From owner-rapd@net.bio.net Thu Jul 01 23:00:00 1993 Path: biosci!YVAX.BYU.EDU!anderswr From: anderswr@YVAX.BYU.EDU (W. Ralph Andersen) Newsgroups: bionet.molbio.rapd Subject: To Toby Bradshaw ("pairs of primers") Message-ID: <01H023Q4NWVO8WYWM6@yvax.byu.edu> Date: 2 Jul 93 17:04:15 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 5 Toby: I tried your E mail address and it did not work for me. Would you kindly send me a protocol? Thanks. Ralph A. From owner-rapd@net.bio.net Thu Jul 01 23:00:00 1993 Path: biosci!uwm.edu!spool.mu.edu!howland.reston.ans.net!noc.near.net!uunet!mcsun!ieunet!tcdcs!vax1.tcd.ie!rpowell From: rpowell@vax1.tcd.ie Newsgroups: bionet.molbio.rapd Subject: biogeographical study Message-ID: <1993Jul2.151014.1@vax1.tcd.ie> Date: 2 Jul 93 15:10:14 GMT Sender: usenet@cs.tcd.ie (NN required at ashe.cs.tcd.ie) Organization: Trinity College Dublin Lines: 9 Nntp-Posting-Host: vax1.tcd.ie Netters, We are about to conduct some RAPD expts on a bacterial fish pathogen. Our problem basically is how to best analyse the results when they (presumably) come. We are interested in the biogeographical distributions of these organisms. Are we too optimistic to assume we can source an outbreak of disease doing RAPDs??? Thanks in advance..... Rich From owner-rapd@net.bio.net Mon Jul 05 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: H.F.J.Bligh@vme.ccc.nottingham.ac.uk Newsgroups: bionet.molbio.rapd Subject: mRNA RAPD Message-ID: <1993Jul6.173823.21641@gserv1.dl.ac.uk> Date: 6 Jul 93 15:58:26 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 11 Original-To: rapd@uk.ac.daresbury Has anyone out there heard of a technique that may or may not be called message mapping? My boss keeps talking about it but is unable to supply any references or methodologies for it. The theory goes that you do a reverse transcriptase reaction on some mRNA and then do RAPD analysis and any differences are caused by differences intranscribed genes. Personally I can see a few problems with this as your chances of getting a hit let alone a difference must be greatly reduced, but then that is probably the whole idea. Any references or experience/advice on this would be welcomed as I would like to try it. thanks Frankie From owner-rapd@net.bio.net Mon Jul 05 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: mRNA RAPD Message-ID: <2770F03736F@uamercury.uark.edu> Date: 6 Jul 93 21:00:35 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 33 >To: rapd@net.bio.net >From: H.F.J.Bligh@vme.ccc.nottingham.ac.uk >Subject: mRNA RAPD >Date: 6 Jul 93 15:58:26 GMT >Has anyone out there heard of a technique that may or may not be called >message mapping? My boss keeps talking about it but is unable to supply any >references or methodologies for it. The theory goes that you do a reverse >transcriptase reaction on some mRNA and then do RAPD analysis and any difference >s >are caused by differences intranscribed genes. Personally I can see a few proble >ms >with this as your chances of getting a hit let alone a difference must be >greatly reduced, but then that is probably the whole idea. >Any references or experience/advice on this would be welcomed as I would >like to try it. >thanks >Frankie The technique you appear to be refering to was described as `differential display' in the paper appearing in Science: Liang and Pardee (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257 (Aug 14) 967- 971. An elegant technique. Does anyone out there have experience with this. We are just starting to do some differential displays. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Tue Jul 06 23:00:00 1993 Path: biosci!MED.PITT.EDU!rapr From: rapr@MED.PITT.EDU (Robert Preston) Newsgroups: bionet.molbio.rapd Subject: Re: UV filter for capturing Ethidium stain gels Message-ID: <9307072159.AA25473@pluto.med.pitt.edu> Date: 7 Jul 93 21:59:03 GMT References: <9307071945.AA18484@net.bio.net> Sender: daemon@net.bio.net Distribution: bionet Lines: 24 > retrieval. We are using a U.V. transilluminator (300 nm) to fluoresce (at 590 > mn) and thereby detect Ethidium Bromide. Our problem is that the image is not > as distinct as traditional polaroid prints because of the sensitivity of the > video camera to UV light. We need a filter that will mask the UV (300 nm) > while allowing the passage of the red-orange Ethidium Bromide fluorescence > (590 nm). We are aware of band pass filters (aka interference filters) with > 600 nm transmittance peak and a 70 nm half-maximal bandwidth but the price is > awfully steep ($181.00 from Corion, Holliston, MA) for a 2-inch (40.5 mm) > filter. Does anyone out there know of any other solutions or have an > interference filter they would like to sell below the above price? > The labs I'm acquainted with have all used relatively inexpensive photo- graphic filters ("Wratten 22A" per Maniatis et al.) to block all but red in EtBr stained gels, even with polaroid lenses. Newcomers sometimes remove the filter, for one reason or another, leaving the next user at a loss to explain why his gels look so good but photograph so lousy. I've often wondered whether an "EtBr-DNA-specific" fancy emission interference bandpass would be any better than the wratten 22A, but never seemed to be sufficiently sensitivity-limited to try to find out. I'll be interested to see whether anyone has significantly improved on the 22A performance. Robert A. Preston UPMC Dept. Pathology rapr@med.pitt.edu From owner-rapd@net.bio.net Tue Jul 06 23:00:00 1993 Path: biosci!YVAX.BYU.EDU!anderswr From: anderswr@YVAX.BYU.EDU (W. Ralph Andersen) Newsgroups: bionet.molbio.rapd Subject: (none) Message-ID: <01H097QN5I0Y8X06KK@yvax.byu.edu> Date: 7 Jul 93 19:14:52 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 4 A piece of academic trivia: Someone has "coped" our acronym. I "pinged" Current Contents for t = RAPD and got an article on "relative afferent pupillary defect"!!!! From owner-rapd@net.bio.net Tue Jul 06 23:00:00 1993 Path: biosci!uwm.edu!math.ohio-state.edu!magnus.acs.ohio-state.edu!rgooding From: rgooding@magnus.acs.ohio-state.edu (Robert W Gooding) Newsgroups: bionet.molbio.rapd Subject: UV filter for capturing Ethidium stain gels Message-ID: <21f96b$k6n@charm.magnus.acs.ohio-state.edu> Date: 7 Jul 93 19:46:19 GMT Organization: The Ohio State University Lines: 19 NNTP-Posting-Host: bottom.magnus.acs.ohio-state.edu We are setting up a laboratory photodocumentation device to 'capture' images of Ethidium stain gels and direct them to a microcomputer for storage and retrieval. We are using a U.V. transilluminator (300 nm) to fluoresce (at 590 mn) and thereby detect Ethidium Bromide. Our problem is that the image is not as distinct as traditional polaroid prints because of the sensitivity of the video camera to UV light. We need a filter that will mask the UV (300 nm) while allowing the passage of the red-orange Ethidium Bromide fluorescence (590 nm). We are aware of band pass filters (aka interference filters) with 600 nm transmittance peak and a 70 nm half-maximal bandwidth but the price is awfully steep ($181.00 from Corion, Holliston, MA) for a 2-inch (40.5 mm) filter. Does anyone out there know of any other solutions or have an interference filter they would like to sell below the above price? We would appreciate any information. Thanks Rob Gooding From owner-rapd@net.bio.net Tue Jul 06 23:00:00 1993 Path: biosci!UNLINFO.UNL.EDU!rfrench From: rfrench@UNLINFO.UNL.EDU (roy french) Newsgroups: bionet.molbio.rapd Subject: Re: UV filter for capturing Ethidium stain gels Message-ID: <9307072300.AA02161@unlinfo.unl.edu> Date: 7 Jul 93 23:00:58 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 30 > > > We are setting up a laboratory photodocumentation device to 'capture' images > of Ethidium stain gels and direct them to a microcomputer for storage and > retrieval. We are using a U.V. transilluminator (300 nm) to fluoresce (at 590 > mn) and thereby detect Ethidium Bromide. Our problem is that the image is not > as distinct as traditional polaroid prints because of the sensitivity of the > video camera to UV light. We need a filter that will mask the UV (300 nm) > while allowing the passage of the red-orange Ethidium Bromide fluorescence > (590 nm). We are aware of band pass filters (aka interference filters) with > 600 nm transmittance peak and a 70 nm half-maximal bandwidth but theprice is > awfully steep ($181.00 from Corion, Holliston, MA) for a 2-inch (40.5 mm) > filter. Does anyone out there know of any other solutions or have an > interference filter they would like to sell below the above price? > > We would appreciate any information. > > Thanks > > Rob Gooding We are using a CCD camera to capture Ethidium stained gel images using an ordinary 'UV (0)' filter. Even with 6-second exposures the transilluminator surface appears black, so I guess the UV filter is enough. My guess is that the UV filter will also be all you need for a video camera as well. Roy French USDA, ARS, Univ. of Nebraska rfrench@unl.edu From owner-rapd@net.bio.net Sat Jul 10 23:00:00 1993 Path: biosci!agate!howland.reston.ans.net!darwin.sura.net!news.duc.auburn.edu!ducvax.auburn.edu!nichoki From: nichoki@ducvax.auburn.edu Newsgroups: bionet.molbio.rapd Subject: Are RAPDs REALLY reproducible? Message-ID: <1993Jul11.090519.1@ducvax.auburn.edu> Date: 11 Jul 93 14:05:19 GMT Sender: usenet@news.duc.auburn.edu (News Account) Organization: Auburn University, AL Lines: 22 Nntp-Posting-Host: ducvax I have a question conerning the reproducibility of RAPD banding patterns. Of the articles that I have read that specifically state that reproducible results were obtained, none state just exactly HOW they went about replicating their results. By this I mean, did they isolate DNA from one tissue sample from an organism, and use that sample to run RAPDS several times, or did they isolate DNA from say, five tissue samples from the same individual organism and then run RAPDs on those, getting the same banding pattern every time? It seems to me that this distinction could possibly produce slightly different results, as depending upon your DNA extraction method, you might shear your dna, leaving you with fragments, so that your banding patterns might differ. Am I way off base here? Does anyone know how they are reproducing their results in the literature? Replies are very welcome as this would help my thesis out no end. KIrsten NIcholson Auburn University exit yb From owner-rapd@net.bio.net Sun Jul 11 23:00:00 1993 Path: biosci!uwm.edu!ux1.cso.uiuc.edu!howland.reston.ans.net!noc.near.net!nic.umass.edu!hamp.hampshire.edu!ycheah From: ycheah@hamp.hampshire.edu Newsgroups: bionet.molbio.rapd Subject: Re: Are RAPDs REALLY reproducible? Message-ID: <1993Jul11.201431.1@hamp.hampshire.edu> Date: 12 Jul 93 00:14:31 GMT References: <1993Jul11.090519.1@ducvax.auburn.edu> Organization: Hampshire College Lines: 22 NNTP-Posting-Host: hamp.hampshire.edu I do RAPDs on inbred mice. RAPDs are reproducible. DNA from different mouse of the same strain shows the same banding patterns so much so that I can tell the diffrent strains apart just from their banding patterns. This have worked over 2.5 years now using DNA from different mice of the same strain. However, there are many ways to fall of this bandwagon. The concentration of the DNA needs to be measured accurately, and a consistant amount used everytime. The volume of the DNA used must also be consistant because the EDTA in the TE can alter the amount of free Mg2+ in your PCR reaction. Of course, the PCR machine, buffer (ie gelatin or not), enzyme etc must be the same each time. Due to the low temperature of RAPD PCR, one must be very careful to avoid contamination. (More so than SSLP PCR.) Also, the method of DNA prep is crucial (for reasons I haven't quite figured out yet.) DNA from spleen and liver works, but DNA from tails don't. Probably the amounts of salts co-precipitating with the DNA. It is for the above caviets that RAPDs lost its appeal to the mouse community. We now mostly use the SSLP (simple sequence length polymorphisms, or CA repeats polymorphisms). Hope this helps. YinChai From owner-rapd@net.bio.net Sun Jul 11 23:00:00 1993 Path: biosci!esvax.dnet.dupont.com!rafalski From: rafalski@esvax.dnet.dupont.com Newsgroups: bionet.molbio.rapd Subject: ME Review appeared! Message-ID: <9307121450.AA06340@esds01.es.dupont.com> Date: 12 Jul 93 14:50:06 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 12 I am happy to report that an extensive discussion of RAPD techniques written by us two years ago or so, finally appeared (Williams, Hanafey, Rafalski and Tingey, Methods in Enzymology vol. 218, p.704-740). The review circulated widely in a preprint form, but the final version contains some additional material, esp. discussion of the choince of mapping populations. By the way, all three volumes of ME devoted to Recombinant DNA (216, 217, 218) appeare almost simultaneously and contain a lot of useful information. Antoni Rafalski From owner-rapd@net.bio.net Sun Jul 11 23:00:00 1993 Path: biosci!esvax.dnet.dupont.com!rafalski From: rafalski@esvax.dnet.dupont.com Newsgroups: bionet.molbio.rapd Subject: Reprint requests Message-ID: <9307121926.AA12609@esds01.es.dupont.com> Date: 12 Jul 93 19:26:34 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 9 To those of you who requested a reprint of the Methods in Enzymology article: Unfortunately we do not have reprints, and copyright laws prevent us from large scale copying of the article for distribution. However, Methods in Enzymology is a widely available book series, and your library should have no problem procuring a copy for you. If we ever get reprints (I am not sure what ME policy is), I will announce their availability. Antoni Rafalski From owner-rapd@net.bio.net Mon Jul 12 23:00:00 1993 Path: biosci!MBCRR.HARVARD.EDU!swiss From: swiss@MBCRR.HARVARD.EDU (Karen Swisshelm) Newsgroups: bionet.molbio.rapd Subject: Re: Reprint requests Message-ID: <9307132109.AA27243@mbcrr.harvard.edu> Date: 13 Jul 93 21:09:30 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 1 From owner-rapd@net.bio.net Mon Jul 19 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: dec@aber.ac.uk (David Edward Cooke) Newsgroups: bionet.molbio.rapd Subject: RAPD info services Message-ID: <1993Jul20.140614.12195@gserv1.dl.ac.uk> Date: 20 Jul 93 14:02:42 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 10 Original-To: rapd@uk.ac.daresbury Please could you log me into the RAPD bulletin board to recieve current information? Thank you. David Cooke University of Wales, Aberystwyth, Dyfed SY23 3DD From owner-rapd@net.bio.net Mon Jul 19 23:00:00 1993 Path: biosci!RAVEL.UDEL.EDU!pgaffney From: pgaffney@RAVEL.UDEL.EDU (Patrick Gaffney) Newsgroups: bionet.molbio.rapd Subject: Sequencing query Message-ID: Date: 20 Jul 93 14:54:16 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 12 We'd like to sequence various PCR products, and are wondering whether to use nonradioactive detection methods, and if so, which ones. Secondly, we'd like to hear opinions on the route from PCR product to sequence - i.e., direct sequencing of DS product, SS amplification, cloning, etc. Your advice to novices greatly appreciated. Patrick Gaffney | pgaffney@ravel.udel.edu College of Marine Studies | (302) 645-4364 University of Delaware | FAX: (302) 645-4028 Lewes, DE 19958 | - This space for rent - From owner-rapd@net.bio.net Mon Jul 19 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: WOLFF@VOEDING.TNO.nl Newsgroups: bionet.molbio.rapd Subject: thermocycler Message-ID: <1993Jul20.184053.24501@gserv1.dl.ac.uk> Date: 20 Jul 93 16:18:00 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 15 X-Envelope-To: rapd@daresbury.AC.UK Content-Identifier: thermocycler Original-To: rapd@uk.ac.daresbury Content-Transfer-Encoding: 7BIT X-Vms-To: tnoit1::in%"rapd@daresbury.ac.uk" We are planning to buy a new thermocycler. We are thinking of a MJ Research PTC 100, 96 wells, V-shaped. We will use it for RAPDs and for PCR with specific primers. It is not expensive and I know of one laboratory that is very satisfied with the results. Are there people that have experience with this machine and the longevity of the Peltier element? Is it advisable to buy the heated lid too? Any advise on duration of the cycling times? I would be grateful for any information. Kirsten Wolff TNO, Dept. of Microbiology PO Box 360 3700 AJ Zeist, the Netherlands e-mail: wolff@voeding.tno.nl From owner-rapd@net.bio.net Mon Jul 19 23:00:00 1993 Path: biosci!daresbury!daresbury!news From: pcm@aber.ac.uk (Paul Metcalfe) Newsgroups: bionet.molbio.rapd Subject: RAPD info service Message-ID: <1993Jul20.140614.12140@gserv1.dl.ac.uk> Date: 20 Jul 93 13:58:32 GMT Sender: list-admin@daresbury.ac.uk Distribution: bionet Lines: 1 Original-To: rapd@uk.ac.daresbury PLEASE PROVIDE INFORMATION ON THE E-MAIL RAPD BULLETIN BOARD TO PCM From owner-rapd@net.bio.net Tue Jul 20 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!mcsun!uunet!elroy.jpl.nasa.gov!usc!usc!not-for-mail From: xuxianz@hsc.usc.edu (Xuxian Zhang) Newsgroups: bionet.molbio.rapd Subject: coverage of genome using rapd Message-ID: <22jqr3$mer@hsc.usc.edu> Date: 21 Jul 93 16:28:19 GMT Sender: xuxianz@hsc.usc.edu Organization: University of Southern California, Los Angeles, CA Lines: 1 NNTP-Posting-Host: hsc.usc.edu Does anyone know how to calculate the coverage of genome using rapd. It seems to me the result of rapd depends on the luck. You just randomly pick up some primers and pcr it. The result may just show very small part of the genome. From owner-rapd@net.bio.net Tue Jul 20 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: thermocycler Message-ID: <3D919BF3716@uamercury.uark.edu> Date: 21 Jul 93 15:00:50 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 26 >To: rapd@net.bio.net >From: WOLFF@VOEDING.TNO.nl >Subject: thermocycler >Date: 20 Jul 93 16:18:00 GMT >We are planning to buy a new thermocycler. We are thinking of a MJ Research >PTC 100, 96 wells, V-shaped. We will use it for RAPDs and for PCR with >specific primers. It is not expensive and I know of one laboratory that is >very satisfied with the results. >Are there people that have experience with this machine and the longevity >of the Peltier element? >Is it advisable to buy the heated lid too? >Any advise on duration of the cycling times? >I would be grateful for any information. > >Kirsten Wolff We tried out the MJ cycler without the heated lid. We used it for RAPDs and PCRs. We had no problems and got excellent results in RAPDs when we slowed down the heat ramping for the first 10 degrees from 35 to 45 to about 0.25 degrees per second. With fast ramps it didn't work. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Tue Jul 20 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: coverage of genome using rapd Message-ID: <3DEC1A1585B@uamercury.uark.edu> Date: 21 Jul 93 20:40:10 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 18 >To: rapd@net.bio.net >From: xuxianz@hsc.usc.edu (Xuxian Zhang) >Subject: coverage of genome using rapd >Date: 21 Jul 93 16:28:19 GMT >Does anyone know how to calculate the coverage of genome using rapd. It seems to > me the result of rapd depends on the luck. You just randomly pick up some prime >rs and pcr it. The result may just show very small part of the genome. > Since each band represents a `novel' region of the genome, ten bands are ten loci. If one has 30 primers with an average of 10 bands that is 300 loci. For a genome of 3 billion b.p. that would be one marker every 1 million base pairs (approximately in the centiMorgan range). Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Tue Jul 20 23:00:00 1993 Path: biosci!esvax.dnet.dupont.com!scolnipa From: scolnipa@esvax.dnet.dupont.com Newsgroups: bionet.molbio.rapd Subject: RAPD Message-ID: <9307211724.AA02354@esds01.es.dupont.com> Date: 21 Jul 93 17:24:52 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 6 In re Paul Miller's article, we recently had to move our reactions to a Perkin Elmer 9600 from a toaster over cycler. We got good results by using a thermocouple to mimic conditions in the 9600. Previous attempts were not very satisfactory. I would suggest that in future papers people include thermocouple profiles of their RAPD reactions because the same program in different instruments may be quite different. From owner-rapd@net.bio.net Tue Jul 20 23:00:00 1993 Path: biosci!daresbury!doc.ic.ac.uk!pipex!sunic!uunet!cs.utexas.edu!convex!constellation!aardvark.ucs.uoknor.edu!aardvark.ucs.uoknor.edu!broe From: broe@aardvark.ucs.uoknor.edu (Bruce Roe) Newsgroups: bionet.molbio.rapd Subject: Re: Sequencing query Message-ID: <21JUL199308564802@aardvark.ucs.uoknor.edu> Date: 21 Jul 93 13:56:00 GMT References: Distribution: bionet Organization: University of Oklahoma - University Computing Services Lines: 66 Nntp-Posting-Host: loopback.uoknor.edu News-Software: VAX/VMS VNEWS 1.41 Lines: 66 In article , pgaffney@RAVEL.UDEL.EDU (Patrick Gaffney) writes... >We'd like to sequence various PCR products, and are wondering whether to >use nonradioactive detection methods, and if so, which ones. Secondly, >we'd like to hear opinions on the route from PCR product to sequence - >i.e., direct sequencing of DS product, SS amplification, cloning, etc. >Your advice to novices greatly appreciated. > >Patrick Gaffney | pgaffney@ravel.udel.edu >College of Marine Studies | (302) 645-4364 >University of Delaware | FAX: (302) 645-4028 >Lewes, DE 19958 | - This space for rent - > Hi all, This has been debated for a while and the answer seems to be that some folks get all of the above to work and some get none to work, while others get one to work, and that's from experiences in my lab. My 2cents worth is that the majority of problems with sequencing off pcr products mainly depends on the quality of the product itself and herein lies the difficulty. Direct sequencing of DS product is the most difficult to do because you may not get the template/primer concentrations optimal, have nicked pcr products, etc. Second most difficult is to do something like assymetric pcr or a nested pcr reaction. Here too, the quality of the sequence data depends greatly on the quality of the product (remember too that whatever purification step you use might cause product degredation). The most efficient way that works well for most (in my lab), is to do the pcr reactions, clean up the product some way (agarose gel and/or phenol) and then CLONE it into M13. Cloning into pUC or other double stranded vectors has it's own problems (i.e. sequencing off double stranded templates is more difficult than sequencing off single stranded templates). Thus, my overall advice is to KEEP IT SIMPLE. If you are experienced with single stranded template sequencing and get good reads, them clone into M13 (or derivatives). If double stranded templates work for you then clone into the appropriate vectors for that approach. If you get really good pcr products by any method, then try direct sequencing off them. If you are NOT very experienced in sequencing, do some control experiments first. Try simple experiments with know templates that others in your lab or down the hall find work well. Try isolating single stranded templates and get that to work first, then move on to double stranded templates and finally to pcr products. Remember we learned to "crawl before we walked, and walked before we ran". Three final notes: One - when sequencing off double stranded templates, if you are using a label that is incorporated into the synthesized strand (alpha-35S dATP for example), will give high background if the template is nicked. Best results with nicked templates usually are obtained with 5'-end labeled primers (kinase catalyzed labeling with gamma labeled 32P rATP or fluorescent labeled primers, for examples). Two - for the really novice sequencer in a lab that does little or no sequencing, start out by buying a KIT (most of you know I hate the KIT mentality but for novices, kits are a great place to start learning a new technique and they usually include control templates, well written tried and true procedures, and allow you access to technical support). Three - as for non-radioactive detection methods, they look nice in the adds in Science, but we've never tried them, so can't give any advice on them. Cheers and best of luck.........bruce - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - \ Bruce A. Roe Professor of Chemistry and Biochemistry / / Dept. of Chem. and Biochem. INTERNET: BROE@aardvark.ucs.uoknor.edu \ \ University of Oklahoma BITNET: BROE@uokucsvx / / 620 Parrington Oval, Rm 208 AT&TNET: 405-325-4912 or 405-325-7610 \ \ Norman, Oklahoma 73019 FAXnet: 405-325-6111 / - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - From owner-rapd@net.bio.net Tue Jul 20 23:00:00 1993 Path: biosci!daresbury!buzz.bmc.uu.se!corax.udac.uu.se!sunic!uunet!biosys!news From: DAK@apldbio.com (Dave Knorr) Newsgroups: bionet.molbio.rapd Subject: RAPD profiles Keywords: RAPD Message-ID: <531@biosys.apldbio.COM> Date: 21 Jul 93 20:09:43 GMT Sender: news@biosys.apldbio.COM Reply-To: DAK@apldbio.com (Dave Knorr) Organization: Applied Biosystems Lines: 20 Ouch... In the recent thread here on reproducibility of RAPDs, scolnipa hit a raw nerve. The manner of temperature control and measurement can be highly variable for different thermal cyclers. Correlation between a thermocouple placed in a tube and one embedded in the block can be quite different, as can the location of the two devices. Also, there are very different type of "philosophies" for controlling temperature. This is particularly seen at the ends of ramping times. It can lead to things like overshooting or undershooting target temperatures. TC patterns may not be quite reproducible on different brands of cyclers. I don't necessarily disagree that TC profiles be included for publication, it's just that such information MAY not be particularly useful for others trying to replicate your results. Dave Knorr DAK@apldbio.com Dave (DAK) Knorr From owner-rapd@net.bio.net Tue Jul 20 23:00:00 1993 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: thermocycler Message-ID: <3DBE5FB639E@uamercury.uark.edu> Date: 21 Jul 93 17:48:39 GMT Sender: daemon@net.bio.net Distribution: bionet Organization: University of Arkansas Lines: 34 >To: DRHOADS@mercury.uark.edu >From: LIN@botany.utoronto.ca >Date: Wed, 21 Jul 1993 12:26:04 -0400 >Subject: Re: thermocycler > >> >> We tried out the MJ cycler without the heated lid. We used it for >> RAPDs and PCRs. We had no problems and got excellent results in RAPDs when >> we slowed down the heat ramping for the first 10 degrees from 35 to 45 to >> about 0.25 degrees per second. With fast ramps it didn't work. >> >> Doug Rhoads || Dept. of Biological Sciences > > >Hi, > I use a Cetus DNA thermal cycler to do RAPDs, and I found the >same thing as you did with the ramping. Going up from anealing (36C) >to extention (72C) ASAP (about 35 seconds) didn't work, but work when >paused at 55C for 30 seconds. I wonder why? Hints appreciated. > Jingzhong Lin > U of Toronto My guess is that if the heating is faster than the Taq Pol extends the products (remember this must be a MAJORITY of the product) to sufficient length to increase thermal stability, then the targets will melt off. Thus, you get no extension. If you slow down the initial heating then more of the priming events are extended to >30 bp and therefore won't melt at temperatures <70C. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Tue Jul 20 23:00:00 1993 Path: biosci!MCMAIL.CIS.MCMASTER.CA!millerp From: millerp@MCMAIL.CIS.MCMASTER.CA (Paul Miller) Newsgroups: bionet.molbio.rapd Subject: RAPD reproducibility Message-ID: Date: 21 Jul 93 16:37:36 GMT Sender: daemon@net.bio.net Reply-To: Paul Miller Distribution: bionet Lines: 36 The newsletter seems somewhat preoccupied with reproducibility of RAPD profiles and the performance of specific thermocyclers (justly so). A recent article by Penner et al (1993), in the May issue of PCR Methods and Applications, addresses both of these issues. They investigate the reproducibility of RAPD profiles obtained both within and between different labs using the same template, primers and protocols but using different thermocyclers. They found that each lab had to customize the reaction conditions to produce any results at all and that the size class of bands produced differed between investigator. This result was largely attributed to the variation in temperature profiles of the thermocycler. The suggestion was also made, that some bands are more reproducible than others. Within lab reproducibility was reported as good. The thermocyclers used were Coy, MJ Research, Techne PHC-3 and the Perkin Elmer 4800. Temperature profiles for the Coy and Techne are displayed. On a personal note, I have been using a Perkin Elmer 480 for the last two years for RAPDs and other PCR applications. The between reaction reproducibility for RAPDs is excellent for dark bands, however lighter 'ghost' bands tend to appear and disappear between reactions. Therefore I do all reactions at least twice and score only repeatable bands. I hope this information was useful ! Paul Miller McMaster University Hamilton Ont. Millerp@mcmail.cis.mcmaster.ca From owner-rapd@net.bio.net Wed Jul 21 23:00:00 1993 Path: biosci!POOH.UCSD.EDU!andy From: andy@POOH.UCSD.EDU (Andy Dizon) Newsgroups: bionet.molbio.rapd Subject: Re: Sequencing query Message-ID: <9307221429.AA22876@pooh.UCSD.EDU> Date: 22 Jul 93 14:29:52 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 33 In article , pgaffney@RAVEL.UDEL.EDU (Patrick Gaffney) writes... >We'd like to sequence various PCR products, and are wondering whether to >use nonradioactive detection methods, and if so, which ones. Secondly, >we'd like to hear opinions on the route from PCR product to sequence - >i.e., direct sequencing of DS product, SS amplification, cloning, etc. >Your advice to novices greatly appreciated. > >Patrick Gaffney | pgaffney@ravel.udel.edu >College of Marine Studies | (302) 645-4364 >University of Delaware | FAX: (302) 645-4028 >Lewes, DE 19958 | - This space for rent - Netters... We're novices also (Marine Science types -- not molecular biologists) but have evolved efficient means for sequencing PCR products. We're doing population surveys so we do lots of samples. We use biotinylated primers so we can attach the amplified product to magnetic micro beads for cleaning and removal of the complementary strand. See Hultman et al. "Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support." Nuc. Acids. Res. 17:4937-4945. Then we just use USB's Sequenase kit with S35. The only thing we recommend is to use internal sequencing primers rather than re-using one of the PCR primers. See also Rosel, P. 1992. Genetic Population Structure... UC, San Diego PhD Thesis. Patty worked out the techniques and protocols for us, and her thesis gives all the details. By the way the Hultman article is 1989. Andrew Dizon National Marine Fisheries Service Southwest Fisheries Center La Jolla CA 92038 (619) 546-7089 From owner-rapd@net.bio.net Wed Jul 21 23:00:00 1993 Path: biosci!esvax.dnet.dupont.com!rafalski From: rafalski@esvax.dnet.dupont.com Newsgroups: bionet.molbio.rapd Subject: Marker distribution Message-ID: <9307221127.AA28405@esds01.es.dupont.com> Date: 22 Jul 93 11:27:22 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 21 >Does anyone know how to calculate the coverage of genome using rapd. It seems t o > me the result of rapd depends on the luck. You just randomly pick up some prim e >rs and pcr it. The result may just show very small part of the genome Any markers chosen essentially at random (RFLPs using random low copy number genomic or cDNA clones, microsatellites randomly picked from a genomic library, etc) will be distributed as if you were blindly throwing darts at a dartboard - assuming that potential polymorphic sites are available throughout the genome (there may be some regions where you would find no markers- for example no RFLPs in highly repetitive regions). Distribution can be calculated (to answer the question how many markers you need to put on the genome so that there are no gaps larger then X centimorgans) - the details could be found in one of the early Botstein and Lander papers (I do not recall the exact reference). Antoni Rafalski From owner-rapd@net.bio.net Wed Jul 21 23:00:00 1993 Path: biosci!esvax.dnet.dupont.com!russelsh From: russelsh@esvax.dnet.dupont.com Newsgroups: bionet.molbio.rapd Subject: re: RAPD profiles Message-ID: <9307222058.AA11489@esds01.es.dupont.com> Date: 22 Jul 93 20:58:54 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 9 Concerning the discussion on thermal profiles and reproducibility: Dave Knorr is correct that temperature control and measurement can be variable. The temperature of the sample is the important measurement, not the temperature of the block, temperature readout on the instrument, or the numbers punched into the instrument. Even the container (i.e. plate vs tube) can make a difference. Sandra Russell russelsh@esvax.dnet.dupont.com From owner-rapd@net.bio.net Thu Jul 22 23:00:00 1993 Path: biosci!daresbury!bioftp.unibas.ch!rc1!ub4b!mcsun!uunet!psinntp!nstn.ns.ca!dragon.acadiau.ca!axe.acadiau.ca!901106c From: 901106c@axe.acadiau.ca (TIM CHIPMAN) Newsgroups: bionet.molbio.rapd Subject: Re: thermocycler Message-ID: <901106c.547.743351531@axe.acadiau.ca> Date: 22 Jul 93 15:32:11 GMT References: <1993Jul20.184053.24501@gserv1.dl.ac.uk> Sender: news@dragon.acadiau.ca Distribution: bionet Organization: Acadia University Lines: 15 Nntp-Posting-Host: 131.162.69.36 >Kirsten Wolff >TNO, Dept. of Microbiology >PO Box 360 >3700 AJ Zeist, the Netherlands >e-mail: wolff@voeding.tno.nl We use the model you describe. However, I have trouble with your email address...it keeps bouncing back. Please send me email at 901106c@axe.acadiau.ca if you wish to know about our experince with this equipment, finding plates for use in it, and cleaning protocols for re-use of plates. Tim Chipman 901106c@axe.acadiau.ca From owner-rapd@net.bio.net Wed Jul 28 23:00:00 1993 Path: biosci!MCMAIL.CIS.MCMASTER.CA!millerp From: millerp@MCMAIL.CIS.MCMASTER.CA (Paul Miller) Newsgroups: bionet.molbio.rapd Subject: analysis of RAPD data Message-ID: Date: 29 Jul 93 14:08:32 GMT Sender: daemon@net.bio.net Reply-To: Paul Miller Distribution: bionet Lines: 29 I am interested in using RAPDs to detect genetic structure within a population of brood-parasitic Brown-headed cowbirds. In the literature so far, it appears that RAPDs have been used only sparingly for population level studies, especially for organisms other than plants or insects. So far people seem to be taking a couple of different approaches, Russel et al (1993) construct a similarity matrix for three populations of cocoa and then use both Principal Component Analysis (Digby and Kempton 1987) and single linkage cluster analysis (Kempton and McNicol 1990) to look for differentiation between the three populations. They (and others have also used Shannon's diversity index to estimate within and between population variation but I'm not quite clear on how to test the significance of population differentiation with this information (Kruskal-Wallis test?). Recently, Andy Clark and Caroline Lanigan developed a program to estimate sequence diversity within populations and divergence between populations. This program corrects for the inability of RAPDs to detect heterozygotes in a diploid organism. I would like to take a quick census on how people are currently analysing RAPD data, especially with respect to population level phenomenon. Thanks for taking the time to read and hopefully respond to this message ! Paul Miller From owner-rapd@net.bio.net Thu Jul 29 23:00:00 1993 Path: biosci!MCMAIL.CIS.MCMASTER.CA!millerp From: millerp@MCMAIL.CIS.MCMASTER.CA (Paul Miller) Newsgroups: bionet.molbio.rapd Subject: Re: literature Message-ID: Date: 30 Jul 93 15:18:32 GMT References: <23073008591868@vms2.macc.wisc.edu> Sender: daemon@net.bio.net Distribution: bionet Lines: 20 On Fri, 30 Jul 1993, Raul O Castillo wrote: > Dear Paul: > > Could you please send me the complete reference of Russel et al (1993). > > Thank you > > Raul Castillo > > (Oaul) The complete reference for the paper I mentioned yesterday concerning RAPD analysis is... Russel, J.R., Hosein,F. Johnson, E., Waugh, R. and Powell, W. 1993. Molecular Ecology 2: 89-97. From owner-rapd@net.bio.net Thu Jul 29 23:00:00 1993 Path: biosci!UICVM.UIC.EDU!CAMPALX%BRUFMG From: CAMPALX%BRUFMG@UICVM.UIC.EDU (Alexandro Carvalho) Newsgroups: bionet.molbio.rapd Subject: RAPD and TG-EDTA Message-ID: <9307302221.AA12374@net.bio.net> Date: 30 Jul 93 22:21:54 GMT Sender: daemon@net.bio.net Distribution: bionet Lines: 10 I am interested in work with spiral microorganisms and RAPD. At hte momente I have some problems with DNA extration methods and RAPD. I tried to work with the tecnique of Guanidium Thiocianate-EDTA and AP_PCR. If someone have some informations or results with this methods give me a tip. Thanks in advance, Alexandro Carvalho - Department of Microbiology_UFMG, Belo Horizonte, MG. BRAZIL. FAX 031 4411412 CAMPALX@BRUFMG.