From owner-rapd@net.bio.net Mon Aug 01 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!doc.ic.ac.uk!daresbury!trane.uninett.no!eunet.no!nuug!EU.net!uunet!munnari.oz.au!newsroom.utas.edu.au!ml.csiro.au!mac-1302.ml.csiro.au!chris.bolch From: Christopher J. S. Bolch Subject: Analysis of RAPD binary matrices Message-ID: <1994Jul28.044014.5141@ml.csiro.au> X-Xxmessage-Id: X-Xxdate: Thu, 28 Jul 94 14: 42:14 GMT Sender: news@ml.csiro.au Organization: CSIRO Division of Fisheries, Hobart, Tasmania X-Useragent: Version 1.1.3 Date: Thu, 28 Jul 1994 04:40:14 GMT Lines: 27 hey you RAPD fans, I would like some advice regarding the analysis of binary matrices generated from presence/absence of RAPD bands. I suspect that this has been churned over before so if ther is an archive then can someone give me a quick pointer on how to get to it!! I use Nuntius as my network news reading software. I have a copy pf PAUP 3.0s which can do cladistic analyses of such data but I have read a few papers which state that cladistic analyses are not valid with RAPD markers. Incidently, anyone know where I can get hold of PAUP 3.1 or later. (I can't get a response from Dave Swofford via e-mail. Last I heard he was having distribution problems.) Distance and similarity measures calculated from the matrices could be used, however I do not have any software which does this. Can someone suggest Mac (or IBM PC at a pinch) software available that will do this efficiently ? Any advice appreciated (especially if accompanied by some reasoning on why the proposed method is preferable.) Cheers Chris. From owner-rapd@net.bio.net Mon Aug 01 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!lyra.csx.cam.ac.uk!warwick!unicorn.nott.ac.uk!pmbcaj.nott.ac.uk!Chris.Jones From: Chris.Jones@nottingham.ac.uk (Chris Jones) Newsgroups: bionet.molbio.rapd Subject: AFLP Kits? Date: Tue, 2 Aug 1994 09:14:56 Organization: Univ. of Nottingham Biochemistry Dept Lines: 5 Message-ID: NNTP-Posting-Host: pmbcaj.nottingham.ac.uk X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Apologies if you all read this on the .plants board but does anyone know if there is a kit for AFLP available yet? I know the patent was issued but I haven't seen a kit appear yet. Chris Jones From owner-rapd@net.bio.net Wed Aug 03 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: Extra long PCR Date: 4 Aug 1994 04:51:23 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <9408041154.AA04037@gw1.phibred.com> NNTP-Posting-Host: net.bio.net About a month ago I had enquired about long PCR. NOw I have found that] Perkin Elmer has, or will be producing a kit for extra long PCR. The buffer has been changed to Tricine instead of tris, and acetate salts are used. Disclaimer: I do not stand to benefit financially from the sharing of this information. This information was obtained during a seminar organized by Perkin Elmer. From owner-rapd@net.bio.net Wed Aug 03 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!usenet.ins.cwru.edu!nigel.msen.com!zib-berlin.de!ceres.fokus.gmd.de!nntp.gmd.de!urmel.informatik.rwth-aachen.de!newsserver.rrzn.uni-hannover.de!mac3.marbot.gu.se!user From: bengt.oxelman@systbot.gu.se (Bengt Oxelman) Subject: Re: Analysis of RAPD binary matrices Message-ID: Followup-To: bionet.molbio.rapd Sender: news@newsserver.rrzn.uni-hannover.de (News Service) Organization: Systematic Botany, Univ. Goeteborg, Sweden References: <1994Jul28.044014.5141@ml.csiro.au> Date: Thu, 4 Aug 1994 13:03:01 GMT Lines: 70 In article <1994Jul28.044014.5141@ml.csiro.au>, Christopher J. S. Bolch wrote: > hey you RAPD fans, > > > I would like some advice regarding the analysis of binary matrices > generated from presence/absence of RAPD bands. I suspect that this has > been churned over before so if ther is an archive then can someone give > me a quick pointer on how to get to it!! I use Nuntius as my network > news reading software. > > I have a copy pf PAUP 3.0s which can do cladistic analyses of such data > but I have read a few papers which state that cladistic analyses are not > valid with RAPD markers. Incidently, anyone know where I can get hold of > PAUP 3.1 or later. > (I can't get a response from Dave Swofford via e-mail. Last I heard he > was having distribution problems.) > > Distance and similarity measures calculated from the matrices could be > used, however I do not have any software which does this. Can someone > suggest Mac (or IBM PC at a pinch) software available that will do this > efficiently ? > > Any advice appreciated (especially if accompanied by some reasoning on > why the proposed method is preferable.) > > Cheers > > Chris. I think there is an PAUP 3.1.1. updater available at anonymous ftp at onyx.si.edu. I am not sure if it works on version 3.0 though. I think you must decide what kind of relationships you are trying to infer from your RAPD presence/absence matrix. If you want to describe is as a dichotomously branching tree with minimal numbers of changes among nodes, parsimony is the method. This also, in my opinion, the method of choice if phylogenetic inference is the objective, because it simply chooses the simplest explanation (=fewest number of steps) for the data. Distance and similarity can be measured in many ways. Moreover, they must be transformed to a tree in order to represent hierarchical relations. There is no way of telling why any such method should be preferable to another, so your conclusions will be very much influenced by arbitrary decisions. As you note, some people have trouble with using RAPD patterns in parsimony analysis. As I have understood this, their main arguments are: - Co-migrating RAPD fragments may not be homologous. This is of course true, but pure similarity is never evidence for homology. You may view the cladistic analysis as a second test of homology (after passing the similarity test). Of course, if the genomes you compare are very diverse, there will be much noise, but at least 20-30 % band sharing (using 10mer primers and standard EtBr agarose gels) should theoretically keep the noise (which probably is unusually unbiased for RAPD bands) low. - For diploid organisms, heterozygous individuals will on average be more similar than homozygous individuals. This is a point of concern, but is so in all cases where dominant markers are used as characters. The degree of this error can be reduced by careful sampling schemes. - Presence/absence of bands may depend on competition among priming sites and/or differential staining. This truly introduces errors into your matrix which are of kind that are quite unique to RAPD. They are however probably relatively unbiased because of the pseudorandom sampling of primers. Hope this is food for thought! -- Bengt Oxelman Dept. of Systematic Botany Carl Skottsbergs Gata 22 S-413 19 Goeteborg SWEDEN bengt.oxelman@systbot.gu.se From owner-rapd@net.bio.net Sat Aug 06 23:00:00 1994 Path: biosci!daresbury!not-for-mail From: "Naresh C. Pancholi" Newsgroups: bionet.molbio.rapd Subject: SUMMARY: Help on DNA quantification and RAPD... Date: 7 Aug 1994 15:43:28 +0100 Lines: 419 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <322rug$7u5@mserv1.dl.ac.uk> Reply-To: "Naresh C. Pancholi" Original-To: Methods-and-Reagents , RAPD Hello everybody, Thanks for your reply to my questions regarding DNA quantification as well as RAPD. I asked the following questions: 1. I am using CTAB DNA extraction method for banana leaves. I tried to quantify DNA through UV spectrophotometer but this reading is not reliable as CTAB also absorbs fluorescence. Then I tried fluorimeter but I am still not happy. It seems that my samples have very low DNA because my starting leaf material is only 50-70 mg. Can someone tell me a reliable, quick and cheap method for DNA quantification? 2. On the basis of this, I am going to use RAPD and I have a big sample size (300 plants) and less time (2 months only). I use primer kits from Operon. I noticed a strange phenomenon in my RAPD reaction. I tried 10, 25 and 50 ng of template DNA (based on fluorimeter readings) per 25 ul reaction. There was an increase in the total number of bands as the concentration of DNA increased. What I understand that this should not happen, though I am a novice for this technique and therefore, would like to receive your advice on this matter. 3. Is any one there who is comparing a big sample size like mine, through RAPD? If so, would you please write general or any specific advice for it? To throw more light on these questions I also posted an additional question later on and, it was: I am still confused about spectrophotometer and fluorimeter readings. For example, one of my samples gave 760 ng/ul (spectrophotometer) but only 64 ng/ul (fluorimeter) So the differences between two methods are so great that I am in a complete confusion. I tried to run a minigel with a known DNA standards and it tells the concentration about 75 ng/ul. Now the question is on which method should I rely to finalize the required concentration of template DNA for RAPD? I have summarised below the replies I received from many people. ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From aweber@biolan.uni-koeln.de Ukn Jul 27 17:03:22 1994 In case you use CTAB as a reference this should not be a problem. In my experiments I always use ethidium bromide stained gels for the estimation of DNA, I think it is reliable. You only see your DNA stained by EtBr, there is no background by other substances. You may want to try DNA Dipstick which is sold by Invitrogen. They claim that it is possible to quantitate DNA, RNA, Oligos etc. at concentrations down to 0,1 ng/ul in 10min. >3. Is any one there who is comparing a big sample size like mine, through >RAPD? If so, would you please write general or any specific advice for it? You could use the Perkin-Elmer 9600 - it can handle 96well plates, and 96 reactions are completed in 60min. ************************************************************* * Andreas Weber Tel.: (49)-221-470-5664 * * University of Cologne Fax.: (49)-221-470-5181 * * Department of Botany II * * Gyrhofstr. 15 * * 50931 Cologne * * Germany email:aweber@biolan.uni-koeln.de * ************************************************************* +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From wagnerr@ccmail.orst.edu Ukn Jul 27 21:49:33 1994 Did you phenol:chloroform:IAA extract your sample? this should remove most of the CTAB. There are also some SDS methods that might give you better yields than CTAB. I personal don't like using CTAB. I'm am never happy with fluorimeter readings myself but if you run some of your sample out on a .5% agarose gel and visualize it you can feel more comfortable. You could also quant it very crudely using an ethidium dot procedure--it is in Maniatis but if you don't have I could email it You have a lot of work to do in 2 months.. Your observation is not strange but typical at the concentrations your working with usually one does get more bands until you titrate to a certain concentration and then it can become inhibitory. What I would do if were you is too use an amount that will give you reproducible, clearly separated bands and you might find the higher concentration the better. But if you have limited sample, you'll have figure out how many reactions you have to do this might limit your amount. I actually use 5ng/25ul rxn. +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From MAL5@cornell.edu Ukn Jul 27 22:49:25 1994 Dear Naresh: As far as DNA quantification is concerned, I did some work on it with different models of spectrophotometer, fluorometer and gel quantification. The results in all cases were quite different. I compared the results of DNA quantification via different methods, with RAPD amplification. DNA was amplified satisfactorily with all other methods except quantified with spec., generally spec was reading about 5-10 times more DNA than the actual amount. Some of these results are mentioned in my thesis that is ready to be submitted. The only published thing that I have, is my DNA extraction protocol that has produced good results in several different plant species. The protocol was published in Plant Molecular Biology Reporter(1994), 12(1):6-13. I can send you a reprint if you don't have access to it. If you have access to fluorometer, it is the very reliable if you don't then you can stick even with spec. but simply use about 5-10 times more DNA. Regarding the effect of DNA conc. on amplification, it is again not a simple problem. I think that with increased concentration one should see changes in the amplification of bands. These changes could also be in the form of new bands because at lower conc. some of the target sites were not amplified enough to be detectable with EtBr. I would refer you to our article on this issue which may be little difficult to get but again I can send you a xerox copy of this article if you are unable to get it. Weeden NF, Timmerman GM, Hemmat M, Kneen BE, Lodhi MA (1992) Inheritance and reliability of RAPD markers. In: Proc. Joint Plant Breeding Symposium Series. Applications of RAPD Technology to Plant Breeding. Crop Science Society of America, American Society for Horticultural Science and American Genetic Association, pp 12-17. Hope it will be helpful Muhammad A. Lodhi From MAL5@cornell.edu Ukn Jul 28 13:20:30 1994 Dear Naresh: The difference in the readings you are getting from Spec. and Fluor. are almost the same that I was. As you mentioned that you don't have access to fluor. as myself so I started using DNA (about 300-500 ng/reaction) based on the spec. reading. This is also consistent with your observations 760 vs. 64 ng DNA. Amplification was very uniform and repeatable all the time. This is due to the reason that DNA quantity in a certain range doesn't make drastic change in amplification. For standardization purposes I did use fluor. and gel and these two results were very close, as you have mentioned too. Muhammad A. Lodhi NYSAES, Cornell University Geneva, NY 14456 +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From hobbs@unixg.ubc.ca Ukn Jul 27 17:05:20 1994 Dear Naresh, I cannot answer all of your queries, but with regard to DNA quantification using UV spectrophotometry, I do not see why CTAB should be a problem. What you measure is the absorption of UV light, not fluorescence. CTAB may interfere with fluorescence measurements, certainly, but it possesses no pi-bonded orbitals or non-bonded pairs of electrons likely to be excited by long-wavelength UV (at least as far as I am aware) so it should not give you any UV absorption and thus not interfere with your DNA quantification. I note that you obtained your RAPD primers from Operon. Is this the usual source for RAPD primers in the UK? I ask because here at UBC we also sell RAPD primer kits, currently to over 20 countries worldwide, but relatively few in Europe, and I am not sure whether this is because of a lack of awareness of the fact that we have them available, or for some other reason. We are significantly cheaper than Operon on a per-primer basis; what we don't do is advertise commercially, although I have posted notices on our primers to the RAPD newsgroup from time to time. I'd be interested in your comments. John Hobbs From hobbs@unixg.ubc.ca Ukn Jul 28 17:53:49 1994 Dear Naresh, Me again. I note that Muhammad Lodhi thinks UV quantification is likely to give you errors, and prefers fluorimetry: I have also just read your further query. Simply, if your DNA is homogeneous and is the only component in your preparation with a significant absorption at 260 nm, then it should give you a perfectly reliable quantitation of your DNA. If these conditions are not met, then the estimate of your DNA is likely to be high, due to the extra absorption at 260 nm of non-DNA material. If you have any components in your mixture likely to quench fluorescence, then your estimate by fluorimetry is likely to be low. Have you tried running DNA standards of known concentration in the presence of the same concentration of CTAB as you are using, to get an estimate of the degree of quenching you are experiencing? You might be able to get a ball-park figure for a correction factor. Otherwise, maybe you could treat a small, sacrificeble sample of a DNA preparation with a deoxyribonuclease to effect exhaustive degradation of your DNA to mono- or short oligonucleotides (monitoring the increase in the absorption at 260 nm with time). It should plateau when digestion is complete. The increase should be due to the hyperchromicity of the DNA on digestion, and is typically around 25% of the absorption due to the undigested DNA (depending on its base analysis). Knowing this figure should allow you to estimate the component of your original absorption at 260 nm which was due to the DNA. It's not a great answer, but should narrow your figure down within the 10-fold range of answers you've got at present, and allow you to estimate what proportion of your absorption at 260 nm is due to components other than DNA. You could then probably apply this correction to your readings for other samples which were prepared identically. I hope this is of some help. Let me know if you try it and it works OK (or, doesn't work!). Regards, 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 rafalski@medicine.cambridge.ac.uk Ukn Jul 28 08:29:19 1994 We use a dimeric intercalating dye called YoPro (from Molecular Probes, Inc.) to quantitate DNA. Take 10 miroliters of DNA and add 190 microliters of solution containing (1 micromolar UpPro, 10 mM Tris-HCl pH7.5, 1 mM EDTA, 50 mM NaCl and 10 micrograms per ml RNase A, DNase-free). Incubate at room temperature 30 min. Measure fluorescence. At the same time do a standard curve at concentrations of DNA 0.15-35 micrograms / ml. Antoni Rafalski (DuPont, currently on sabbatical in Cambridge) ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From wmoens@rc4.vub.ac.be Ukn Jul 28 10:51:17 1994 We have done two RAPD studies in microbial intra-species diversity with a sampling size of 164 and 374 strains respectively. DNA concentration is estimated by eye on standardized baby gels using BRL ladder as quantitative standard. Obviously, good eyes and experience are necessary in order not to bias the data. It is not the best system but it works with RAPD even with contaminating RNA. There is no ideal RAPD format neither concerning the number of bands nor the type of primer to use. What seems to be clear is that the multiplication of bands above 2-2.5kb seems to be more PCR recombinants than products of allele amplification. The way to find out an "good" format might be the following: 1. Use 20 ng of DNA and titer the primer concentration. You should see a progressive shift down of the range of molecular weights with increasing primer concentration. The shift stabilizes when the right primer concentration is reached. 2. If a faint smear is seen in the background of the track this may mean several, things: - you have used too much template, or you have more template than what you have guessed to use - the annealing temperature is too low - the primer specificity is at the border of recognizing less specific templates than the targetted one, less specific templates which are much more abundant than the targetted one. If you know the law of michaelis-menten you should understand what I mean. Solutions to the two last points: try thermocycling conditions with two annealing steps: for example: 10 first cycles at a annealing temperature 5C above the temperature used for the next 30-35 cycles: if smear reduces then it is a problem of primer specificity versus ratios among targets concentrations. 3. If no smear or acceptable smear is obtained, fix the primer concentration established in (1) and play with Taq polymerase units. Doing that, the number of bands should increase and the amount of DNA in those bands obtained at the lowest Unit numbers should increase. Once again a plateau is reached. Fix the Taq concentration. 4. Produce 3-6 independent DNA batches from the same leave; test your conditions in triplicates per DNA using a common mix containing everything except DNA. If the RAPD pattern is identical among triplicates and DNA batches you can start to produce real results. But do not touch anything further till publication!!! Be confident that competitors will get different patterns. What is important later is the data processing. Clustering techniques of the data are less sensitive to RAPD pattern variations that the naked eye. Even if competitors have different RAPD patterns on gels, they will not find different results in terms of population diversity or at least not results which infer your results. I hope this might help you, feel free to further contact me, I like very much bananas, Cheers, William Moens Ph.D. IHE- Biotechnology Brussels BE +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Dear Naresh, Regarding your DNA quantification problem, I have observed similar problems. Firstly, does it really matter if you are able to determine the DNA quantity accurately? If you assess all the samples using the same method, perhaps you can assume that they all contain the same degree of error. Therefore you should be able to dilute your samples accordingly and use the same amount of DNA in each tube. I would suspect that the fluorimeter reading is more accurate, especially as it agrees with the mini-gel result. Ethidium bromide fluorescence in the gel is unlikely to be affected by contaminants such as CTAB and protein, which will be in other parts of the gel. I am not surprised that increasing the DNA concentration alters the number of bands produced by RAPDs. Yours sincerely, Nigel Blackhall, Plant Genetic Manipulation Group, Dept Life Science, University of Nottingham. plznb@pln1.life.nottingham.ac.uk +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From wolff@voeding.TNO.nl Ukn Jul 28 13:23:40 1994 Use the ethidium bromide stained gel electrophoresis for quantifying your plant DNA concentration! Easy and reliable and always comparable if you make (polaroid) photographs. Good luck Kirsten Wolff +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From LIN@botany.utoronto.ca Ukn Jul 28 17:45:52 1994 After the first a few runs, I don't quantify my DNA at all for RAPDs. I just add 1 ul (15-30 ng, judgement based on gel comparison) of my concentrated DNA in a 15-ul reaction mix. They all work well. In fact you probably don't need to know how much DNA you put in, but how many ul to give you the good results. Cheers! Jingzhong Lin U of Toronto ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From corley@sfu.ca Ukn Jul 28 20:26:49 1994 Hi, Spectrophotometer: Reads absorbance of Uv light. Must be done in quartz cuvette. RNA and protein absorb uv light at 260 nm (approximately the wavelength at which DNA absorbs maximally). Thus O.D. 260 measurements can give over estimation of DNA concentration if sample contains RNA and/or protein. Fluorometer: Hoechst 33258 binds preferentially to a specific tetranucleotide sequence in double stranded DNA. Thus, protein and RNA contamination will have less affect on the fluorometric reading. Thus, this method is preferable if you have 1) A good set of standards of known DNA concentration to calibrate instrument, 2) a DNA sample with RNA and/or protein contamination. I use tissue homogenates for my RAPD reactions and thus fluorometry is more appropriate than spectrophotometry to determine DNA concentration. Ethidium Bromide Dot Blots: Quick and dirty method. Ethidium Bromide intercalates between the stacked bases of RNA and DNA. Thus, you DNA concentration will be affected by RNA contamination. As to your question. Spec reading will should be greater than fluorometer reading when RNA and DNA are also in sample. Is it possible that you have lots of RNA in your sample? More importantly than determining accurately your DNA concentration, is finding a method that you can reuse to get the same concentration of DNA each time you run your RAPD reaction. Hope this information helps. 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 e-mail: corley@sfu.ca FINAL WORDS: AT LAST I HAVE DECIDED TO USE FLUOROMETER AS I HAVE LIMITED TIME LEFT OVER. I SINCERELY THANK EVERYONE WHO SPENT SOME TIME IN RESPONSE TO MY QUESTION. +-----------------------------------------------+ | Naresh Pancholi abrpanch@reading.ac.uk | | Agricultural Botany Phone: +44 734 318092 | | Reading University Fax: +44 734 316577 | | Reading RG6 2AS | | UK | +-----------------------------------------------+ From owner-rapd@net.bio.net Sat Aug 06 23:00:00 1994 Path: biosci!daresbury!not-for-mail From: "Naresh C. Pancholi" Newsgroups: bionet.molbio.rapd Subject: SUMMARY-2: Help on DNA quantification and RAPD... Date: 7 Aug 1994 16:07:26 +0100 Lines: 69 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <322tbe$95n@mserv1.dl.ac.uk> Reply-To: "Naresh C. Pancholi" Original-To: RAPD , Methods-and-Reagents Somehow, I forgot to include the following messages in my summary. From rafalski@medicine.cambridge.ac.uk Sun Aug 7 15:48:40 1994 It is likely that your DNA prep contains both DNA and RNA (most miniprep methods isolate a mixture of both, with about 10x more RNA then DNA), so you measure both DNA and RNA with the spectrophotometer, and mostly DNA with a fluorimeter. It is best to calibrate your RAPDs by running a dilution series over a wide range of concentrations, and use whatever works best for your particular DNA. Antoni Rafalski +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From J.T.Behnam@reading.ac.uk Sun Aug 7 15:48:54 1994 Dear Naresh We had similar problems with our spectrophotometer but we solve it by believing the flurometer reading only!! Its reading is reliable as far as all other conditions were standard. i.e. fresh TNE buffer filtered through ..2u filter, fresh Hoechst 33258 sol. and correct pH. For measuring low concentration of DNA, Some spectrophotometers are not very accurate ( in the range below 100ng) May I suggest the following: 1. Calibrate your flurometer with standard DNA i.e. Lambda phage or Salmon sperm etc.. 2. Draw a standard curve of serial dilution of standard DNA , plot the graph and find out your RAPD concentration from it . Best regards Joseph +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From DEMEKE@ABRSLE.AGR.CA Sun Aug 7 15:49:32 1994 I will use the fluorometer and the gel estimation. The spectrophotometer reading is usually exaggerated. I did not have any problem with fluorometer quantification. So if I were you I would use fluorometer. Good luck! +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ From pezmen@gate.net Sun Aug 7 15:49:37 1994 I had a similar problem with crustacean DNA and solved it by using a different extraction protocol. I extracted the DNA after grinding in liquid nitrogen and that removed whatever was quenching the fluorescence. Also, you my want to try to quantify the DNA using a DipStick (Invitrogen). If you send me your address Ill send you a copy of my extraction protocols (too long to type here). I process about 150-175 samples a month with this method (and Southerns) without any difficulties (70-90% results scorable). good luck Hector Cruz-Lopez (pezmen@gate.net) +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ NARESH PANCHOLI ABRPANCH@READING.AC.UK From owner-rapd@net.bio.net Sat Aug 06 23:00:00 1994 Path: biosci!rutgers!netnews.upenn.edu!news.amherst.edu!not-for-mail From: walin@unix.amherst.edu (Athena Wen-chuan Lin) Newsgroups: bionet.molbio.rapd Subject: test Date: 7 Aug 1994 15:44:24 -0400 Organization: Amherst College, Amherst MA, USA Lines: 2 Message-ID: <323dio$c90@amhux3.amherst.edu> NNTP-Posting-Host: amhux3.amherst.edu X-Newsreader: TIN [version 1.2 PL0] just to see if I can post here From owner-rapd@net.bio.net Tue Aug 09 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!europa.eng.gtefsd.com!emory!swrinde!pipex!jura.sasa.gov.uk!news From: odonnell@sasa.gov.uk (Kevin O'Donnell) Subject: RAPDs as dominant markers Organization: Scottish Agricultural Science Agency Date: Wed, 10 Aug 1994 15:54:24 GMT Message-ID: X-Newsreader: WinVN 0.91.3 Sender: news@jura.sasa.gov.uk (Usenet) Lines: 9 I have seen several references to RAPDs (in relation to plant breeding) as dominant markers. Does 'dominant' as used in this context have a different meaning from the dominant/recessive gene context? Kevin O'Donnell Scottish Agricultural Science Agency Edinburgh Scotland From owner-rapd@net.bio.net Tue Aug 09 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!europa.eng.gtefsd.com!uhog.mit.edu!news.mtholyoke.edu!news.amherst.edu!news.umass.edu!nic.umass.edu!usenet From: AAT@UCSVAX.UCS.UMASS.EDU (Gus Trautweiler) Newsgroups: bionet.molbio.rapd Subject: Re: RAPDs as dominant markers Date: 10 Aug 1994 17:18:28 GMT Organization: University of Massachusetts Lines: 23 Distribution: world Message-ID: <32b254$j1h@nic.umass.edu> References: NNTP-Posting-Host: phobos.ucs.umass.edu X-News-Reader: VMS NEWS v1.25 In-Reply-To: odonnell@sasa.gov.uk's message of Wed, 10 Aug 1994 15:54:24 GMT In odonnell@sasa.gov.uk writes: :Q I have seen several references to RAPDs (in relation to plant breeding) :Q as dominant markers. Does 'dominant' as used in this context have a :Q different meaning from the dominant/recessive gene context? :Q :Q :Q Kevin O'Donnell :Q Scottish Agricultural Science Agency :Q Edinburgh :Q Scotland This refers to dominant as not codominant. In other words you can't tell whether the organism is homozygous or heterozygous for the marker as in RFLPs. Gus Trautweiler ########* *##* *##* *##* *##* Plant & Soil Science Dept. * : : * * : : * : : * * : : University of Massachusetts * : : * * : : * * : : * * : : * Amherst, MA 01002 : * * : : * : : * * : : * : : * AAT@BIO.UMASS.EDU ###* *##* *##* *##* *##* From owner-rapd@net.bio.net Wed Aug 10 23:00:00 1994 Path: biosci!CSCGPO.ANU.EDU.AU!lce652 From: lce652@CSCGPO.ANU.EDU.AU (Livinus Emebiri) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 11 Aug 1994 00:39:05 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <9408110740.AA08394@cscgpo.anu.edu.au> NNTP-Posting-Host: net.bio.net Dear Friends, I am presently optimizing the RAPD techingue for my research work, and running into a lot of flak. I wish to increase the number of specific amplifications, while reducing the amount of non-specific amplifications. The method I'm currenting trying out involves digesting the DNA prior to the amplification reaction, but non-specific amplification (seemly) create a lot of background smearing that is driving me nuts! What can I do? Suggestions, no matter how trivial would be most appreciated. This may be a FAQ, but getting answers from the Archive is equally frustrating. Livinus Emebiri From owner-rapd@net.bio.net Wed Aug 10 23:00:00 1994 Path: biosci!agate!doc.ic.ac.uk!warwick!bham!bcs88.bham.ac.uk!user From: A.C.Hilton@bham.ac.uk (Wiggy) Newsgroups: bionet.molbio.rapd Subject: Re: (none) Followup-To: bionet.molbio.rapd Date: 11 Aug 1994 09:00:06 GMT Organization: University of Birmingham Lines: 26 Distribution: world Message-ID: References: <9408110740.AA08394@cscgpo.anu.edu.au> NNTP-Posting-Host: bcs88.bham.ac.uk In article <9408110740.AA08394@cscgpo.anu.edu.au>, lce652@CSCGPO.ANU.EDU.AU (Livinus Emebiri) wrote: > Dear Friends, > I am presently optimizing the RAPD techingue for my research work, and > running into a lot of flak. I wish to increase the number of specific > amplifications, while reducing the amount of non-specific amplifications. > The method I'm currenting trying out involves digesting the DNA prior to > the amplification reaction, but non-specific amplification (seemly) create > a lot of background smearing that is driving me nuts! What can I do? > Suggestions, no matter how trivial would be most appreciated. > > This may be a FAQ, but getting answers from the Archive is equally frustrating. > > Livinus Emebiri Hi Livinus, You could try to increase the annealing temperature of your primer in the PCR towards that of its melting temperature resulting in the binding to the template being more stringent. This should reduce non-specific amplification initiated by a bound mismatched primer. Hope this helps, it worked for me! Wig. From owner-rapd@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!UNIXG.UBC.CA!yongbifu From: yongbifu@UNIXG.UBC.CA (Yong-Bi Fu) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 12 Aug 1994 13:46:49 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net sub Yong-Bi Fu Biotechnology laboratory University of British Columbia From owner-rapd@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!sfu.ca!corley From: corley@sfu.ca ("Graham Corley-Smith") Newsgroups: bionet.molbio.rapd Subject: RE: (none) Date: 11 Aug 1994 21:32:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 49 Sender: daemon@net.bio.net Distribution: world Message-ID: <77364.corley@sfu.ca> Reply-To: corley@sfu.ca NNTP-Posting-Host: net.bio.net In Message 11 Aug 1994 00:39:05 -0700, lce652@cscgpo.anu.edu.au (Livinus Emebiri) writes: >Dear Friends, >I am presently optimizing the RAPD techingue for my research work, and >running into a lot of flak. I wish to increase the number of specific >amplifications, while reducing the amount of non-specific amplifications. >The method I'm currenting trying out involves digesting the DNA prior to >the amplification reaction, but non-specific amplification (seemly) create >a lot of background smearing that is driving me nuts! What can I do? >Suggestions, no matter how trivial would be most appreciated. > >This may be a FAQ, but getting answers from the Archive is equally frustrating. > >Livinus Emebiri I use annealing temp of 42!C. A simplistic answer, but it may help. Also, high Mg++ conc can lead to non-specific priming. Choose one primer that gives you a fair number of bands, and run a series to optimize your amplification conditions. Templates: 1. 25 ng DNA 2. control (water or what ever you DNA is in) Temperature 1. 32 degrees Celcius 2. 36 3. 40 4. 42 5. 44 Magnesium concentration 1. 1 mM 2. 2 mM 3. 3 mM 4. 4 mM This will require making 2 x 5 x 4 = 40 reactions, but in one day you can optimize for these important conditions. Best of Luck, 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 e-mail: corley@sfu.ca From owner-rapd@net.bio.net Thu Aug 11 23:00:00 1994 Path: biosci!rutgers!gatech!newsxfer.itd.umich.edu!newsrelay.iastate.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!cs.utexas.edu!news.tamu.edu!mam4339 From: mam4339@tamsun.tamu.edu. (mam4339) Newsgroups: bionet.molbio.rapd Subject: Old question, but important. Date: 12 Aug 1994 18:16:55 GMT Organization: Texas A&M University, College Station, TX Lines: 21 Message-ID: <32gean$r8k@news.tamu.edu> NNTP-Posting-Host: medmicrotb.tamu.edu X-Posted-From: InterNews 1.0@news.tamu.edu. This is probably a question that gets asked alot here, but I need some help so I'm going to ask it again. My blanks have been showing bands since the first time I tried RAPDs in my lab. In another building I could do them without contamination, apparently, but here, or with the reagents here, I find big, bright, numerous bands in my blanks. I have received advice on the matter and have tried to make use of it but to no avail. I realize that the technique I use is not the absolute best, in terms of contamination, as I have heard of the incredibly sterile, careful facilities and procedures of other, successful labs. But I don't think that kind of care is feasible in the lab I work in, both monetarily and in the interest of time. My blanks include Taq polymerase, MgCl2, dNTPs, PCR buffer, dH20 and primer. I have tried using alternate sources of all of these but the primer, and have had no luck. (The same aliquots of primer produced no contamination in the other lab, and I don't think they've had the opportunity to be contaminated here). I started doing all parts of the procedure under a laminar flow hood; no luck. New gloves, new tubes, cleaned Pipetmen, stoppered tips: no luck. Any suggestions? Thanks! Michael From owner-rapd@net.bio.net Thu Aug 11 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!rutgers!gatech!newsxfer.itd.umich.edu!nntp.cs.ubc.ca!news.bc.net!news.mic.ucla.edu!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: Re: RAPDs as dominant markers Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] Date: Fri, 12 Aug 1994 22:24:28 GMT Lines: 61 Subject: Re: RAPDs as dominant markers Newsgroups: bionet.molbio.rapd References: Distribution: I tried to post this earlier but don't think it worked. Guess I'm not too good at this! I hope this one works, please disregard my last post. Kevin O'Donnell (odonnell@sasa.gov.uk) wrote: : I have seen several references to RAPDs (in relation to plant breeding) : as dominant markers. Does 'dominant' as used in this context have a : different meaning from the dominant/recessive gene context? : Kevin O'Donnell : Scottish Agricultural Science Agency : Edinburgh : Scotland Dear All: One problem I have come across regarding dominance of RAPDs is that they are not always dominant. About 10% of our RAPDs turn out to be co-dominant. This is a bit higher than generally reported, but I think that most people don't bother to verify the independence of each bands using southern hybridization, as we do sometimes. The problem of with co-dominant RAPDs we have encountered is when using these bands in a diversity study. The computer program we have been using (NTSYS) assumes all data are dichotomous and independent (presence/absence of bands) and we found if we enter all our data this way, the co-dominant loci are giving in essence the same information two times (not exactly true, but alternate homozygotes get entered twice, once as presence of one band, once as absence of the other one). When we generated a dendrogram of this data matrix (after computing similarity coefficients) it was apparent that the data were skewed because of this. I thought perhaps simply to remove one allele of each co-dominant RAPD from the data matrix, but the heterozygotes prevented this because depending which allele you threw out, you made the heteros look more like one or the other group of homozygotes. I brought this question up at the joint ASHS/CSSA Plant Breeding Symposium, and it generated some discussion later, but no difinitive answers. Some suggestions were: 1. Throw out all data from co-dominant loci (I'd rather not, it took awhile to generate them!) 2. Weight the data so that co-dominant bands are weighted 1/2 of what dominant bands are (I would need another computer program, NTSYS does not allow this; does anyone know a program that does?) 3. Use a LOT of data points so that the co-dominant bands are "swamped out" (but if 10% of my bands are always co-dominant, this won't help). I would appreciate any input you may have on this subject. Sorry the post is so long, but it is hard to describe the problem! Also, I posted it as a reply to this post so people are aware that RAPDs are not ALWAYS dominant, as the current dogma states. Marilyn Warburton grad student, UCD From owner-rapd@net.bio.net Thu Aug 11 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!bcm!cs.utexas.edu!swrinde!gatech!newsxfer.itd.umich.edu!nntp.cs.ubc.ca!news.bc.net!news.mic.ucla.edu!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: Re: RAPDs as dominant markers Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] References: Date: Fri, 12 Aug 1994 22:05:10 GMT Lines: 10 Kevin O'Donnell (odonnell@sasa.gov.uk) wrote: : I have seen several references to RAPDs (in relation to plant breeding) : as dominant markers. Does 'dominant' as used in this context have a : different meaning from the dominant/recessive gene context? : Kevin O'Donnell : Scottish Agricultural Science Agency : Edinburgh : Scotland From owner-rapd@net.bio.net Sun Aug 14 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: osp086@clss1.bangor.ac.uk Newsgroups: bionet.molbio.rapd Subject: bands in the blank Date: 15 Aug 1994 10:56:47 +0100 Lines: 31 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <32ne4v$4p3@mserv1.dl.ac.uk> Original-To: rapd@dl.ac.uk Michael (?) [mam4339@tamsun.tamu.edu] wrote: This is probably a question that gets asked alot here, but I need some help so I'm going to ask it again. My blanks have been showing bands since the first time I tried RAPDs in my lab. In another building I could do them without contamination, apparently, but here, or with the reagents here, I find big, bright, numerous bands in my blanks. I have received advice on the matter and have tried to make use of it but to no avail. I realize that the technique I use is not the absolute best, in terms of contamination, as I have heard of the incredibly sterile, careful facilities and procedures of other, successful labs. But I don't think that kind of care is feasible in the lab I work in, both monetarily and in the interest of time. [stuff deleted] I remember reading a similar query sometime ago on the newsgroup (no idea who from), and one potential reason for these bands in the blanks concerned autoclaving. The gist of it was that if you autoclave tips, tubes etc. in the same autoclave that microbiological samples are done in then their DNA may get coated over your stuff and amplify up later. The sender had said that he/she stopped autoclaving and hey presto! the bands went. No idea if this was true or whether I presented their answer properly but hope it helps. ********************************************************************* * CRAIG WILDING, E-mail: OSP086@BANGOR.AC.UK * * SCHOOL OF OCEAN SCIENCES, Phone: (0)248 351151 ext.2899 * * UNIVERSITY OF WALES (BANGOR), Fax:(0)248 761367 * * MENAI BRIDGE, * * GWYNEDD, LL59 5EY, * * WALES, U.K. * ********************************************************************* From owner-rapd@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!COMP.UARK.EDU!wetges From: wetges@COMP.UARK.EDU (William J. Etges) Newsgroups: bionet.molbio.rapd Subject: Re: dominant vs. codominant Date: 16 Aug 1994 09:50:53 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 38 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408161650.LAA06384@comp.uark.edu> NNTP-Posting-Host: net.bio.net At 2:18 PM 8/16/94 +0000, Toby Bradshaw wrote: >In article <940816162843.6f50@nefelh.imbb.forth.gr>, > wrote: >>Now, after M. Warburton's message on co-dominant RAPDs I'm completely lost! >>What does one mean by "codominant" in this case? If a band can be observed >>in the heterozygous condition, it is *DOMINANT* by definition. > >If an F2 of a cross between red- and white-flowered plants segregates >1:2:1 for red:pink:white, is the red/white marker dominant or codominant? >This is the situation described for RAPD band intensity. While I myself >wouldn't score "codominant" RAPDs based on intensity, band intensity >is as potentially valid a codominant marker as a length polymorphism. >The original Williams et al. manuscript had something to say about this, >but I don't find it in the published version. > >-Toby Bradshaw >toby@u.washington.edu The example of pink heterozygotes is neither "dominant or codominant". This is incomplete dominance. Codominance is defined as inheritance producing heterozygotes which express both parental phenotypes with no mixing, eg allozyme phenotypes, MN blood group polymorphisms in man, and roan coloration in shorthorn cattle. Please everyone, to your closest Strickberger. Bill ------------------------------------------------------------------------- William J. Etges "Rural Arkansas is Department of Biological Sciences poor. Although you University of Arkansas do see better-off areas. Fayetteville, AR 72701 USA You can tell because wetges@comp.uark.edu the trailers are voice: (501) 575-6358 double-wides." FAX (501) 575-4010 P.J. O'Rourke ------------------------------------------------------------------------- From owner-rapd@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!EMUNIX.EMICH.EDU!bio_hannan%emuvax.dnet.emich.edu From: bio_hannan%emuvax.dnet.emich.edu@EMUNIX.EMICH.EDU Newsgroups: bionet.molbio.rapd Subject: bands in the blank Date: 16 Aug 1994 06:12:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <9408161311.AA19541@emunix.emich.edu> NNTP-Posting-Host: net.bio.net Craig, We use a shared mol bio lab and an autoclave that is normally used only for clean equipment and media and we get bands in the negative control about 30-50% of the time. Fortunately, the bands we have found have not overlapped with the bands we get in our Iris lacustris samples. We think that we are very careful about contamination (taking aliquots of most things, sterilizing forceps, etc.) but it shows up anyway. It is possibly a result of sharing the lab with many others, some of whom are culturing Salmonella, Strep, etc. Gary Hannan From owner-rapd@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!VTVM1.CC.VT.EDU!TURNER From: TURNER@VTVM1.CC.VT.EDU (Bruce Turner) Newsgroups: bionet.molbio.rapd Subject: Codominance and rapds Date: 16 Aug 1994 09:48:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408161648.JAA19985@net.bio.net> NNTP-Posting-Host: net.bio.net C'mon guys, the definition of "dominance" in the sense that it is used by RAPD workers is that a presumptive homozygous genotype class can't be distinguishe d from a heterozygous one. It is of importance to RAPDS because often one deal s with presence/absence of a band as two alternative character states. Often, crossing a "presence" with and "absence" gets you a "presence" in the F1, so th at the band is said to be "dominant." Thus, if you score the frequency of a pa rticular band in a population survey, some of the individuals with the band may be homozygotes while others may be heterozygotes. This means, in turn, that o ne can't accurately estimate allele frequencies from phenotypic distribution da ta. Similar problems arise in the interpretation of multilocus VNTR fingerprin ts and other techniques as well. By the way, I'm not a timid soul, but it woul d take a braver person than me to attempt to assign genotypic classes in RAPDs by the INTENSITY of the bands. Wow... From owner-rapd@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!netnews.nwnet.net!news.u.washington.edu!toby From: toby@u.washington.edu (Toby Bradshaw) Newsgroups: bionet.molbio.rapd Subject: Re: dominant vs. codominant Date: 16 Aug 1994 14:18:33 GMT Organization: University of Washington, Seattle Lines: 16 Message-ID: <32qhrp$kra@news.u.washington.edu> References: <940816162843.6f50@nefelh.imbb.forth.gr> NNTP-Posting-Host: stein1.u.washington.edu In article <940816162843.6f50@nefelh.imbb.forth.gr>, wrote: >Now, after M. Warburton's message on co-dominant RAPDs I'm completely lost! >What does one mean by "codominant" in this case? If a band can be observed >in the heterozygous condition, it is *DOMINANT* by definition. If an F2 of a cross between red- and white-flowered plants segregates 1:2:1 for red:pink:white, is the red/white marker dominant or codominant? This is the situation described for RAPD band intensity. While I myself wouldn't score "codominant" RAPDs based on intensity, band intensity is as potentially valid a codominant marker as a length polymorphism. The original Williams et al. manuscript had something to say about this, but I don't find it in the published version. -Toby Bradshaw toby@u.washington.edu From owner-rapd@net.bio.net Mon Aug 15 23:00:00 1994 Path: biosci!NEFELH.IMBB.FORTH.GR!LOUIS From: LOUIS@NEFELH.IMBB.FORTH.GR Newsgroups: bionet.molbio.rapd Subject: dominant vs. codominant Date: 16 Aug 1994 06:30:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <940816162843.6f50@nefelh.imbb.forth.gr> NNTP-Posting-Host: net.bio.net Now, after M. Warburton's message on co-dominant RAPDs I'm completely lost! What does one mean by "codominant" in this case? If a band can be observed in the heterozygous condition, it is *DOMINANT* by definition. I can con- ceivably see situations in which RAPDs would be codominant, but to be able to make a statement like this, one would have to be certain that two bands of a different size (amplified with the same primer) are indeed colinear for a "long" stretch. This would also imply that all amplifications from a given primer have been cloned and sequenced. If this is not what M. Warburton means, then we don't have a situation of codominance! Kitsos Louis From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: codominance take 2 (or more!) Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] Date: Wed, 17 Aug 1994 02:43:24 GMT Lines: 36 Subject: Re: Codominance and rapds Newsgroups: bionet.molbio.rapd References: <199408161648.JAA19985@net.bio.net> Subject: Re: dominant vs. codominant Newsgroups: bionet.molbio.rapd References: <940816162843.6f50@nefelh.imbb.forth.gr> Distribution: world LOUIS@NEFELH.IMBB.FORTH.GR wrote: : Now, after M. Warburton's message on co-dominant RAPDs I'm completely lost! : What does one mean by "codominant" etc. AAH, I cant stand it, my posts arent working! I will try ONE more time. Please desregard MULTIPLE posts to the other thread. Co-dominant RAPDs are as follows: One homozygote has one band, the other has ANOTHER band of different molecular weight, and the heterozygote has BOTH bands (and sometimes a third heteroduplex band). To test for co-dominance, we look for segregation patterns in an F2 and look for a 1:2:1, then cut one band out of the gel and southern hybridize it back to the other band and the heterozygote. If there is a heteroduplex, it hybridizes to that too. Pretty neat, huh? Marilyn Warburton From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!sznew From: sznew@chip.ucdavis.edu () Subject: Codominance Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] Date: Wed, 17 Aug 1994 01:27:22 GMT Lines: 1 Sorry for the blank posts; will try again tomorrow From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: Re: dominant vs. codominant Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] References: <940816162843.6f50@nefelh.imbb.forth.gr> Date: Wed, 17 Aug 1994 00:59:46 GMT Lines: 0 From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: Re: dominant vs. codominant Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] References: <940816162843.6f50@nefelh.imbb.forth.gr> Date: Wed, 17 Aug 1994 00:55:29 GMT Lines: 0 From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: Re: dominant vs. codominant Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] References: <199408161650.LAA06384@comp.uark.edu> Date: Wed, 17 Aug 1994 00:46:20 GMT Lines: 1 ez007415@chip.ucdavis.edu wrote: From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Path: biosci!UKCC.UKY.EDU!VSC003 From: VSC003@UKCC.UKY.EDU (Ernie Bailey) Newsgroups: bionet.molbio.rapd Subject: codominance Date: 17 Aug 1994 09:03:11 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408171603.JAA02299@net.bio.net> NNTP-Posting-Host: net.bio.net Dominant simply means the gene is expressed (detected) whenever it is present. Codominance means that genotype can be inferred from the phenotype,e.g., heterozytgotes can be distinguished from homozygotes. From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!netnews.nwnet.net!news.u.washington.edu!toby From: toby@u.washington.edu (Toby Bradshaw) Newsgroups: bionet.molbio.rapd Subject: Re: dominant vs. codominant Date: 17 Aug 1994 14:40:22 GMT Organization: University of Washington, Seattle Lines: 22 Message-ID: <32t7gm$kkh@news.u.washington.edu> References: <940817145951.744f@nefelh.imbb.forth.gr> NNTP-Posting-Host: stein1.u.washington.edu In article <940817145951.744f@nefelh.imbb.forth.gr>, wrote: >In answer to my comments on dominance/codominance Toby Bradshaw wrote: > >>If an F2 of a cross between red- and white-flowered plants segregates >>1:2:1 for red:pink:white, is the red/white marker dominant or codominant? >>This is the situation described for RAPD band intensity. While I myself >>wouldn't score "codominant" RAPDs based on intensity, band intensity >>is as potentially valid a codominant marker as a length polymorphism. > >While I also think that band intensity can be of value for certain purposes, >this is *NOT* an example of co-dominance. Which is why I put "codominant" in quotes. The marker may be *scored* as though it is codominant (assuming one has confidence in determining the 3 phenotypes), even though (strictly speaking) the inheritance can be described as partial dominance. Of course, strictly speaking, RAPD bands are phenotypes and not genotypes, but that doesn't stop (most of) us from determining "genotypes" with them. -Toby Bradshaw toby@u.washington.edu From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Path: biosci!NEFELH.IMBB.FORTH.GR!LOUIS From: LOUIS@NEFELH.IMBB.FORTH.GR Newsgroups: bionet.molbio.rapd Subject: Re: dominant vs. codominant Date: 17 Aug 1994 05:01:42 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 33 Sender: daemon@net.bio.net Distribution: world Message-ID: <940817145951.744f@nefelh.imbb.forth.gr> NNTP-Posting-Host: net.bio.net In answer to my comments on dominance/codominance Toby Bradshaw wrote: >If an F2 of a cross between red- and white-flowered plants segregates >1:2:1 for red:pink:white, is the red/white marker dominant or codominant? >This is the situation described for RAPD band intensity. While I myself >wouldn't score "codominant" RAPDs based on intensity, band intensity >is as potentially valid a codominant marker as a length polymorphism. While I also think that band intensity can be of value for certain purposes, this is *NOT* an example of co-dominance. 1) The Bar locus of D. melanogaster (to use a classical example) has, in the homozygous condition, a different phenotype from the heterozygous one. Yet, the mutation is a classical example of dominance, and if you wish, it correlates fully with the case where one can distinguish between a RAPD having a different intensity when heterozygous versus the homozygous condition. 2) "Introduction to Genetic Analysis" (Suzuki et al.) calls the example of red/pink/white petals "incomplete dominance" while "codominance" is defined as the condition in which the phenotypes of *both* the homozygotes are seen in the heterozygote. The M-N bloodgroups are given as examples. In the case of RAPDs this would imply that codominant RAPDS both "have the band present and absent". The absurdity of this is obvious. On the other hand, RFLPs and SSRs are indeed bona fide codominant markers since the definition of codominance describes what we see. 3) As I said in my original posting, the only case of real codominance in RAPDs can be obtained when, using the same primer, one identifies two different bands amplified from the same DNA and differing in length. In this case, we will either see either one of the bands (in the two homozygous cases) or both, when heterozygous. Strictly speaking of course, this example is one of two RAPDs, and not one! While this can be the case (we have identified at least one such RAPD in An. gambiae), the work involved in identifying such helpful RAPDs is, very mildly put, tedious, and we should stick to RAPDs being dominant, with all of their advantages and disdvantages. Kitsos Louis From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: Re: Codominance and rapds Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] References: <199408161648.JAA19985@net.bio.net> Date: Wed, 17 Aug 1994 00:16:05 GMT Lines: 4 True, I would never trust a "less intense" band to mean heterozygous; there is too many factors affecting amplification! Marilyn Warburton From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: Re: dominant vs. codominant Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] References: <199408161650.LAA06384@comp.uark.edu> Date: Wed, 17 Aug 1994 00:29:21 GMT Lines: 0 From owner-rapd@net.bio.net Tue Aug 16 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!library.ucla.edu!news.ucdavis.edu!chip.ucdavis.edu!ez007415 From: ez007415@chip.ucdavis.edu () Subject: Re: dominant vs. codominant Message-ID: Sender: usenet@ucdavis.edu (News Guru) Organization: University of California, Davis X-Newsreader: TIN [version 1.2 PL2] References: <199408161650.LAA06384@comp.uark.edu> Date: Wed, 17 Aug 1994 00:13:44 GMT Lines: 0 From owner-rapd@net.bio.net Wed Aug 17 23:00:00 1994 Newsgroups: bionet.molbio.rapd Path: biosci!agate!howland.reston.ans.net!vixen.cso.uiuc.edu!news.uoregon.edu!netnews.nwnet.net!ns1.nodak.edu!plains!mcclean From: mcclean@plains.NoDak.edu (Phillip McClean) Subject: Re: dominant vs. codominant Sender: usenet@ns1.nodak.edu (Usenet login) Message-ID: Date: Thu, 18 Aug 1994 04:18:00 GMT References: <199408161650.LAA06384@comp.uark.edu> Nntp-Posting-Host: plains.nodak.edu Organization: North Dakota Higher Education Computing Network X-Newsreader: TIN [version 1.2 PL2] Lines: 35 I'd like to pipe in on this topic. I teach genetics and as hard as we might try, the term codominance will be with us. The term is classically applied to the red/white/pink situation. Newer terms that have been proposed, but never widely adopted, are no dominance and incomplete dominance. Codominace and no dominance are equivalent terms and mean that the both alleles are expressed equally in the F1. RFLPs are a great molecular example as are the blood types that can be distingushed immunologically. If the the amount of red and whitle pigments were equal in the pink F1 then the classical example would be codominance. Unfortunately, the two pigments are not expressed equally. Thus the term incomplete dominance was born. With regards to codominant RAPD alleles, you can simply score them in a segregating population. We are scoring a recombinant inbred population of common bean. We often have several bands that differ between the two parents used in the initial cross to develop the population. If this is the case we determine if any two bands, one present in one parent and a second that is present in the other parent are alleles, in the following manner. If they are alleles you will never see a line in the segregating population in which both bands are missing. You will see lines that contain one or the other band and a line that contains both bands. Those with both bands are heterozygous lines. Our population consists of F2 derived F5 lines and we expect that 5% of the lines will be heterozygous. To date we have identified five codominant loci and statistically (by chi-square analysis) 5% of the lines in the population are heterozygous. This was reassuring --- genetics actually works as predicted. As far as using RAPDs and trying to score intensity, I think you are getting garbage data. The technique is just not clean enough for that. I know that those doing variability studies need as much data as possible, so I agree that the best thing to do for those experiments is just run through a lot of primers and minimize the possible codominace effects. Phil McClean mcclean@plains.nodak.edu From owner-rapd@net.bio.net Wed Aug 17 23:00:00 1994 Path: biosci!YVAX.BYU.EDU!FARMERJ From: FARMERJ@YVAX.BYU.EDU Newsgroups: bionet.molbio.rapd Subject: Re: dominant vs. codominant Date: 18 Aug 1994 11:42:41 -0700 Organization: Brigham Young University Lines: 53 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HG1ZPLVVK28Y8Z1S@yvax.byu.edu> NNTP-Posting-Host: net.bio.net RE: dominant vs. codominant This discussion is getting far afield from RAPD per se, but it is clearly relevant to the intelligent application of names to things that we study. I looked up "dominant" and "codominant" in the two editions of Rieger, Michaelis, and Green's GLOSSARY OF GENETICS. Various forms of dominance, including incomplete dominance, are listed under "dominant". "Codominant" has its own entry. Unfortunately, the definitions in the two editions differ. The definition in the newer edition follows. codominant-- of two alleles when there are three phenotypes corresponding to the three genotypes (A1 A1, A1 A2, A2 A2). This definition is almost like that of semi-dominance (partial dominance or incomplete dominance), except that it does not specify that the heterozygote be intermediate in phenotypic expression. I looked up the definition in several genetics textbooks and the consensus seems to be the following. Semi-dominance occurs when the phenotype of the heterozygote is intermediate between the phenotypes of the two homozygotes. The classical example is red, pink, and white flowers when that phenotype is controlled by a single gene with two alleles. Codominance occurs when the phenotype of each allele is fully expressed in the heterozygote. That is impossible, of course, in the case of flower colors since a flower cannot be both fully red and fully white at the same time. The classical example is MN blood type. There are very few good examples of codominance. Along these lines, something that has always annoyed me is calling a human disorder such as Achondroplastic dwarfism "dominant". Like everyone else, I call it dominant for lack of a better word, but the three genotypes are normal, achondroplastic dwarf, and lethal. The heterozygote is clearly unlike either homozygote. It is amazing how hard it is for many students to understand that point. They confuse labels with reality. James L. Farmer Department of Zoology 571 WIDB Brigham Young University Provo, Utah 84602, USA (801) 378-2153 (voice, phone mail) (801) 378-7499 (FAX) FARMERJ@YVAX.BYU.EDU From owner-rapd@net.bio.net Thu Aug 18 23:00:00 1994 Path: biosci!CFL.FORESTRY.CA!HAMELIN From: HAMELIN@CFL.FORESTRY.CA Newsgroups: bionet.molbio.rapd Subject: terminology Date: 19 Aug 1994 06:50:37 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 4 Sender: daemon@net.bio.net Distribution: world Message-ID: <940819094927.18648@CFL.FORESTRY.CA> NNTP-Posting-Host: net.bio.net I have read some people call RAPD patterns (i.e. the set of DNA bands one visualizes after amplification and electrophoresis) amplitypes. I have also read (heard?) the term amplicon. Is there any concensus out there regarding these terms? From owner-rapd@net.bio.net Thu Aug 18 23:00:00 1994 Path: biosci!CHRISTA.UNH.EDU!sbenard From: sbenard@CHRISTA.UNH.EDU (Susan A Benard) Newsgroups: bionet.molbio.rapd Subject: Quality of RAPDs Date: 19 Aug 1994 08:20:58 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 31 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net My banding patterns are usually reproducible but I often get a background smear that may obscure some of the bands making the gels hard to score. I also find variability in the tightness of the bands, occasionally I get great looking bands but of late most of my experiments have yielded bands that are more diffused. I should also add that my results used to be consistently good but that RAPD amplification quality and quantity have fallen off during the past 8 months. I have tried changing amounts and types of Taq, buffer(lots and types), making new template and changing concentrations of it, looking at different MgCl2 and dNTP concentrations and adding BSA to reactions. I have sent my thermocyclers in to be checked and check them frequently with my own thermocouple I've also tried decreasing denaturation temps and times to avoid Taq damage. I'm currently using 34 as my annealing temp but have also looked at 33, 35, and 36. The upshot of all this fooling around is that on two consequtive days with identical conditions I cannot reproduce the occasionally good looking gel. I have also tried altering my gel running conditions ie voltage, thickness of gel, with and without EtBr in the gel, depth of running buffer over the gel. I cannot find a single parameter that consistently changes my results. I am getting primers from UBC and will be trying some from Operon soon. Has anyone had trouble with primers from UBC Thanks for any suggestions! Susan Benard University of New Hampshire Biochemistry Dept. Sbenard@christa.unh.edu ^X From owner-rapd@net.bio.net Fri Aug 19 23:00:00 1994 Path: biosci!UNIXG.UBC.CA!hobbs From: hobbs@UNIXG.UBC.CA Newsgroups: bionet.molbio.rapd Subject: Re: Quality of RAPDs Date: 19 Aug 1994 17:37:49 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 52 Sender: daemon@net.bio.net Distribution: world Message-ID: <9408200037.AA22671@unixg.ubc.ca> NNTP-Posting-Host: net.bio.net On 19th. August 1994, Susan Benard wrote: >My banding patterns are usually reproducible but I often get a background >smear that may obscure some of the bands making the gels hard to score. I >also find variability in the tightness of the bands, occasionally I get >great looking bands but of late most of my experiments have yielded >bands that are more diffused. I should also add that my results used to >be consistently good but that RAPD amplification quality and quantity have >fallen off during the past 8 months. I have tried changing amounts and >types of Taq, buffer(lots and types), making new template and changing >concentrations of it, looking at different MgCl2 and dNTP concentrations >and adding BSA to reactions. I have sent my thermocyclers in to be checked >and check them frequently with my own thermocouple I've also tried decreasing >denaturation temps and times to avoid Taq damage. I'm currently using 34 as >my annealing temp but have also looked at 33, 35, and 36. The upshot of all >this fooling around is that on two consequtive days with identical >conditions I cannot reproduce the occasionally good looking gel. I have also >tried altering my gel running conditions ie voltage, thickness of gel, >with and without EtBr in the gel, depth of running buffer over the gel. >I cannot find a single parameter that consistently changes my results. >I am getting primers from UBC and will be trying some from Operon soon. > >Has anyone had trouble with primers from UBC >Thanks for any suggestions! >Susan Benard >University of New Hampshire >Biochemistry Dept. >Sbenard@christa.unh.edu I am puzzled by her observations, and have e-mailed to ask the history in terms of age, storage, etc. of the primers she is using, and also how general (restricted to just a few primers, to many, to re-ordered primers) is her observed deterioration in banding patterns . This may throw some light on the problem. Meantime, if others have made similar observations using our primers, I would like to hear about it also. There will be a delay in responding to e-mail while I am on vacation, but I would like feedback if recipients of our primers are having problems (or successes, come to that!) which they think are primer-related. John. 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 Sun Aug 21 23:00:00 1994 Path: biosci!agate!doc.ic.ac.uk!daresbury!not-for-mail From: "Dijk van, Peter" Newsgroups: bionet.molbio.rapd Subject: DNA software Date: 22 Aug 1994 11:16:10 +0100 Lines: 11 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <339tta$gda@mserv1.dl.ac.uk> Encoding: 11 TEXT Original-To: rapd At our institute we want to buy DNA software for multiple sequence allignments , database searches, restriction site analysis etc. We are considering PCgene or Lasergene for Windows. Who has experience with Lasergene for Windows? (or for Apple Macinthosh?). Are there alternatives? Thanks in advance, Peter van Dijk Netherlands Institute for Ecological Research Email: pjvandijk@nioo.nl From owner-rapd@net.bio.net Sun Aug 21 23:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: Poor RAPD band quality Date: 22 Aug 1994 11:55:55 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <9408221855.AA16150@gw1.phibred.com> NNTP-Posting-Host: net.bio.net Dear Susan Benard, Seeing that you have checked everything form Thermocyclers, gels and reagents (except primer) you should check the tubes that you perform the reactions in. I have had failed reactions using the falcon 96 well plates (U bottom). If you wash with BSA, it should be non-acetylated- (Perkin_Elmer sales rep recommended non-actylated BSA). If the reactions still fail with new tubes (or autoclaved or treated with UV overnight- a past discussion addressed background and bands in negative controls.), you could try calling one of the technical service lines, such as: Perkin Elmer or Boehringer Mannheim. Hope that this helps. Ian Murray Research Associate Pioneer Hi-Bred, Georgetown, Canada From owner-rapd@net.bio.net Tue Aug 23 23:00:00 1994 Newsgroups: bionet.plants,bionet.molbio.gdb,bionet.immunology,nz,molbio,bionet.women-in-bio,bionet.virology,bionet.journals.note,umn.bioc.class.5025,umo.biomed.inroads,umo.biomed.engrg,bionet.n2-fixation,bionet.biology.n2-fixation,bionet.molbio.rapd Path: biosci!rutgers!gatech!howland.reston.ans.net!europa.eng.gtefsd.com!library.ucla.edu!csulb.edu!nic-nac.CSU.net!charnel.ecst.csuchico.edu!csusac!csus.edu!netcom.com!syllabus From: syllabus@netcom.com (Syllabus Press) Subject: Free Syllabus magazine subscription Message-ID: Organization: NETCOM On-line Communication Services (408 261-4700 guest) Date: Wed, 24 Aug 1994 19:43:08 GMT Lines: 166 Xref: biosci bionet.plants:3878 bionet.molbio.gdb:230 bionet.immunology:1887 bionet.women-in-bio:1374 bionet.virology:771 bionet.journals.note:312 bionet.biology.n2-fixation:205 bionet.molbio.rapd:757 Educators are qualified to receive Syllabus magazine 9 times per year FREE! 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(check all that apply) (A) o Productivity software (B) o Multimedia software (C) o Utility software (D) o Math & statistics software (E) o Computer upgrades/expansion cards (F) o Networking/communications software/hardware (G) o Audio/visual hardware (H) o Visualization/graphics products (I) o Educational content/information software (J) o Personal/desktop computers (K) o Printers (L) o Laptop computers (M) o Workstations (N) o Simulation software (O) o Scanners (P) o Disk drives/memory storage units (Q) o Classroom presentation software (R) o Monitors & projection devices (S) o CD-ROM Signature required _________________________________________________________ (not valid without signature) Date ____________________________________________________ -- syllabus@netcom.com From owner-rapd@net.bio.net Tue Aug 23 23:00:00 1994 Path: biosci!HARPO.TNSTATE.EDU!GAWELN From: GAWELN@HARPO.TNSTATE.EDU Newsgroups: bionet.molbio.rapd Subject: ALTERNATIVES TO CTAB-BASED PLANT DNA EXTRACTIONS Date: 24 Aug 1994 12:17:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HGAGUE820I8WYI51@HARPO.TNSTATE.EDU> NNTP-Posting-Host: net.bio.net CTAB-based extraction protocols have never failed me until now. I am trying to run RAPDs on a heavily-pigmented woody ornamental (Loropetalum). I either get no DNA or an oily red precipitate. If any one has a good alternative/modification extraction protocol, please let me know. Thanks. Nick Gawel GAWELN@ACAD.TNSTATE.EDU From owner-rapd@net.bio.net Wed Aug 24 23:00:00 1994 Path: biosci!agate!library.ucla.edu!csulb.edu!nic-nac.CSU.net!gopher.sdsc.edu!news.tc.cornell.edu!travelers.mail.cornell.edu!newsstand.cit.cornell.edu!NewsWatcher!user From: wfl1@cornell.edu (Warren Frank Lamboy) Newsgroups: bionet.molbio.rapd Subject: Next PLANT GENOME meetings Followup-To: bionet.molbio.rapd Date: Thu, 25 Aug 1994 00:25:41 -1200 Organization: Cornell University Lines: 10 Sender: wfl1@cornell.edu (Verified) Message-ID: NNTP-Posting-Host: 132.236.3.90 Does anyone know when the next Plant Genome meetings will be held, and whom I might contact for more information? Thank you. -- Warren F. Lamboy Dept. of Horticultural Sciences and USDA-ARS Plant Genetic Resources Unit Cornell University Geneva, NY 14456-0462 warren_lamboy@cornell.edu From owner-rapd@net.bio.net Wed Aug 24 23:00:00 1994 Path: biosci!internet!biosci!not-for-mail From: kristoff (David Kristofferson) Newsgroups: bionet.molbio.rapd Subject: UNSUBSCRIBING, BIOSCI ARCHIVES, ADDRESS DATABASE & BIOSCI FAQ Date: 25 Aug 1994 02:00:29 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 322 Sender: daemon@net.bio.net Distribution: world Message-ID: <199408250900.CAA16843@net.bio.net> NNTP-Posting-Host: net.bio.net Four important items follow: How to cancel e-mail subscriptions to BIOSCI newsgroups, BIOSCI archive searching, the BIOSCI FAQ, and the BIOSCI User Address Directory form. If you have not yet listed yourself in our BIOSCI user directory, please take a few minutes to complete and return the form below. 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Thanks again for your cooperation! --------------- please cut here and return portion below --------------- New information or Update to old record (enter N or U): date (DD-MM-YY): first name: middle initial: family name: job title: e-mail address: e-mail network: phone number: FAX number: institution: address1: address2: address3: city: state/province: country: postal code: research interest: research interest: comment: comment: comment: comment: comment: From owner-rapd@net.bio.net Thu Aug 25 23:00:00 1994 Path: biosci!EMUNIX.EMICH.EDU!bio_hannan%emuvax.dnet.emich.edu From: bio_hannan%emuvax.dnet.emich.edu@EMUNIX.EMICH.EDU Newsgroups: bionet.molbio.rapd Subject: alternative to CTAB-based plant DNA extractions Date: 25 Aug 1994 19:57:01 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <9408260250.AA23008@emunix.emich.edu> NNTP-Posting-Host: net.bio.net Nick, I had problems getting clean, amplifiable DNA from Iris lacustris (got gooey gunk, and got no amplification). We had to resort to a mild extraction method using CTAB, but instead of grinding the leaf tissue in liquid N, we mashed the previously frozen leaf tissue in a 1.5ml tube using a pipette tip, then adding CTAb, press the trapped air out of the tissue with the same pipette tip, freezing the tube at -20 for 45-60 min (or storing at -20 in CTAB for days), then lysis at 65C for 45 min, followed with sodium acetate & ethanol (not isopropyl) precipitation, then Bioclean (US Biochemical product). Low yield (ca 150-700 ng from .02-.15g leaf tissue), but it seems to be clean and it does amplify using Operon Kit A primers. The freeze/thaw extraction method worked best using -20C. At -70 and with liquid N, yield seemed to be much lower. Good luck. Gary HAnnan From owner-rapd@net.bio.net Mon Aug 29 23:00:00 1994 Path: biosci!agate!ihnp4.ucsd.edu!munnari.oz.au!newsroom.utas.edu.au!mg1_98.plant.utas.edu.au!user From: Kath.Nesbitt@plant.utas.edu.au () Newsgroups: bionet.molbio.rapd Subject: Re: codominance take 2 (or more!) Followup-To: bionet.molbio.rapd Date: Tue, 30 Aug 1994 15:51:14 +1000 Organization: University of Tasmania Lines: 27 Message-ID: References: NNTP-Posting-Host: mg1_98.plant.utas.edu.au > Co-dominant RAPDs are as follows: > One homozygote has one band, the other > has ANOTHER band of different molecular > weight, and the heterozygote has BOTH > bands (and sometimes a third heteroduplex > band). To test for co-dominance, we look > for segregation patterns in an F2 and look > for a 1:2:1, then cut one band out > of the gel and southern hybridize it back to > the other band and the heterozygote. > If there is a heteroduplex, it hybridizes > to that too. Pretty neat, huh? > > Marilyn Warburton What is meant by a heteroduplex? What size woud you expect it to be? In my lab we are having trouble with the cutting oout and re-amplification bit, do you have any suggestions? People are also trying to clone the band for sequencing. Kath Nesbitt P.s. It seems that I have to make more text than I'm replying to for this news-server to post my questions. Sometimes I just don't understand computers!!! I wonder if that's enough text for it.