOT added and the vector circularizes, transformants are blue, while insert-positive transformants are white. 2. We use "lots" of vector DNA, at least a microgram or two. 3. We use quite a high concentration of dTTP: 1 mM. 4. We use a microliter, i.e. a few units, of thermostable polymerase in a 100-microliter reaction, and incubate at 72C for 2 hrs, under mineral oil. Note: it doesn't always work, but then, what does? Jon Jon Woods Department of Molecular Microbiology Box 8230 Washington University School of Medicine 660 S. Euclid Av. St. Louis, MO 63110-1093 phone (314) 362-2741 FAX (314) 362-1232 e-mail: woods@borcim.wustl.edu From owner-rapd@net.bio.net Fri May 20 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!spool.mu.edu!howland.reston.ans.net!europa.eng.gtefsd.com!emory!cs.utk.edu!martha.utcc.utk.edu!bme!gaoli From: gaoli@bme.utmem.edu (gaoli) Subject: cDNA probes for stromelysin and collagenase from bovine tissue Message-ID: <1994May21.040735.3782@martha.utcc.utk.edu> Keywords: cDNA probes for stromelysin and collagenase Lines: 11 Sender: gaoli@bme (gaoli) Reply-To: gaoli@bme.utmem.edu (gaoli) Organization: University of Tennessee, Memphis Date: Sat, 21 May 1994 04:07:35 GMT I am looking for cDNA probes for stromelysin and collagenase from bovine tissue. Any help would be deeply appreciated. Please E-mail me at below address: gaoli@bme.utmem.edu gaoli@eeg1.lcmc.edu Thanks in advance. gaoli From owner-rapd@net.bio.net Sun May 22 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!ihnp4.ucsd.edu!swrinde!sgiblab!munnari.oz.au!news.Hawaii.Edu!uhunix!stiles From: stiles@uhunix.tmc.edu (John Stiles) Subject: Re: Tm and Operon Technologies Message-ID: Sender: news@news.Hawaii.Edu Organization: University of Hawaii References: <2qqprm$dc4@mserv1.dl.ac.uk> Distribution: bionet Date: Fri, 13 May 1994 18:48:34 GMT Lines: 24 In article <2qqprm$dc4@mserv1.dl.ac.uk> Julian Paul Robinson writes: >Does any one out-there know how to calculate >Tm for primers of 10 bp?? > >Also, does anyone know the address/fax no. >of Operon Technologies, Inc. >(or distributor for the UK) > >:-) > >Julian > Julian, Operon Technologies, Inc. 1000 Atlantic Ave. Alameda CA 94501 USA Phone: (415) 865-8644 Fax: (415) 865-5255 Hope this helps. John Stiles stiles@uhunixa.uhcc.hawaii.edu From owner-rapd@net.bio.net Mon May 23 23:00:00 1994 Path: biosci!COASMAIL.PHYSICS.DREXEL.EDU!lab_gealt From: lab_gealt@COASMAIL.PHYSICS.DREXEL.EDU ("Lab Gealt") Newsgroups: bionet.molbio.rapd Subject: sequencing PCR products Date: 24 May 1994 15:10:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <199405242206.PAA23055@net.bio.net> NNTP-Posting-Host: net.bio.net REGARDING sequencing PCR products Has anyone tried sequencing their PCR product directly. I have been trying to do it by dideoxy method using Sequenase 2.0 but have only got a smear. My product is double-stranded and I used boiling at 100 C for denaturing it. I take the PCR product, gel purify it, denature it as above and then follow the manufacturer's (USB) instructions to do the sequencing. The labelling reaction was performed on ice instead of RT. Anyone who has faced similar problem or has any other protocol for direct sequencing (without cloning into a plasmid) of PCR product please let me know. Thanx. Vandana saboovm@duvm.ocs.drexel.edu From owner-rapd@net.bio.net Mon May 23 23:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!EU.net!howland.reston.ans.net!vixen.cso.uiuc.edu!phylo!smith From: smith@phylo.life.uiuc.edu (Steven Smith) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD program Date: 24 May 94 19:25:56 GMT Organization: University of Illinois at Urbana Lines: 30 Message-ID: References: <199405041510.IAA26183@net.bio.net> <1994May12.225448.466@ml.csiro.au> NNTP-Posting-Host: phylo.life.uiuc.edu mes@zoo.toronto.edu (Mark Siddall) writes: >In article <1994May12.225448.466@ml.csiro.au> Christopher J. S. Bolch writes: >>In article <199405041510.IAA26183@net.bio.net> , EQUIGENE@UKCC.UKY.EDU >>writes: >>>I would also like information on a computer program for systematics of >>>RAPDs. Thanks. >> >>So would I. I have seen analyses of data with other programs (e.g. PAUP) >>but would like to know if there are other approaches available. >I (think) I have responded to this once already regarding the software >available out there for phylogenetic work. Upon re-reading the query it >appears as though the poster might be under the impression that there is >RAPD-systematics oriented software. There is not. Nor is there any >need as such. Phylogenetic software (e.g., PAUP, Hennig86, PHYLIP etc) .... >I'm probably going to be unpopular around here for having said this. Mark, While it is remarkable easy to make such off the cuff statements, you mislead people in the reliability of character based methods. All suffer considerably from false matches. I suggest you read Hillis et. al. in April 29, 1994 Science (vol 264). Pay particular attention to Figure 7 demonstrating comparing sequence based phylogenetic reconstruction to restriction site data. Sincerely, Steven Smith From owner-rapd@net.bio.net Wed May 25 23:00:00 1994 Path: biosci!agate!spool.mu.edu!torn!nermal.cs.uoguelph.ca!herman.cs.uoguelph.ca!mlagace From: mlagace@uoguelph.ca (Mark C Lagace) Newsgroups: bionet.molbio.rapd Subject: Re: rapd problems Date: 26 May 1994 21:10:55 GMT Organization: University of Guelph Lines: 65 Message-ID: <2s338v$fhg@nermal.cs.uoguelph.ca> References: <2s2sme$slc@canopus.cc.umanitoba.ca> NNTP-Posting-Host: herman.cs.uoguelph.ca X-Newsreader: TIN [version 1.2 PL2] Shaukat Ali (shaukat@cc.umanitoba.ca) wrote: : Sorry if this is a FAQ. We are just starting to do some rapds in our : lab to distinguish between different isolates of pyrenophora tritici repentis. : In our first reaction we tested 6 primers under the following conditions: : dNTPs: 100mM : primer: 0.36 mM : DNA: 25 ng : Mgcl2 1.5 mM : Taq 1.5 U : in a 25 ul final vol. Pcr conditions : 94 1' : 36 1' : 72 2' : But we did not get any bands. None at all. So we played around a bit with : different conc. of Mgcl2 and DNA. We got just one reaction working for us : with just one primer. The other two primers we tested didn't work. The : conditions under which one primer worked were Mgcl2 2.5 mM and 50 ng total : DNA. : We are wondering what we should do now. Any suggestions will be welcome. : Thanks. I have been doing some RAPD work for a few weeks now with P. vulgaris (common white bean) DNA. What I have found to work well is the following reaction mixture: (N.B. ul = microlitre) per 24ng DNA (in 3ul H2O) 3 ul H2O 10 ul 2 mM 4dNTP 2.5 ul 10X PCR buffer (eg Promega or Gibco BRL) 1.5 ul 100mM MgCl2 3.0 ul primer (conc: 10 ug in 1 ml H2O) 2.0 ul 1U/ul Taq The PCR conditions are: 94 C 1 min 94 C 15 sec ---| 36 C 45 sec |-- 35 cycles 72 C 1 min ---| 72 C 5 min then keep at 4 C overnight if necessary. I suggest you read "Optimization of the PCR program for RAPD analysis", by Kangfu Yu and K.P. Pauls, in Nucleic Acids Research Vol 20, No 10, 1992, pp 2606 for a more detailed explanation. (Note, however, that the PCR program has changed slightly from that described in the article). Unfortunately, different DNA works best under different conditions, so trial and error is the only method to determine what will work for your samples. Sincerely, Mark. -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- Mark Lagace, Molecular Biology and Genetics University of Guelph Guelph, Ontario, Canada email: mlagace@uoguelph.ca NSERC Student working for Dr. Peter Pauls, University of Guelph Dept. of Crop Science. -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- From owner-rapd@net.bio.net Wed May 25 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!europa.eng.gtefsd.com!newsxfer.itd.umich.edu!nntp.cs.ubc.ca!unixg.ubc.ca!quartz.ucs.ualberta.ca!tribune.usask.ca!canopus.cc.umanitoba.ca!shaukat From: shaukat@cc.umanitoba.ca (Shaukat Ali) Newsgroups: bionet.molbio.rapd Subject: rapd problems Date: 26 May 1994 19:18:38 GMT Organization: University of Manitoba, Winnipeg, Manitoba, Canada Lines: 34 Message-ID: <2s2sme$slc@canopus.cc.umanitoba.ca> NNTP-Posting-Host: pollux.cc.umanitoba.ca Newsgroups: bionet.molbio.rapd Subject: problems with rapd. HELP! Summary: Expires: Sender: Followup-To: Distribution: bionet.molbio.rapd Organization: University of Manitoba, Winnipeg, Manitoba, Canada Keywords: Sorry if this is a FAQ. We are just starting to do some rapds in our lab to distinguish between different isolates of pyrenophora tritici repentis. In our first reaction we tested 6 primers under the following conditions: dNTPs: 100mM primer: 0.36 mM DNA: 25 ng Mgcl2 1.5 mM Taq 1.5 U in a 25 ul final vol. Pcr conditions 94 1' 36 1' 72 2' But we did not get any bands. None at all. So we played around a bit with different conc. of Mgcl2 and DNA. We got just one reaction working for us with just one primer. The other two primers we tested didn't work. The conditions under which one primer worked were Mgcl2 2.5 mM and 50 ng total DNA. We are wondering what we should do now. Any suggestions will be welcome. Thanks. From owner-rapd@net.bio.net Wed May 25 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!news.cac.psu.edu!news.tc.cornell.edu!travelers.mail.cornell.edu!newsstand.cit.cornell.edu!CU-DIALUP-0232.CIT.CORNELL.EDU!bl14 From: bl14@cornell.edu (Barbara E. Liedl) Newsgroups: bionet.molbio.rapd Subject: Post-doc position: Plant Genetics Date: Thu, 26 May 1994 13:08:07 Organization: Cornell University Lines: 49 Sender: bl14@cornell.edu (Verified) Message-ID: NNTP-Posting-Host: cu-dialup-0232.cit.cornell.edu POSTDOCTORAL POSITION: PLANT GENETICS A post-doctoral position is available immediately at CORNELL UNIVERSITY, Ithaca, NY. This project is to illucidate the molecular genetic control of the biosynthetic pathway for acylsugars, a class of compounds mediating multiple pest resistance in the wild tomato L. pennellii. The research is directed at determining the role of the genes in identified genomic regions on the biosynthesis of acylsugars and using this information in the transfer of acylsugar-mediated multiple pest resistance to cultivated tomato. The research involves all or part of the following: *Use of an existing map and additional populations to determine the role of each of identified regions in acylsugar biosynthesis *Testing to determine whether any additional regions not yet identified affect types or levels of acylsugars produced *Use of lines segregating for only one of the acylsugar-related genomic regions to improve fine mapping of these regions as a precursor to mapped based cloning. The position is funded by a UDSA/NRI genome grant for up to two years, although application for renewal is expected. QUALIFICATIONS: Applicants MUST have a Ph.D. degree by the time of appointment, along with experience in molecular genetics and plant molecular biological techniques and/or biochemistry (DNA extraction, RFLP techniques, PCR methodology, etc.) and training and/or experience in either statistics or quantitative genetics. An interest in plant improvement is also highly desirable. Experience with QTL mapping is not required. APPLICATION: Send curriculum vitae, brief statement of research accomplishments and goals (less than one page), and list of three references (with phone and FAX numbers) to Dr. Martha A. Mutschler Cornell University Dept. of Plant Breeding and Biometry 303 Bradfield Hall Ithaca, NY 14853 tel: 607-255-1660 fax: 607-255-6683 Email: mam13@cornell.edu PLEASE don't contact me, I am only posting the position!!! From owner-rapd@net.bio.net Wed May 25 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: dudy bar-zvi Newsgroups: bionet.molbio.rapd Subject: RAPD of DNA from dry tissue Date: 26 May 1994 15:38:20 +0100 Lines: 16 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2s2c8s$8ee@mserv1.dl.ac.uk> Original-To: rapd@dl.ac.uk Hello netters, I wonder if anyone has the knows if it is possible to get good quality DNA finger print or RAPD from DNA isolated from dried mammalian tissue (e.g. dried remaining of muscle or bone marrow tissues). Thanks for your help, D. Bar-Zvi Life Sciences Ben-Gurion University Beer-Sheva, Israel E-mail: dbarzvi@bgumail.bgu.ac.il From owner-rapd@net.bio.net Thu May 26 23:00:00 1994 Path: biosci!A1.TCH.HARVARD.EDU!FREEMAN_M From: FREEMAN_M@A1.TCH.HARVARD.EDU ("Michael R. Freeman,Ph.D.") Newsgroups: bionet.molbio.rapd Subject: Southerns of RNA RAPDs Date: 27 May 1994 15:10:00 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <199405272209.PAA11149@net.bio.net> NNTP-Posting-Host: net.bio.net Hello Netters, We are entertaining the idea of attempting to confirm differential gene expression data obtained by RNA RAPD (differential display) by using Southern blots of the RAPD products hybridized to 32P-labeled cDNA probes generated by reverse transcription. I did something like this a long time ago (before PCR) to obtain genomic clones corresponding to genes expressed in rat brain but not in other tissues. The trick in this case was to use Cot 1 DNA as competitor to suppress hybridization to repetitive DNA sequences. I'd like any comments/predictions/bets/advice about this approach, specifically whether it would be advisable to generate the cDNA primer extension probe from total RNA or polyA+ RNA, and what conditions for probe labeling or hybridization might be preferable. Thanks in advance. Michael Freeman Children's Hospital, Boston freeman_m@a1.tch.harvard.edu From owner-rapd@net.bio.net Thu May 26 23:00:00 1994 Path: biosci!agate!library.ucla.edu!europa.eng.gtefsd.com!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Wirote Tuntiwechapikul Newsgroups: bionet.molbio.rapd Subject: Subscription Date: 27 May 1994 08:58:50 +0100 Lines: 7 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2s497q$s46@mserv1.dl.ac.uk> Original-To: rapd@dl.ac.uk subscribe Wirote Tuntiwechapikul Department of Biochemistry Faculty of Medicine Chiangmai University Chiangmai, Thailand. From owner-rapd@net.bio.net Mon May 30 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!news.cac.psu.edu!news.tc.cornell.edu!travelers.mail.cornell.edu!newsstand.cit.cornell.edu!CU-DIALUP-0312.CIT.CORNELL.EDU!bl14 From: bl14@cornell.edu (Barbara E. Liedl) Newsgroups: bionet.molbio.rapd Subject: Re: What type of gel is best for RAPD? Date: Tue, 31 May 1994 08:40:38 Organization: Cornell University Lines: 13 Sender: bl14@cornell.edu (Verified) Message-ID: References: NNTP-Posting-Host: cu-dialup-0312.cit.cornell.edu >Subject: What type of gel is best for RAPD? We use a 2% agarose gel to separate our RAPDs. In addition, we use regular agarose in our lab. Several labs have used other combinations, so I tried using NuSieve agarose and 1/2 NuSieve:1/2 regular agarose and found no differences. So we use regular agarose (it is lots cheaper than NuSieve) and find it separates our samples (300-1500 bp) well. If you look in Maniatis (or Sambrook), 2% gels work well for separating 0.1 - 3 kb. Hope this helps. Barbara Liedl Cornell University Dept. of Plant Breeding From owner-rapd@net.bio.net Mon May 30 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!ihnp4.ucsd.edu!munnari.oz.au!metro!angis.su.oz.au!kanemats From: kanemats@angis.su.oz.au () Subject: What type of gel is best for RAPD? Message-ID: Sender: news@ucc.su.OZ.AU Nntp-Posting-Host: morgan.angis.su.oz.au Reply-To: kanemats@angis.su.oz.au () Organization: ANGIS, The University of Sydney, Australia Date: Tue, 31 May 1994 07:08:02 GMT Lines: 8 ÿWPCÛ Ákk* Many thanks Francisca Supple kanemats@morgan.angis.su.oz.au From owner-rapd@net.bio.net Mon May 30 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!howland.reston.ans.net!wupost!crcnis1.unl.edu!newsfeed.ksu.ksu.edu!moe.ksu.ksu.edu!osuunx.ucc.okstate.edu!vms.ucc.okstate.edu!shufran From: shufran@vms.ucc.okstate.edu Subject: temp. slopes Message-ID: <1994May31.100413.1@vms.ucc.okstate.edu> Lines: 26 Sender: news@osuunx.ucc.okstate.edu (USENET News System) Nntp-Posting-Host: vms.ucc.okstate.edu Organization: Oklahoma State University Computer Center Date: Tue, 31 May 1994 15:04:13 GMT I've been lurking for a few days now and here is my first attempt to post a message. I have a question conerning annealing and extension times in RAPD-PCR. I've just started doing some amplifications with aphid DNA and have tried 2 programs. They are bascially the same, except for the time of heating from annealing temp (37) to extension temp (72). The first program has the temp increase as fast as possible. The second uses a slope with a 1 dergree increase per 8 sec (I got this from Black et al 1992). My results were quite different. There was one band well amplified that was common to both, but the long ramp time had more bands of a larger size. The short ramp time had smaller size products. Bothe progams yielded products which were consistently amplified with the same template, but the products differed greatly between the two programs. Q.1. If I am to continue optimizing, which program should I follow? Q.2. Is there an advantage or reason why the long slope time would be better? Any advice would be appreciated. Kevin A Shufran USDA-ARS Stillwater, OK From owner-rapd@net.bio.net Tue May 31 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!sunic!EU.net!uunet!senior.nectec.or.th!mars.mahidol.ac.th!mucc!scbst From: scbst@mucc.mahidol.ac.th (Burachai Sonthayanon - SCBC) Newsgroups: bionet.molbio.rapd Subject: Reducing reaction volume to 10 ul cause problem. Suggestion ? Date: 1 Jun 1994 11:36:00 GMT Organization: Mahidol University, Thailand. Lines: 16 Message-ID: <2shrr0$jbr@mars.mahidol.ac.th> NNTP-Posting-Host: mucc.mahidol.ac.th X-Newsreader: TIN [version 1.2 PL2] Dear colleagues, I need some advise from you. Since we started to survey a no. of DNA samples by reducing rapd reaction volume to 10 ul per tube lately, we started to get problem : in many of the reactions, rapd failed to produced bands. We have never had problem when using 25 ul reaction during the past 6 months. My condition was 10 mM Tris-HCl 8.3, 50 mM KCl, 2 mM MgCl2, 0.001% gelatin, 200 uM dNTPs, 0.2 uM primer, 10 ng rice genomic DNA, 0.2 U AmpliTaq, running 35 cycles of 94 C (15 sec), 36 C (45 sec), 72 C (90 sec). We did not use hot start. I would appreciate all comments. Thanks. Burachai Sonthayanon From owner-rapd@net.bio.net Tue May 31 23:00:00 1994 Path: biosci!DEAKIN.EDU.AU!huangbx From: huangbx@DEAKIN.EDU.AU Newsgroups: bionet.molbio.rapd Subject: How do you manage your working sample DNA in RAPD? Date: 31 May 1994 20:29:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <199406010329.NAA07640@sol.ccs.deakin.edu.au> NNTP-Posting-Host: net.bio.net Hello netters, I am having difficulty in the Abalone DNA. Once the DNA put into -20 or -70 or 4C frige, there was NO RAPD bands immediately. I presume that the DNA was broken down during the frozen processing. Normally how do you arrange the DNA if you will use the same DNA in several RAPD reactions? Heart-felt thanks. With regards. Bixing Huang huangbx@deakin.edu.au From owner-rapd@net.bio.net Tue May 31 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!msuinfo!harbinger.cc.monash.edu.au!news.cs.su.oz.au!metro!angis.su.oz.au!kanemats From: kanemats@angis.su.oz.au () Subject: What type of gel is best for RAPD: 2? Message-ID: Sender: news@ucc.su.OZ.AU Nntp-Posting-Host: morgan.angis.su.oz.au Reply-To: kanemats@angis.su.oz.au () Organization: ANGIS, The University of Sydney, Australia Date: Wed, 1 Jun 1994 04:43:19 GMT Lines: 10 HELP! (that's just a small "help"). I'm just beginning to use RAPD PCR. My PCR is going O.K. but I'm not getting good resolution on my 6% polyacrylamide gels/TBEgels (non-denaturing). The band sizes I'm interested in are 1kb or smaller (100bp). So what I want to know is what sort of gel is going to be good for this? Many thanks Francisca Supple kanemats@morgan.angis.su.oz.au From owner-rapd@net.bio.net Tue May 31 23:00:00 1994 Path: biosci!CORNELL.EDU!MAL5 From: MAL5@CORNELL.EDU (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: Conversion of RAPD primers to co-dominant markers? Date: 1 Jun 1994 11:36:17 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: <199406011836.OAA06769@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net In response to Ian, There are several articles on cleaved amplified polymorphic sequences (CAPS), amplification fragment length polymorphisms (AFLPs), sequence characterized amplified regions (SCARs) and may be some others that could be used in converting RAPD markers to co-dominant markers. If you need references about any of these, please let me know. Muhammad A. Lodhi Cornell University ============================================================================= >Hello again, > >Does anyone know how to or has information concerning converting >RAPD markers to co-dominant markers? > >Thanks for you help in advance. > >Murrayian@phibred.com > >Ian Murray From owner-rapd@net.bio.net Tue May 31 23:00:00 1994 Path: biosci!CORNELL.EDU!MAL5 From: MAL5@CORNELL.EDU (Muhammad A. Lodhi) Newsgroups: bionet.molbio.rapd Subject: Re: How do you manage your working sample DNA in RAPD? Date: 1 Jun 1994 11:28:41 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Distribution: world Message-ID: <199406011828.OAA04773@postoffice.mail.cornell.edu> NNTP-Posting-Host: net.bio.net In response to Bixing Huang, I have kept grape DNA for more than 2 years at -20C without any problem. I saw problem in RAPD amplification when the color of the extracted DNA was not clear before storage. What I normally do is, just after extraction I aliquat DNA into several small tubes (25-100 ng/uL dH2O) and keep it at -20C and the rest of the concentrated stock is also kept at the same temp. I keep on using the aliquat tubes without thawing the conc. stock. This way if one tube goes bad I have others to work with and also the stock tube. This procedure did very well for grapes hope it works with other species. Muhammad A. Lodhi >Hello netters, > >I am having difficulty in the Abalone DNA. Once the DNA put into -20 or -70 >or 4C frige, there was NO RAPD bands immediately. I presume that the DNA >was broken down during the frozen processing. Normally how do you arrange >the DNA if you will use the same DNA in several RAPD reactions? > >Heart-felt thanks. > >With regards. > >Bixing Huang >huangbx@deakin.edu.au From owner-rapd@net.bio.net Tue May 31 23:00:00 1994 Path: biosci!agate!ihnp4.ucsd.edu!swrinde!pipex!uknet!daresbury!not-for-mail From: Louis van de Zande Newsgroups: bionet.molbio.rapd Subject: Re: Reducing reaction volume to 10 ul cause problem. Sugges Date: 1 Jun 1994 15:21:58 +0100 Organization: Department of Biology, RUGroningen Lines: 18 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2si5i6$i18@mserv1.dl.ac.uk> Reply-To: ZANDELPW@BIOL.RUG.nl Return-receipt-to: ZANDELPW@BIOL.RUG.NL Original-To: rapd@dl.ac.uk > Dear colleagues, > I need some advise from you. Since we started to survey a no. of DNA > samples by reducing rapd reaction volume to 10 ul per tube lately, we > started to get problem : in many of the reactions, rapd failed to > produced bands. We have never had problem when using 25 ul reaction > during the past 6 months. > > My condition was 10 mM Tris-HCl 8.3, 50 mM KCl, 2 mM MgCl2, > 0.001% gelatin, 200 uM dNTPs, 0.2 uM primer, 10 ng rice genomic DNA, 0.2 > U AmpliTaq, running 35 cycles of 94 C (15 sec), 36 C (45 sec), 72 C (90 > sec). We did not use hot start. > > I would appreciate all comments. Thanks. > > Burachai Sonthayanon > Well, switch back to 25 ul and life is beautiful again. From owner-rapd@net.bio.net Tue May 31 23:00:00 1994 Path: biosci!UNIXG.UBC.CA!hobbs From: hobbs@UNIXG.UBC.CA Newsgroups: bionet.molbio.rapd Subject: UBC DDRT-PCR Kit Date: 1 Jun 1994 16:36:07 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 57 Sender: daemon@net.bio.net Distribution: world Message-ID: <9406012332.AA25824@unixg.ubc.ca> NNTP-Posting-Host: net.bio.net We were recently asked to make up the primers required to perform DDRT-PCR (or "mRNA RAPD") by colleagues here at UBC. The cost for the 38 primers they required was fairly substantial based on (even our modest) custom primer rates, and in view of the wide interest in the technique, it seemed a reasonable idea to construct an affordable "Kit", in the same way that we have been putting together Primer kits for RAPD work, and to offer them to the community in the same way. So, I have a modest number of kits for DDRT-PCR work available, if anyone is interested. If demand is significant, no doubt we could make more. Details are as follows: The UBC primer set for Differential Display Reverse Transcription PCR contains 30 nmoles each of 12 primers of general sequence d[(T11)VN] and 3 nmoles each of 26 primers numbered mRNA 1 through mRNA 26. These latter sequences are the 26 "optimised" sequences described by Bauer et al. in Nucleic Acids Research, 1993, 21, 4270-4280. These quantities should suffice to perform DDRT-PCR in 20 tissue samples, according to the published protocols (e.g. Bauer et al., and Chang and Pardee). The primers were purified by elution with Tris/EDTA buffer through NAP-5 drip columns. The yields were determined spectrophotometrically. Aliquots corresponding to the quantities indicated above were made near the bottom of sterile Eppendorf tubes and air dried. In the case of the mRNA 1-26 primers, the volume aliquotted was 10 microlitres; for the d[(T11)VN] primers the volume of the aliquot varied but was always less than 60 microlitres. All manipulations were conducted in a sterile laminar flow hood. These primers are functioning well in laboratories at U.B.C. The price of the UBC DDRT-PCR Primer Set is Can.$400.00 or US$ 315.00, plus carriage: by airmail, US$4 to most parts of the world, or, for courier delivery $7Can. for shipments within Canada, US$ 11 for shipments to the US, US$16 to the UK, US$16 to Western Europe, US$17 to Australasia, US$17 to the Far East, US$25 to China, South America, and Africa. I can accept purchase orders by fax, mail or e-mail. Orders must specify your mailing address, the name of the person to be billed (i.e. grant holder), your preference as to shipment via courier or air mail, and either an official Purchase Order Number or your cheque in advance (made out to "University of British Columbia") and sent to me at the address below. If an order is phoned in, a copy of the P.O. for our records is still required. Our RAPD primer sets #1 through #8 are still available: stocks are low on some of them , but we are restocking imminently, and intend to maintain their availability. Anyone requiring details of these is requested to contact me by e-mail, phone or fax. For those who are wondering, we are presently at work on Primer Set #9: it is designed to target microsatellite sequences. I will give more details when the set is available. John Hobbs Dr. John Hobbs Nucleic Acid - Protein Service (NAPS) Unit Biotechnology Laboratory Room 237, Wesbrook Building 6174 University Boulevard University of British Columbia Vancouver, V6T 1Z3 Canada. FAX (604)822-5437 or (604)822-0676; Tel. (604)822-6373 From owner-rapd@net.bio.net Tue May 31 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: Conversion of RAPD primers to co-dominant markers? Date: 1 Jun 1994 06:53:34 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <9406011358.AA25934@gw1.phibred.com> NNTP-Posting-Host: net.bio.net Hello again, Does anyone know how to or has information concerning converting RAPD markers to co-dominant markers? Thanks for you help in advance. Murrayian@phibred.com Ian Murray