From owner-rapd@net.bio.net Thu Jan 02 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!news-peer.gsl.net!news.gsl.net!howland.erols.net!news.nacamar.de!news.icm.edu.pl!uw.edu.pl!news.nask.pl!lublin.pl!biopc07.umcs.lublin.pl!tchorz From: tchorz@biotop.umcs.lublin.pl (Marek Tchorzewski) Newsgroups: bionet.molbio.rapd Subject: DNA software Date: Fri, 3 Jan 1997 12:36:42 GMT Organization: Marie Curie-Sklodowska University Lines: 20 Message-ID: NNTP-Posting-Host: biopc07.umcs.lublin.pl Dear Netters, First of all, Happy New Year 1997 to all of you !!! On the other hand, I need advice concerning programs for DNA and protein analysis. I am going to buy such program however I am not fully determined which program I should purchase. So far, I have seen DNAsis and DNAstar, however I am not sufficiently confident that these programs are reliable, and can provide me with the best options. So, could you advice me, which programs is actually the best on the market. Marek Tchorzewski PhD Univ. of Maria Curie-Sklodowska Dept. of Molecular Biology Lublin, Poland From owner-rapd@net.bio.net Sat Jan 04 22:00:00 1997 Path: biosci!HORTUS.AR.LUBLIN.PL!tyran From: tyran@HORTUS.AR.LUBLIN.PL (Tyrka) Newsgroups: bionet.molbio.rapd Subject: wheat Date: 5 Jan 1997 04:22:14 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <32D01B9A.3EAD@hortus.ar.lublin.pl> NNTP-Posting-Host: net.bio.net subscribe rapd sub rapd From owner-rapd@net.bio.net Wed Jan 08 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!news-peer.gsl.net!news.gsl.net!news-penn.gsl.net!news.gsl.net!news.NetVision.net.il!news From: Mark Klein Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins,bionet.molbio.rapd Subject: FREE pH measurement booklet Date: 9 Jan 1997 10:49:48 GMT Organization: NetVision LTD. Lines: 9 Message-ID: <5b2igc$2bp@news.NetVision.net.il> NNTP-Posting-Host: ts006p13.pop2a.netvision.net.il Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.2N (Windows; I; 16bit) Xref: biosci bionet.molbio.methds-reagnts:53275 bionet.molbio.proteins:9679 bionet.molbio.rapd:1770 A free pH booklet is available which contains valuable information on basic pH measurement theory, pH measurement techniques, selecting the proper pH electode for a particular application, and a pH troubleshooting guide. The booklet is available from Lazar Research Labs. Inc. by emailing service@lazarlab.com or faxing 1-213-931-1434. The booklet can also be obtained from the Lazar web site at http://www.lazarlab.com From owner-rapd@net.bio.net Sat Jan 11 22:00:00 1997 Path: biosci!DIRECT.CA!amayrs From: amayrs@DIRECT.CA (Andrw Mayrs) Newsgroups: bionet.molbio.rapd Subject: QUESTIONS??? Date: 11 Jan 1997 20:50:36 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: world Message-ID: <32D86BED.3362@direct.ca> NNTP-Posting-Host: net.bio.net I'm looking for anything and everything surrounding the sexing of cetacea (especially killer whales) using PCR. I have DNA obtained through biopsy sampling of free ranging juveniles and wish to begin ASAP. Any information/ideas/links on the topic would be greatly appreciated. Thanks--Valentina Mendoza vallymun@unixg.ubc.ca From owner-rapd@net.bio.net Wed Jan 15 22:00:00 1997 Path: biosci!daresbury!not-for-mail From: Stephane.Pasteau@pasteur-lille.fr (Stephane Pasteau) Newsgroups: bionet.molbio.rapd Subject: Duck female specific nucleic probe Date: 16 Jan 1997 11:10:55 -0000 Lines: 17 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5bl2bv$f42@mserv1.dl.ac.uk> Original-To: rapd@dl.ac.uk I'm looking for a nucleic probe that I could use for sexing a duck female. I know that it exists specic probes on chicken W chromosome. Does anybody know if probes like that exist for the duck and if not, can I use chicken probes on the duck. Last question, could anybody give me such a probe. Thank you in advance for answers (you can use my email) Dr Stephane PASTEAU Institut Pasteur de Lille Service de Recherche et D=E9veloppement Domaine du CERTIA 369, rue Jules Guesde - B.P. 39 59651 VILLENEUVE D'ASQ cedex tel. : (33) 03-20-43-86-67 fax. : (33) 03-20-43-86-66 e.mail : Stephane.Pasteau@pasteur-lille.fr From owner-rapd@net.bio.net Thu Jan 16 22:00:00 1997 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.rapd Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 17 Jan 1997 03:43:49 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199701171000.CAA28881@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. C) In the body of your message put one or more of the following commands with an "end" command on the last line, e.g., subscribe methods unsubscribe methods end Do NOT put your e-mail address or other text on these lines. The server only allows you to cancel your subscription if the address on your mail header matches the address on our mailing list. Please ask for help at biosci-help@net.bio.net if your address has changed, e.g., if you know you are on the list but the server tells you that you are not a member. Users in Europe, Africa, and Central Asia who use the BIOSCI node at -------------------------------------------------------------------- computer daresbury.ac.uk (also known as dl.ac.uk): ------------------------------------------------- To subscribe and unsubscribe to/from the BIOSCI lists, you need to specify the full USENET newsgroup name with "bionet-news." prepended. The USENET newsgroup names are listed in the BIOSCI Information sheet on the Web at http://www.bio.net/. For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the body of the message as text on the Subject: line is ignored. To unsubscribe from all the lists at the UK node, use unsub bionet-news Please note that if the address in the list is different than the one in your mail message header, you will not be able to unsubscribe by this method. If you have problems, please mail biosci@daresbury.ac.uk. 4) The BIOSCI user address and research interest directory. ----------------------------------------------------------- Please take this opportunity to add your name, address, and research interest information to the BIOSCI User Address Database if you have not already done so. You can fill out the address form directly through our Web page at URL http://www.bio.net/adrform.html. The address database is reindexed nightly for WWW access (the URL is http://www.bio.net/). If you are not directly on the Internet but can reach it by e-mail, please use our waismail server to access the user directory. waismail use is described above. You can also request a user address form by e-mail from biosci-help@net.bio.net. Please check your database entry from time-to-time to see if your address information is still up-to-date. Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-rapd@net.bio.net Mon Jan 20 22:00:00 1997 Path: biosci!rutgers!gatech!csulb.edu!hammer.uoregon.edu!news-xfer.netaxs.com!newshub2.home.com!news.home.com!newshub1.home.com!news.home.com!ames!purdue!news.bu.edu!ppp-77-8.bu.edu!user From: gilbride@bu.edu (Kevin Gilbride) Newsgroups: bionet.molbio.rapd Subject: ? Viral ID using RAPD ? Date: Tue, 21 Jan 1997 16:02:15 -0500 Organization: Mallory Institute of Pathology Lines: 6 Message-ID: NNTP-Posting-Host: ppp-77-8.bu.edu I am interested in any work, published or ongoing, involving the use of RAPD in viral detection and or typing. Thanks, Kevin From owner-rapd@net.bio.net Thu Jan 23 22:00:00 1997 Path: biosci!USERS.AFRICAONLINE.CO.KE!wellcome From: wellcome@USERS.AFRICAONLINE.CO.KE ("Wellcome Trust Research Labs, Nairobi") Newsgroups: bionet.molbio.rapd Subject: Some Advice about PCR: Dr. NZILA ALEXIS Date: 24 Jan 1997 01:23:36 -0800 Organization: Wellcome Trust Research Labs Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <32E8D960.1CE2@users.africaonline.co.ke> NNTP-Posting-Host: net.bio.net To the news group I am amplifying 2 fragments of an unique DNA sequence of malaria; pfmdr1 gene with 2 different sets of primers. All primers are 21 nucleotides length .Set1 primers have Tm of 59 and 61 °C and Set2 have 61.7 and 51.2°C. PCR is carried out in standard condition and the Tm=40 °C. Using a same amount of DNA template (5 ul) of purified culture DNA, only Set2 give an amplification. Set1 give an amplification if the template is 20 to 50 times more. How can someone explain this difference in the PCR efficiency. The gene target is the same only the site of primers annealing differs. Moreover, Tm primers are quite similar and I used very low Tm (40 °C) for amplification. So, when amount of DNA is not high (naturally infected sample), Set2 can not amplify the gene however Set1 does. Is there a problem in my PCR condition? As the PCR conditions are the same for the 2 sets, why this difference? Alexis FOR ANSWER: PLEASE PRECISE MESSAGE FOR ALEXIS NZILA From owner-rapd@net.bio.net Mon Jan 27 22:00:00 1997 Path: biosci!ASRR.ARSUSDA.GOV!abartlet From: abartlet@ASRR.ARSUSDA.GOV ("Alan C. Bartlett") Newsgroups: bionet.molbio.rapd Subject: Re: Destruction of DNA contamination by Microwave Date: 28 Jan 1997 12:28:29 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <01IEQNSUI5EQCHHS6Q@tay.ac.uk> NNTP-Posting-Host: net.bio.net This problem has come up on the list before and I think it is an almost universal problem with RAPD's. I believe that it is a contamination of DNA in the TAQ (although the manufacturers stoutly deny this). It seems that if there is any sample DNA present, it will overshadow the contaminating DNA from the TAQ so that we never get matching bands in our sample lanes and the control lanes for a given primer are often very similar or identical. I have come to believe that bands in my control lanes are proof of the cleanliness of our techniques :^) Alan C. Bartlett "GENES-R-US" "ALTERATIONS AVAILABLE!^" "^some restrictions may apply:)" From owner-rapd@net.bio.net Mon Jan 27 22:00:00 1997 Path: biosci!tay.ac.uk!MLTAJS From: MLTAJS@tay.ac.uk Newsgroups: bionet.molbio.rapd Subject: Destruction of DNA contamination by Microwave Date: 28 Jan 1997 00:45:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <01IEQNSUI5EQCHHS6Q@tay.ac.uk> NNTP-Posting-Host: net.bio.net Hello there. I am using RAPD PCR to detect fungal DNA extracted from wood samples. The system does work and by comparing the banding pattern to that obtained from pure fungal DNA I can map the spread of a particular fungus through wood. The problem is I get bands appearing in the control (no DNA samples) which do not appear in any of the sample tracks. All the equipment (pipette tips, eppendorf tubes, etc.) have been autoclaved (121'C) twice for thirty minutes. The only equipment that has not been cleaned are the pipettes, which were used for normal lab purposes previous to being used specifically for PCR. So, I have a sneaking suspicion that the DNA contamination is coming from them. What I would like to ask is whether DNA contamination on equipment could be removed by microwaving them?. Also, can pipettes be cleaned to remove any contaminating DNA that may be present inside them?. Thanks, in advance, for any help that you can provide. Alan Score. From owner-rapd@net.bio.net Mon Jan 27 22:00:00 1997 Path: biosci!agate!howland.erols.net!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed1.bbnplanet.com!ox.mc.edu!usenet From: Robert Hamilton Newsgroups: bionet.molbio.rapd Subject: Re: Destruction of DNA contamination by Microwave Date: 28 Jan 1997 15:30:24 GMT Organization: Mississippi College Lines: 23 Message-ID: <5cl62g$rut@ox.mc.edu> References: <01IEQNSUI5EQCHHS6Q@tay.ac.uk> NNTP-Posting-Host: mobo.mc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) We have the same problem, and it is not the tips for us. It is either complexes of the primers or DNA in the DNA polymerase. We have used clonetechs antitaq to try to deal with primer complexes, and reduced the problem a little. We have not been able to destroy DNA in the DNA polymerase without adversely affecting the reactions with either psoralen or DNAase. I think that this is a widespread problem that goes unreported (as evidenced by the lack of FULL control lanes in many publications involving the use of RAPDs). MLTAJS@tay.ac.uk wrote: >Hello there. I am using RAPD PCR to detect fungal DNA extracted from wood >samples. The system does work and by comparing the banding pattern to that >obtained from pure fungal DNA I can map the spread of a particular fungus >through wood. The problem is I get bands appearing in the control (no DNA >samples) which do not appear in any of the sample tracks. All the equipment >(pipette tips, eppendorf tubes, etc.) have been autoclaved (121'C) twice >for thirty minutes. >Thanks, in advance, for any help that you can provide. > >Alan Score. From owner-rapd@net.bio.net Tue Jan 28 22:00:00 1997 Path: biosci!ACS.UCALGARY.CA!twhidden From: twhidden@ACS.UCALGARY.CA (Troy Whidden) Newsgroups: bionet.molbio.rapd Subject: subscribe Date: 29 Jan 1997 08:56:19 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net subscribe From owner-rapd@net.bio.net Tue Jan 28 22:00:00 1997 Path: biosci!pbi.nrc.ca!tdemeke From: tdemeke@pbi.nrc.ca ("Demeke, Tigst") Newsgroups: bionet.molbio.rapd Subject: Destruction of DNA contamination by microwave Date: 29 Jan 1997 13:20:31 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <32EFDB75@gate1.pbi.nrc.ca> NNTP-Posting-Host: net.bio.net Comments for the questions posed: 1. Most pipets are autoclavable. Check with the manufacturer if yours can be autoclaved. 2. DNA in the control lanes may be from the Taq DNA polymerase you are using. Several people have noticed this. Change your Taq source and see if you don't get DNA in the control lanes. Demeke T. tdemeke@pbi.nrc.ca From owner-rapd@net.bio.net Tue Jan 28 22:00:00 1997 Path: biosci!rutgers!gatech!csulb.edu!hammer.uoregon.edu!arclight.uoregon.edu!netnews.nwnet.net!news.nodak.edu!plains!comstock From: comstock@plains.nodak.edu (Clay Comstock) Newsgroups: bionet.molbio.rapd Subject: contamination Date: 29 Jan 1997 01:47:31 GMT Organization: North Dakota Higher Education Computing Network (NDHECN) Lines: 29 Message-ID: <5cma7j$337@daily-planet.nodak.edu> NNTP-Posting-Host: plains.nodak.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit X-Newsreader: TIN [version 1.2 PL2] I don't know how much it is worth but many people have told me as long as the bands in your control are not in your sample lanes then it is ok. I myself find this hard to believe. I have also ran into problems with contamination in the glassware. They say that autoclaving degrades the DNA but I found that washing the glass with acid then heating it in an oven or sterilizing with UV light helps. I have also raninto problems with my primers it is easy to contaminate them, as well as the polymerase(don't rule out bad polymerase from the start). Also the pipetors can pose a problem, use barrier tips if you already haven't or clean with acid( there is a technique but I don't remeber off hand. Hope some of this helps. -- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Clay Comstock e-mail comstock@plains.nodak.edu homepage http://murphy220.phys.dsu.nodak.edu/students/clay/comstock.htm PGP Public Key available through most key servers. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ From owner-rapd@net.bio.net Wed Jan 29 22:00:00 1997 Path: biosci!agate!howland.erols.net!newsxfer3.itd.umich.edu!news.bbnplanet.com!su-news-hub1.bbnplanet.com!csn!nntp-xfer-1.csn.net!magnus.acs.ohio-state.edu!bl128.oardc.ohio-state.edu!user From: dsammata@pop.service.ohio-state.edu (Diana Sammataro) Newsgroups: bionet.molbio.rapd Subject: contamination Date: Wed, 29 Jan 1997 14:16:40 -0500 Organization: The Ohio State University Lines: 15 Message-ID: NNTP-Posting-Host: bl128.oardc.ohio-state.edu I also found contamination a problem, especially with primers. I was having a great deal of trouble with my mite samples, and it ended up that I had too little DNA in my sample. This conflicts with the literature that you need only a little. I did a dilution series on a bee and found that the less DNA in the sample, the more bands appeared. Run your primers to see if they are contaminated and then run differnt taq. Diana Diana Sammataro, Ph.D. The Ohio State University, OARDC/ Dept. Entomology Extension Bee Laboratory, 1680 Madison Avenue Wooster, OH 44691 Phone: (330) 263 3684 Fax: (330) 262 2720 Email: Sammataro.1@osu.edu From owner-rapd@net.bio.net Thu Jan 30 22:00:00 1997 Path: biosci!bioag.byu.edu!JLFarmer From: JLFarmer@bioag.byu.edu ("James L. Farmer") Newsgroups: bionet.molbio.rapd Subject: RE: contamination Date: 30 Jan 1997 16:10:56 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.32.19970130171016.0068b088@ucspop.byu.edu> NNTP-Posting-Host: net.bio.net For newcomers to this news group, I would like to point out that a new discussion of amplified DNA in negative controls appears about once each year. There is a very big library of messages on this subject, including some with real data, in the archives. If you use the bionet web site, it has a search program which would probably allow you to find the old messages fairly quickly. My recollection is that the two most common sources of contaminating DNA are water and Taq. I can get perfect negative controls if I am very careful, but I'm not sure that it is necessary. I have never seen the "negative control bands" in any tube that had template DNA. I assume that the template swamps any contaminant. Remember, this isn't PCR. There is a requirement for a pretty high template-DNA concentration to get reliable RAPD's. Best wishes. James Farmer From owner-rapd@net.bio.net Thu Jan 30 22:00:00 1997 Path: biosci!bioag.byu.edu!JLFarmer From: JLFarmer@bioag.byu.edu ("James L. Farmer") Newsgroups: bionet.molbio.rapd Subject: Re: contamination Date: 31 Jan 1997 08:43:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 32 Sender: daemon@net.bio.net Distribution: world Message-ID: <3.0.32.19970131094309.0068c658@ucspop.byu.edu> NNTP-Posting-Host: net.bio.net At 09:22 AM 1/31/97 -0500, you wrote: >James L. Farmer wrote: > >> Remember, this isn't PCR. There is a >> requirement for a pretty high template-DNA concentration to get reliable >> RAPD's. > >Umm, *how* are RAPD's not PCR, exactly? >-- >Susan > >http://www4.ncsu.edu/unity/users/s/sjhogart/public/home.html >Suzuki GS page: http://homepage.cistron.nl/~peterh/gsresources/ > In PCR, if all is working well, the amplified product will remain the same (perhaps changing in yield) as the amount of template DNA is varied. That is NOT true for RAPD's. PCR primers (in the best of all worlds) anneal to only one site in the genome. RAPD primers anneal to a very large number of sites, although eventually only a small number of amplification products are produced because most annealing sites are not properly oriented to, and sufficiently close to, another site. I'm not sure why this introduces a requirement for a relatively high concentration of template DNA in RAPD reactions, but it does. I and others have titrated all of the components of the RAPD reaction, including template DNA. Changing the concentration of anything changes the band pattern that you see on gels. Some bands decrease in intensity (sometimes they disappear) while others increase in intensity, so that the overall pattern can be quite different as concentrations vary. Fortunately, there appears to be a range of concentration for each component within which the RAPD products are pretty reproduceable. I always try to work within that range. From owner-rapd@net.bio.net Thu Jan 30 22:00:00 1997 Message-ID: <32F2AE85.2F75@nf.sympatico.ca> Date: Fri, 31 Jan 1997 18:46:29 -0800 From: William Byrd X-Mailer: Mozilla 2.02E-SYMPA (Win95; I; 16bit) MIME-Version: 1.0 Newsgroups: bionet.molbio.rapd Subject: get mail Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: 207.164.147.61 Lines: 2 Path: biosci!daresbury!nntp-trd.UNINETT.no!online.no!sn.no!nntp.uio.no!www.nntp.primenet.com!nntp.primenet.com!howland.erols.net!psinntp!news.nstn.ca!newsflash.concordia.ca!sunqbc.risq.net!news1.bellglobal.com!endeavor.newcomm.net!207.164.147.61 my e-mail address is will.byrd@nf.sympatico.ca From owner-rapd@net.bio.net Thu Jan 30 22:00:00 1997 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!cwix!uunet!in1.uu.net!198.82.160.249!solaris.cc.vt.edu!newsgate.duke.edu!news-relay.ncren.net!nntp-xfer.ncsu.edu!news From: Susan Jane Hogarth Newsgroups: bionet.molbio.rapd Subject: Re: contamination Date: Fri, 31 Jan 1997 09:22:28 -0500 Organization: North Carolina State University Lines: 12 Message-ID: <32F20024.5086@unity.ncsu.edu> References: <3.0.32.19970130171016.0068b088@ucspop.byu.edu> NNTP-Posting-Host: sparc5b-2203gardnr.ppath.ncsu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; SunOS 5.4 sun4m) To: "James L. Farmer" James L. Farmer wrote: > Remember, this isn't PCR. There is a > requirement for a pretty high template-DNA concentration to get reliable > RAPD's. Umm, *how* are RAPD's not PCR, exactly? -- Susan http://www4.ncsu.edu/unity/users/s/sjhogart/public/home.html Suzuki GS page: http://homepage.cistron.nl/~peterh/gsresources/ From owner-rapd@net.bio.net Fri Jan 31 22:00:00 1997 Path: biosci!ACD.TUSK.EDU!Prakash From: Prakash@ACD.TUSK.EDU ("C. S. Prakash") Newsgroups: bionet.molbio.rapd Subject: Highlights of the Plant and Animal Genome Conference Date: 31 Jan 1997 19:34:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 138 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Here I reproduce my article on the highlights of the recent Plant & Animal Genome meeting held in San Diego that appeared in the February '96 ISB News Report. You can receive this free newsletter through email or in hard copy. Visit http://www.nbiap.vt.edu or send an email to ---------------------------------------------------- INTERNATIONAL CONFERENCE ON PLANT AND ANIMAL GENOMES While the global effort to map and sequence the massive human genome is cruising well on schedule, scientists are also making impressive strides in understanding the genomes of plants and animals of agricultural importance. This progress in agricultural genome research is especially admirable considering its special challenges: a large number of organisms being studied each with unique complexities; relatively lower funding levels and fewer number of scientists working with any single organism when compared to the human genome project. When more than five hundred scientists from across the world gathered in San Diego between January 12-16, 1997 for the Plant and Animal Genome V Conference, the outcome was a testimony for the achievements made in understanding the genomes of organisms that feed the world. While the San Diego meeting has been an annual ritual for plant genome scientists every January for the past four years, this year s meeting included the animal genome researchers for the first time. More than 125 invited presentations and workshop lectures along with 400 poster presentations marked this year s conference. The workshops were essentially commodity based and provided glimpses into recent developments in the genome studies of plants such as rice, cotton, maize, legumes, fruit trees, sugarcane, forest trees, triticaceae, solanaceae, compositae and also of animals such as cattle/sheep, horse, pig, poultry and fish. Detailed genetic maps of these species have been developed using molecular approaches such as RFLP, RAPD, AFLP and microsatellites, and are providing valuable insights into their genome organization and also helping scientists to identify useful genes. Workshops also featured computer applications such as showcasing new software for genome analysis, the World Wide Web sites offering agricultural genome databases and the use of Java programming language that helps in providing interactive content for graphical genome displays. In his plenary lecture, Bradie Metheny of the Washington FAX newsletter called on agricultural genome researchers to play a more proactive role in explaining the importance of their work to law makers and the general public. He said that such efforts are critical in raising the funding levels for agricultural genome research and in ensuring elevating agricultural productivity through biotechnology. David Cox of Stanford University described the utility of radiation hybrids in developing high resolution genome maps where human cells exposed to radiation are fused to hamster cells to create hybrids with select chromosomes. David Schwartz of New York University talked about optical mapping where DNA molecules are fixed on modified surfaces, cut by restriction enzymes and observed under powerful microscopes to produce high density maps. Glen Evans of University of Texas showed how advanced automation and robotics are being employed by human genome scientists in their quest for high-throughput sequencing. Steven Tanksley of Cornell University explained his group s success in isolating, for the first time, a large segment of tomato chromosome containing quantitative trait loci for fruit weight. Many plant disease resistance genes have been identified recently and thus further advances made in this area were of considerable interest. Gregory Martin of Purdue University used a novel yeast-two hybrid system to isolate genes encoding proteins involved in plant-pathogen specificity while Pamela Ronald of University of California-Davis described further progress in isolating resistance genes in rice against bacterial blight. As many disease resistance genes across plant species appear to share similar nucleotide sequences, scientists have developed degenerate primers corresponding to such sequences and used PCR to isolate resistance gene analogs in plant species. Saghai Maroof of Virginia Tech and Randy Shoemaker of USDA/ARS have exploited this approach to isolate molecular markers for disease resistance gene clusters in soybean. Carol Hamilton of Cornell University described a new vector that enables the transfer of multiple foreign genes into plants. The binary bacterial artificial chromosome (BAC) vector is capable of transferring DNA molecules larger than 150 kilobases using Agrobacterium and represents a significant improvement over current methods. This development paves the way for future introduction of multiple genes such as those controlling yield traits into crop plants. Many BAC libraries containing large chromosome pieces derived from crop or animal species were described during the San Diego meeting. Such libraries are helping researchers to identify useful genes through a positional cloning approach. One clear message that has come across from comparative studies on genetic maps of crop species is that gene order and gene content even among unrelated plants appears to be similar. Andrew Paterson of Texas A & M University and Jeff Bennetzen of Purdue University elaborated on how such unified genetic maps are helping in our understanding of genome evolution in agricultural plants and how a genetic map from one species would help in the development of maps in other species. The weed plant Arabidopsis is definitely the darling of many plant genome researchers because of its short life cycle, extremely small genome size and fewer repeat sequences. An international effort to sequence the complete genome of Arabidopsis is now underway. Genes from Arabidopsis can be isolated with relative ease and their availability would help in the rapid isolation of similar genes from crop plants. Many scientists described their progress with the genome analysis of Arabidopsis including the use of Expressed Sequence Tags (EST), the use of advanced computing techniques such as neural networks in such analysis and the international collaborative ventures. The AFLP markers are clearly gaining popularity among genome researchers as evident by the number of studies using this marker system (See the May 1996 ISB News Report for description of this technique). A workshop on AFLP markers featured Pieter Vos of Keygene, a codeveloper of this technique, who described recent applications of AFLP technology including YAC contig fingerprinting. Keygene also demonstrated their new computer software which helps in the analysis of complex AFLP blots using image analysis. Abstracts of all the invited presentations, workshop lectures and posters presented at the Plant and Animal Genome V Conference can be viewed on the World Wide Web at http://probe.nalusda.gov:8000/otherdocs/pg/pg5/allabstracts.html. C. S. Prakash Center for Plant Biotechnology Research Tuskegee University prakash@acd.tusk.edu --------------------------------------------- C. S. Prakash Prakash@Acd.Tusk.Edu Center for Plant Biotechnology Research Tuskegee University Phone (334) 727 8023 College of Agriculture Fax (334) 727 8552 Tuskegee, AL 36088. USA http://agriculture.tusk.edu ---------------------------------------------- From owner-rapd@net.bio.net Fri Jan 31 22:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!cs.utexas.edu!newshost.convex.com!newsgate.duke.edu!news-relay.ncren.net!nntp-xfer.ncsu.edu!news From: Susan Jane Hogarth Newsgroups: bionet.molbio.rapd Subject: Re: contamination Date: Sat, 01 Feb 1997 01:07:34 -0500 Organization: North Carolina State University Lines: 25 Message-ID: <32F2DDA6.740F@unity.ncsu.edu> References: <3.0.32.19970131094309.0068c658@ucspop.byu.edu> NNTP-Posting-Host: sparc5b-2203gardnr.ppath.ncsu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; SunOS 5.4 sun4m) To: "James L. Farmer" > >James L. Farmer wrote: > > > >> Remember, this isn't PCR. There is a > >> requirement for a pretty high template-DNA concentration to get reliable > >> RAPD's Then Susan Hogarth wrote: > >Umm, *how* are RAPD's not PCR, exactly? ... and James L. Farmer replied: > In PCR, if all is working well, the amplified product will remain the same > (perhaps changing in yield) as the amount of template DNA is varied. That > is NOT true for RAPD's. . Actually, I was just being pedantic. RAPDs are a subset of PCR. Those bands certainly come from the Polymerase Chain Reaction.... Probably no need to continue this discussion. -- Susan http://www4.ncsu.edu/unity/users/s/sjhogart/public/home.html Suzuki GS page: http://homepage.cistron.nl/~peterh/gsresources/