From owner-rapd@net.bio.net Thu Oct 03 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!in1.uu.net!fu-berlin.de!cs.tu-berlin.de!map_1.iae.tu-berlin.de!blehn From: Bettina Lehnhardt Newsgroups: bionet.molbio.rapd Subject: reproducibility of RAPD results Date: Fri, 4 Oct 1996 14:51:08 +0000 Organization: Technical University of Berlin, Germany Lines: 14 Message-ID: References: <525cd9$752@manuel.anu.edu.au> NNTP-Posting-Host: map_1.iae.tu-berlin.de Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <525cd9$752@manuel.anu.edu.au> hi, my question concerns the reproducibility of RAPD results. Does GoldStar DNA polymerase from Eurogentec give reliable results in your experiments? ++++++++++++ PLEASE REPLY THIS MESSAGE FOR RECEIPT ++++++++++++ http://map_1.iae.tu-berlin.de/~blehn/ http://130.149.72.27 (look for"user" and "blehn") Dr. Bettina Lehnhardt / HU Berlin / FG Pflanzenzuechtung / Albrecht-Thaer-Weg 5 / D- 14 195 Berlin / Tel. +49 30 314 71141 / Fax +49 30 314 71211 From owner-rapd@net.bio.net Fri Oct 04 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!vixen.cso.uiuc.edu!news.uoregon.edu!hunter.premier.net!netnews.worldnet.att.net!newsadm From: "Stephen M. Schwartz" Newsgroups: bionet.molbio.rapd Subject: Re: reproducibility of RAPD results Date: 5 Oct 1996 15:49:28 GMT Organization: Schwartz Consulting Lines: 23 Message-ID: <01bbb2d4$9d83ebe0$c984eea5@Anything> References: <525cd9$752@manuel.anu.edu.au> NNTP-Posting-Host: 201.seattle-1.wa.dial-access.att.net X-Newsreader: Microsoft Internet News 4.70.1155 What is RAPD? -- Steve Schwartz Steves@U.Washington.edu RAPD? Bettina Lehnhardt wrote in article ... > hi, > my question concerns the reproducibility of RAPD results. > Does GoldStar DNA polymerase from Eurogentec give reliable results in > your experiments? > > > ++++++++++++ PLEASE REPLY THIS MESSAGE FOR RECEIPT ++++++++++++ > > http://map_1.iae.tu-berlin.de/~blehn/ > http://130.149.72.27 (look for"user" and "blehn") > Dr. Bettina Lehnhardt / HU Berlin / FG Pflanzenzuechtung / > Albrecht-Thaer-Weg 5 / D- 14 195 Berlin / > Tel. +49 30 314 71141 / Fax +49 30 314 71211 > > From owner-rapd@net.bio.net Mon Oct 07 23:00:00 1996 Path: biosci!agate!howland.erols.net!swrinde!news.sgi.com!esiee.fr!jussieu.fr!oleane!in2p3.fr!swidir.switch.ch!01-newsfeed.univie.ac.at!news.cesnet.cz!rhino.cis.vutbr.cz!risc.upol.cz!decsys.vsb.cz!newsmast From: Respondby@Mail.com (LSAT Productions) Newsgroups: bionet.molbio.rapd Subject: !!!!!!!!!!! FREE INTERNET ACCESS !!!!!!!!!!! Date: Fri, 04 Oct 1996 08:24:57 GMT Organization: Home.com Lines: 74 Message-ID: <532hdc$73v@decsys.vsb.cz> Reply-To: Respondby@Mail.com NNTP-Posting-Host: cust104.max22.los-angeles.ca.ms.uu.net X-Newsreader: Forte Agent NS#8 1.0.82 !!!!!!!!!!! FREE INTERNET ACCESS !!!!!!!!!!! NEVER EVER pay for Internet Access AGAIN!!! E-V-E-R! Amazing Course on Audio Tape describes STEP by STEP what your Internet Service Provider doesn't want you to know! * How to get FREE LOCAL DIAL-UP PPP Internet Access! * How to Surf the Web,Newsgroups,and EMAIL Anonymously/Untraceable! * How to Make VOICE Phone Calls ANYWHERE in the World UNTRACEABLE! * Where you can get FREE Email Remailing * Where you can get FREE Email Addresses * Where you can get FREE Access to News Servers * How to get FREE Internet Tools for Email, News, WWW, Etc. * How to get free accounts on BBS's * MUCH MUCH More!!! No matter where you live in the world we guarantee you will get FREE internet access legally and anonymously! TO ORDER: LSAT Productions PO Box 2747-453 DEPT NS-8A Huntington Beach, CA 92648 USA WE ACCEPT Check or Money Order. $24.95 Delivered (WORLDWIDE) (US FUNDS ONLY) Act NOW supplies are in Limited Supply! FAST SERVICE! ORDER FORM - Print out and mail - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Price Each Sub-Total __ Total # of Courses __________ ___________ Shipping (+ 1.00/add. course) ___________ Sales Tax (CA residents 7.75%%) ___________ Order total US $___________ PAYMENT BY: ___ Check ___ Money Order - US FUNDS only! SHIP TO: __________________________ _____________________ Name Phone # ______________________________ Address ______________________________ As it should be written on a mail City,State,Zip piece. ______________________________ Country WE ACCEPT US FUNDS ONLY! Please make checks payable to -> LSAT From owner-rapd@net.bio.net Tue Oct 08 23:00:00 1996 Path: biosci!faseb.org!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!nntp-hub2.barrnet.net!news.sgi.com!news.sprintlink.net!news-peer.sprintlink.net!uunet!in1.uu.net!fu-berlin.de!cs.tu-berlin.de!map_1.iae.tu-berlin.de!blehn From: Bettina Lehnhardt Newsgroups: bionet.molbio.rapd Subject: Re: reproducibility of RAPD results Date: Wed, 9 Oct 1996 12:06:46 +0000 Organization: Technical University of Berlin, Germany Lines: 38 Message-ID: References: <525cd9$752@manuel.anu.edu.au> <01bbb2d4$9d83ebe0$c984eea5@Anything> NNTP-Posting-Host: map_1.iae.tu-berlin.de Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <01bbb2d4$9d83ebe0$c984eea5@Anything> RAPD = random amplified polymorphic DNA ++++++++++++ PLEASE REPLY THIS MESSAGE FOR RECEIPT ++++++++++++ http://map_1.iae.tu-berlin.de/~blehn/ http://130.149.72.27 (look for"user" and "blehn") Dr. Bettina Lehnhardt / HU Berlin / FG Pflanzenzuechtung / Albrecht-Thaer-Weg 5 / D- 14 195 Berlin / Tel. +49 30 314 71141 / Fax +49 30 314 71211 On 5 Oct 1996, Stephen M. Schwartz wrote: > What is RAPD? > > -- > Steve Schwartz > Steves@U.Washington.edu RAPD? > > Bettina Lehnhardt wrote in article > ... > > hi, > > my question concerns the reproducibility of RAPD results. > > Does GoldStar DNA polymerase from Eurogentec give reliable results in > > your experiments? > > > > > > ++++++++++++ PLEASE REPLY THIS MESSAGE FOR RECEIPT ++++++++++++ > > > > http://map_1.iae.tu-berlin.de/~blehn/ > > http://130.149.72.27 (look for"user" and "blehn") > > Dr. Bettina Lehnhardt / HU Berlin / FG Pflanzenzuechtung / > > Albrecht-Thaer-Weg 5 / D- 14 195 Berlin / > > Tel. +49 30 314 71141 / Fax +49 30 314 71211 > > > > > > From owner-rapd@net.bio.net Thu Oct 10 23:00:00 1996 Path: biosci!IRRI.CGNET.COM!JPHAM From: JPHAM@IRRI.CGNET.COM Newsgroups: bionet.molbio.rapd Subject: GelPro-Ultralum Date: 11 Oct 1996 02:19:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <01IAIVVZ9UJ68X029I@IRRI.CGNET.COM> NNTP-Posting-Host: net.bio.net Dear all, I am looking for users'comments on the gel analysis software Gelpro and the gel documentation system Ultralum. Thank you. Jean-Louis Pham ------------------------------------------------------------------ Genetic Resources Center Fax: (63-2) 891-1292 International Rice Research Institute (63-2) 817-8470 P.O. Box 933 (63-2) 761-2406 1099 Manila The Philippines E-mail: J.PHAM@CGNET.COM ------------------------------------------------------------------- From owner-rapd@net.bio.net Thu Oct 10 23:00:00 1996 Path: biosci!PUBLIC.CD.SC.CN!gene From: gene@PUBLIC.CD.SC.CN (Feng Lin) Newsgroups: bionet.molbio.rapd Subject: Help!RAPD Analysis Date: 11 Oct 1996 04:57:13 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <325F08EB.2D6F@public.cd.sc.cn> NNTP-Posting-Host: net.bio.net Hi ! Can anyone tell me the percentage that same-size fragments in RAPD profiles are homologous DNAs at Genus level( among Genera)? Did you check the same percentage at Family level? Can I get any references on this kind of information? Thank you very much for your help. Feng Lin Sichuan Univ. From owner-rapd@net.bio.net Sun Oct 13 23:00:00 1996 Path: biosci!BRIC.POSTECH.AC.KR!mhlee From: mhlee@BRIC.POSTECH.AC.KR (mhlee) Newsgroups: bionet.molbio.rapd Subject: help; DNA sequence of Cotesia glomerata virus (Polydnavirus: Bracoviridae) Date: 13 Oct 1996 18:10:14 -0700 Organization: bric Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <326193B6.4C3A@bric.postech.ac.kr> Reply-To: mhlee@bric.postech.ac.kr NNTP-Posting-Host: net.bio.net Hello! Is there anybody know the DNA sequence of Cotesia glomerata virus? Thanks in advance. Your Sincerely, Min-Ho Lee. Min-Ho Lee; mhlee@bric.postech.ac.kr Web for the Korean Entomologist(my home page); http://bric.postech.ac.kr/about/mhlee/ From owner-rapd@net.bio.net Wed Oct 16 23:00:00 1996 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.rapd Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 17 Oct 1996 02:00:49 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199610170900.CAA19419@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. 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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 Oct 21 23:00:00 1996 Path: biosci!ACAD21.NTU.EDU.SG!na1998401 From: na1998401@ACAD21.NTU.EDU.SG (Lim Chee Whye) Newsgroups: bionet.molbio.rapd Subject: mapping a backcross population Date: 21 Oct 1996 22:37:58 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Dear, Can anyone help me with linkage analysis of a backcross population using MAPMAKER ? After going through the "tutorial", I am still not clear on using the program for linkage analysis and is there any more help on using RAPD with mapmaker. David. From owner-rapd@net.bio.net Tue Oct 22 23:00:00 1996 Path: biosci!CATI.CSUFRESNO.EDU!aelashr From: aelashr@CATI.CSUFRESNO.EDU (Abd elnasser Elashry) Newsgroups: bionet.molbio.rapd Subject: Extrea cultures Date: 23 Oct 1996 09:27:07 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <199610231627.JAA04800@CATI.CSUFresno.EDU> NNTP-Posting-Host: net.bio.net Dear Netters: Hello, I am a master student and my project is about "the molecular characterization of Meloidogyne spp.".In fact, I wanted to identify my mixed cultures to the race-level and purify them by using single egg masses technique.So that, I planned to use 6 infected tomato seedlings, each seedlings have been inoculated by using single egg masses from the same mixed isolate, three of them will be identified for the race-level by using the differential hosts. I will use these purified races for the molecular identification by using RAPD-PCR, but As a result of this plan, their will be purified and identified cultures as extra cultures. I want to ask if there is anyone have any idea to use these extra cultures in my research, or in another way, are these cultures can be useful for my research, or excluding it will be better?. Your suggestions will be very appreciated. Thanks for your cooperation. Nasser From owner-rapd@net.bio.net Mon Oct 28 22:00:00 1996 Path: biosci!LAMAR.COLOSTATE.EDU!antolin From: antolin@LAMAR.COLOSTATE.EDU (Michael Antolin) Newsgroups: bionet.molbio.rapd Subject: PAGE and SSCP Date: 29 Oct 1996 07:49:28 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 175 Sender: daemon@net.bio.net Distribution: world Message-ID: <9610291549.AA70806@lamar.ColoState.EDU> NNTP-Posting-Host: net.bio.net > > You should look at a paper in Genetics in August 1996 where we > describe how we map genomes with SSCP analysis of RAPD's. We use large > format (50 cm) sequencing gels for RAPD, but use the smaller systems for > single fragments and targeted PCR with plenty of resolution. > > I include a copy of our complete protocol below. I use an Owl Scientific > system (about $600 per rig), while Bill Black uses the Bio-Rad apc > system (about $900 per rig). As far as I know, there's no single place > to go, like a web page, for how to do SSCP. Regardless, we have lots > of success with it. > > Cheers, > > Mike Antolin > > ************************************************************************ > > Michael F. Antolin > > Assistant Professor > Department of Biology > Colorado State University > Ft. Collins, CO 80523 U.S.A. > > Phone: 970-491-1911 > FAX: 970-491-0649 > E-mail: ANTOLIN@LAMAR.COLOSTATE.EDU > > ********************************************************************** > September 5, 1996 > > > See: Antolin, et al. 1996. Genetics 148: 1727-1738. > > > > SSCP Protocol (developed by Bill Black, Mike Antolin and Nancy Duteau) > > REAGENTS > > Acrylamide Sigma A-8887 > Bis-acrylamide Sigma M-7256 > TEMED Sigma T-7024 > Bind silane Sigma M-6514 gamma- > methacryloxypropyltrimethoxysilane > Sigmacote Sigma SL-2 (but we use commercial Rain-X) > > Silver nitrate Sigma S-0139 > Sodium thiosulfate Sigma S-8503 > Glacial acetic acid > Formaldehyde (37%) Fisher F79-500 > Sodium carbonate Fisher S263-500 or S263-1 or S262-3 > > > SOLUTIONS > > > Acrylamide solution: > [makes 150 ml--for 35 x 45 cm plates only need 100 > ml -- those amounts for 100 ml are in parentheses] > > 25 ml (16.7 ml) 30% acrylamide, 2% bis-acrylamide > (stock acrylamide: 73.5 g acrylamide, 1.5 g bis-acrylamide to 250 g > with water and then filtered) > 30 ml (20 ml) 5X TBE (4 L of 5X TBE is 216 g Tris, 110 g Boric acid, 80 ml > 0.5 M EDTA pH 8.0) > 87.5 ml (58.3 ml) dd water > 7.5 ml (5 ml) glycerol > > Filter through 2 Whatman 1 filters > > > Ammonium persulfate (Sigma A-9164) > > 25% (250 mg in 1 ml or 50 mg in 200 ul) > > Bind silane solution: > > 5 ul of bind silane in 1 ml ethanol/GAA solution > (95% EtOH, 0.5% GAA: 95 ml 100% EtOH, 0.5 ml GAA, 4.5 ml dd water > to make 100 ml of stock ethanol/GAA solution) > > Silver staining solutions: > > 1. Fix/stop > 10% glacial acetic acid (1800 ml dd/di water + 200 ml GAA) > > 2. Staining solution (make at least 15 minutes before use) > 3 g silver nitrate (0.15%) > 3 ml formaldehyde (0.055%) > 2 L dd/di water > > 3. Developing solution (make night before staining gel) > 60 g sodium carbonate (3.0%) > 2 L dd/di water > > Chill in refrigerator to 4 C > > Immediately before development add 3 ml formaldehyde (0.055%) and 400 ul 1% sodium thiosulfate (1% stock is 0.5 g brought to 50 ml with dd/di water) (0.0002%) > > > Sequencing stop solution: > > 10 mM NaOH 1 ml 1 M NaOH > 95% formamide** 95 ml formamide > 0.05% Bromophenol blue 50 mg > 0.05% xylene cyanol 50 mg > 4 ml dd water > > **Stir in 5 g analytical grade mixed-bed resin beads (Bio-Rad AG 501-X8) > > > > PROTOCOLS > > SSCP plate and gel preparation > > Solid plate (soak in 1 N NaOH after each use to keep silane from building up) > > 1. Wash plate with grease-cutting soap using a plastic scouring pad. > 2. Rinse, rinse, rinse with dd water. > 3. Dry with Kay dry wipes (Kimberly Clark). > 4. Wash with 95% EtOH (or MeOH) 3 times. > 5. Treat with bind silane: (5 ul bind silane in 1 ml ethanol/GAA stock) > a. Pour bind silane solution onto plate. > b. Spread onto the plate in an even coat using Kimwipe Ex-L and > let it dry for 5 minutes. > 6. Wash bind silane plate (solid plate) 3 times with 95% EtOH. > > *** CHANGE GLOVES: do not get bind silane onto notched plate *** > > Notched plate (DO NOT soak in 1 N NaOH) > > 7. Wash plate with grease-cutting soap and a different sponge than used on notched plate. > 8. Rinse, rinse, rinse with dd water. > 9. Wash with 95% EtOH (or MeOH) three times. > 10. Spread 1 ml Rain-X across the top of the plate and spread it evenly onto the plate with Kimpwipes Ex-L. Dry the plate and repeat. > 11. Set up glass plates and spacers. > 12. Add 0.1% TEMED and 0.1% of the 25% APS (whatever volume of gel in ml, add the equal amount in ul of TEMED and 25% APS) > 13. Pour gel evenly and continously to prevent bubbles. Place comb in between plates with flat edge against the gel surface. Clamp comb into place. > 14. Can store gel overnight. Place filter with 1X TBE buffer over comb. Wrap both ends of the gel in saran wrap. > 15. Set up gel apparatus with 1.0 X TBE in lower buffer chamber and 1.5X TBE in upper buffer chamber. Each chamber uses 1 L of solution. > 16. Prepare samples. Use 4.0 ul PCR product and 2.0 ul stop solution. > 17. Run gel at constant voltage (350 V; approx 14 mA and 5 W) for 15-16 hours. > > Staining procedure > > 1. Cover gel (adhered to one plate) with fix solution. Agitate well for 20 > min. or until the tracking dyes are no longer visible. Can store > covered overnight without shaking at this point. SAVE the fix > solution. The fix solution will be used to terminate the developing > reaction. > 2. Rinse the gel 3 X 2 min. with ultrapure water. Drain 10-20 sec before > transferring to the next wash. > 3. Stain the gel in staining solution 30 min. with agitation**. SAVE this > solution to precipitate the silver. Add 10 g NaCl and save the AgCl > precipitate. > 4. Add 3 ml formaldehyde and 400 ul sodium thiosulfate to the developing > solution. > 5. Turn off the lights and then rinse the gel by moving from the staining > solution to ultrapure water. Rinse no longer than 10-15 sec. so that > stain is not removed. > 6. Immediately develop the gel in low light by transferring it to the prechilled developing solution. > 7. Terminate the developing reaction and fix the gel by adding the fix > solution from step 1 directly to the developing solution. Terminate for > about 5 minutes. > 8. Rinse gel in water for at least 10 minutes otherwise salt crystals form > while drying. > 9. Dry at room temperature. > > **The silver can be re-used for a second plate if two gels are run the same day. > > From owner-rapd@net.bio.net Mon Oct 28 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!news-peer.sprintlink.net!newsfeed.internetmci.com!news.uoregon.edu!newsfeed.orst.edu!news.orst.edu!sslab.FSL.ORST.EDU!krutovsk From: krutovsk@fsl.orst.edu (Konstantin Krutovskii) Newsgroups: bionet.molbio.rapd Subject: PAGE equipment to do SSCP Date: Mon, 28 Oct 1996 16:11:04 Organization: Forestry Sciences Lab Lines: 23 Message-ID: NNTP-Posting-Host: sslab.fsl.orst.edu Keywords: PAGE , PCR, SSCP (single strand conformational polymorphism) X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Dear netters, I am looking for good, reliable and easy to assemble PAGE equipment and accessories to do SSCP (single strand conformational polymorphism) analysis. I would appreciate it very much if you could name the best manufacturer or company in terms of price and quality. I would also appreciate all advice on the gel system, size / length of gel, optimal number of samples to run, etc., as well as any hints on buffer and gel contents (for instance, acrylamide/bis ratio), and on staining systems (silver vs. radiolabeling vs. fluorescent). I have found that many researchers use Protein PAGE apparatus instead DNA sequencing systems to separate PCR-SSCP products. They are shorter and thicker, but may be easier to assemble. Do they give resolution comparable with sequencing gel? I also wonder if there is any web site where I can inquire about SSCP. Sincerely, Konstantin krutovsk@ccmail.orst.edu From owner-rapd@net.bio.net Mon Oct 28 22:00:00 1996 Path: biosci!cbr.for.csiro.au!charlie.bell From: charlie.bell@cbr.for.csiro.au (charlie bell) Newsgroups: bionet.molbio.rapd Subject: source of oligonucleotide repeats Date: 28 Oct 1996 17:32:42 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: <199610290136.MAA15845@acacia.cbr.for.csiro.au> NNTP-Posting-Host: net.bio.net Hello all, We are looking for a commercial supplier of tri- and tetra-nucleotide repeats with a range of base compositions to try to find microsatellite sequences. We would like oligos of at least 50 bases in length. Does anyone know if we can buy these "off the shelf" rather than having to have them synthesised? Thanks, ----------------------------------------------------------------------- Charlie Bell Phone 06-2818324 (International) +616-2818324 CSIRO Forestry Fax 06-2818312 (International) +616-2818312 and Forest Products E-mail harlie.bell@cbr.for.csiro.au PO Box E4008 Kingston ACT 2604 Australia ----------------------------------------------------------------------- From owner-rapd@net.bio.net Thu Oct 31 22:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!newsxfer2.itd.umich.edu!portc01.blue.aol.com!newsstand.cit.cornell.edu!news.acsu.buffalo.edu!acsu.buffalo.edu!usenet From: hlasker@acsu.buffalo.edu Newsgroups: bionet.molbio.rapd Subject: JOB POSTING - Asst Prof. - Molecular ecology Date: 1 Nov 1996 23:37:47 GMT Organization: University at Buffalo Lines: 39 Message-ID: <55e1kb$nu2@prometheus.acsu.buffalo.edu> NNTP-Posting-Host: lasker.bio.buffalo.edu Mime-Version: 1.0 Content-Type: Text/Plain; charset=US-ASCII NNTP-Posting-User: hlasker X-Newsreader: WinVN 0.99.7 ECOLOGY/EVOLUTIONARY BIOLOGY Assistant Professor The Department of Biological Sciences, State University of New York at Buffalo invites applications for a tenure track position at the level of assistant professor starting in September 1997. We seek an individual working in the area of MOLECULAR ECOLOGY, whose research addresses ecological and evolutionary questions using molecular genetic techniques such as manipulating the genome and following genetic markers within populations and species. The department has active research groups in ecology and molecular biology (including plant molecular biology) and we have identified molecular ecology as an area of future growth. The successful candidate will be expected to develop an active externally funded research program and contribute to undergraduate and graduate courses in ecology including a course in molecular ecology. Candidates should have a Ph.D. and two years of post-doctoral or equivalent experience. Applications, including a curriculum vitae, statement of research and teaching interests, and three letters of recommendation should be submitted to: Dr. Howard R. Lasker Molecular Ecology Search Committee Department of Biological Sciences State University of New York at Buffalo Buffalo, NY 14260-1300. Application review will begin December 15, 1996. Information about the Department and the University can be viewed at: http://wings.buffalo.edu/academic/department/fnsm/bio-sci/index.html. The State University of New York is an Equal Opportunity/Affirmative Action Employer, and encourages applications from minorities, women, and the disabled. No applicant shall be discriminated on the basis of age, creed, color, disability, nation of origin, religion, ethnicity, sex, sexual orientation, marital or veteran status.