From owner-biotechniques@net.bio.net Sun Aug 11 23:00:00 1996 Path: biosci!biosci!not-for-mail From: tadichp@io.org (Paul Tadich) Newsgroups: bionet.journals.letters.biotechniques Subject: Genomic DNA preps Date: 12 Aug 1996 05:33:00 -0700 Organization: Internex Online (io.org), Toronto, Ontario, Canada Lines: 17 Sender: daemon@net.bio.net Approved: MMCCARTHY@BIOTECHNET.COM Distribution: world Message-ID: <4ul88o$pi5@news1.io.org> NNTP-Posting-Host: net.bio.net Hi. I'm having a bit of trouble making a genomic DNA prep from Brevibacterium sterolicum. I've tried the boiling method and a standard phenol extraction, but these two procedures don't seem to be working very well. My best purification to date has only resulted in a very faint smear. If anyone knows of a better method of purifying bacterial genomic DNA, or if you've had experience working with B. sterolicum, I'd appreciate some help. Also... I'm trying to PCR amplify a 1.7 kb segment from the said DNA. I'm using an Idaho Technologies RapidCycler with 10 ul volume/sample, using an annealing temp. of 45-50 deg. C. for about a minute. These temperatures are consistent with my primer's specifications, but I can't seem to get any product. Any help with this would also be appreciated. From owner-biotechniques@net.bio.net Tue Aug 13 23:00:00 1996 Path: biosci!biosci!not-for-mail From: MMCCARTHY@biotechnet.com Newsgroups: bionet.journals.letters.biotechniques Subject: BIOTECHNIQUES 21(3), SEPTEMBER 1996 Date: 14 Aug 1996 10:14:53 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 185 Sender: daemon@net.bio.net Approved: MMCCARTHY@BIOTECHNET.COM Distribution: world Message-ID: <01I89MOND5HY8ZGK0O@delphi.com> NNTP-Posting-Host: net.bio.net BioTechniques 21(3), September 1996 Table of Contents BENCHMARKS Elimination of sequence ambiguities by a single-step modification of a solid-phase, single-stranded sequencing protocol -Lawson V.A., D.A. McPhee and N.J. Deacon Recovery of unlabeled PCR product from polyacrylamide gel for sequencing -Wu S.-M., L.A. Blomberg and W.-Y. Chan PCR-RFLP genotyping of murine MHC haplotypes -Peng S.L. and J. Craft Cautionary note on the use of dUMP-containing PCR primers with Pfu and VentR DNA polymerases -Sakaguchi A.Y., M. Sedlak, J.M. Harris and M.F. Sarosdy Use of the fluorescent dye PicoGreen for quantification of PCR products after agarose gel electrophoresis -Enger O. Quantitation of T cell receptor transcripts using a wet agarose gel method -Daniel E.S. and D.G. Haegert Simple method for constructing internal standards for competitive PCR -Tang J., G. Lagace and R. Collu Limited growth PCR screening of a plasmid library -Ross L.S. and S.S. Gill Streamlined procedures for screening a P1 library -Shovlin C.L. Hydrogen peroxide decomposes the heme compound in forensic specimens and improves the efficiency of PCR -Akane A. Isolation of multiple mRNAs from a few eukaryotic cells: a fast method to obtain templates for RT-PCR -Noppen C., G.C. Spagnoli and C. Schaefer Improved dicistronic mRNA expression vectors for efficient selection of transfectants highly expressing foreign genes -Kobayashi M., Y. Yamauchi, A. Tanaka and S. Shimamura Method for rapid restriction analysis of YAC clones -Del-Favero J. and M. Jacobs Differential display without radioactivity - a modified procedure -Doss R.P. Method for joining DNA fragments using positive selection -Gubin A.N. Removal of template after random priming yields DNA probes of maximal specific activity -Hammerl P. and J. Thalhamer Repeated probing of Western blots obtained from Coomassie Brilliant Blue-stained or unstained polyacrylamide gels -Suck R.W.L. and K. Krupinska Use of 33P for ribozyme assays: The safe way -Wolff P.R.A. and R. Hull RNA from air-dried frozen sections for RT-PCR and differential display -Castles C.G., D.C. Allred, S.L. Krieg, M.G. Benedix and S.A.W. Fuqua Use of the two-hybrid system and random sonicated DNA to identify the interaction domain of a protein -Stagljar I., J.-P. Bourquin and W. Schaffner THE INTERNET ON-RAMP -Cyberspace for Biologists SHORT TECHNICAL REPORTS Simple strategy for sequencing cDNA clones -Zeng L.-W. and M. Kreitman Automated DNA sequencing requiring no DNA template purification -Chen Q., C. Neville, A. MacKenzie and R.G. Korneluk Isolation of high molecular length DNA from human skin -Bennett P.V., R.W. Gange, H. Hacham, V.S. Hejmadi, M. Moran, S. Ray and B.M. Sutherland Simultaneous detection of microorganisms in soil suspension based on PCR amplification of bacterial 16S rRNA fragments -McGregor D.P., S. Forster, J. Steven, J. Adair, S.E.C. Leary, D.L. Leslie, W.J. Harris and R.W. Titball Efficient long-PCR site-specific mutagenesis of a high GC template -Chouljenko V., S. Jayachandra, G. Rybachuk and K.G. Kousoulas Quantitative Multiple Competitive PCR of HIV- 1 DNA in a Single Reaction Tube -K. Zimmermann, D. Schogl, B. Plaimauer and J.W. Mannhalter RESEARCH REPORTS Fluorescence in situ hybridization method for measuring transfection efficiency -Cheng L., C.D. Bucana and Q. Wei Efficiency of transduction by recombinant Sindis replicon virus varies among cell lines, including mosquito cells and rat sensory neurons -Corsini J., D. Traul, C.L. Wilcox, P. Gaines and J.O. Carlson Detection of cell-surface antigens using antibody-conjugated fluorospheres (ACF): application for six-color immunofluorescence -Beavis A.J. and K.J. Pennline Sequential ELISA for cytokine levels in limited volumes of biological fluids -Steffen M.J. and J.L. Ebersole Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products -Iwahana H., M. Fujimura, Y. Takahashi, T. Iwabuchi, K. Yoshimoto and M. Itakura PRODUCT APPLICATION FOCUS Dual luminescence-based reporter gene assay for luciferase and beta-galactosidase -Martin C.S., P.A. Wight, A. Dobretsova and I. Bronstein Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA -Chenchik A., L. Diachenko, F. Moqadam, V. Tarabykin, S. Lukyanov and P.D. Siebert NEW PRODUCTS INDEX TO ADVERTISERS From owner-biotechniques@net.bio.net Sun Aug 18 23:00:00 1996 Path: biosci!biosci!not-for-mail From: Scott Noggle Newsgroups: bionet.journals.letters.biotechniques Subject: Multiplex PCR problem Date: 19 Aug 1996 12:36:48 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 33 Sender: daemon@net.bio.net Approved: MMCCARTHY@BIOTECHNET.COM Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hello All, I am having a problem with a multiplex PCR application. As I have little experience using multiple primers in on reaction, I was wondering if there is something obvious I am missing. My application involves amplifying two genes at once in transgenic mice. The primers are designed to be specific for an Ig heavy chain transgene that is present in about 30 tandem copies, and for the GSalpha gene, a single copy gene. I am using a nested protocol, because eventually I want to be able to use these together to amplify from a single cell. Also the primers were designed to be free of interactions with eachother and have Tms within half a degree. Hot start is also used to ensure specificity. My problem is this: Thus far I have only seen the predicted signal from the transgene primers, the GSalpha signal has not shown itself yet with the flanking set of primers. I am using 100ng of template DNA so that I will be able to see the signal from the first round.[this is rational, isn't it?] To get the transgene signal, I had to reduce the transgene primer concentration to 25-100nM--both worked well as did 5nM. The GSalpha primer concentration was also titrated from 25nM in ten-fold increments to .025nM without any signal(the transgene signal was great). What can I do to get the GS alpha signal to show? Any suggestions would be greatly appreciated, and thankyou for reading this long-winded explaination. Your comments can be sent to me directly or to the news group. Thankyou, Scott Noggle Immunology Department of Biological Sciences University of Arkansas at Fayetteville email:snoggle@comp.uark.edu From owner-biotechniques@net.bio.net Mon Aug 19 23:00:00 1996 Path: biosci!biosci!not-for-mail From: BIOSCI Administrator Newsgroups: bionet.journals.letters.biotechniques Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 20 Aug 1996 06:36:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Approved: MMCCARTHY@BIOTECHNET.COM Distribution: world Message-ID: <199608200900.CAA06360@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