From owner-biotechniques@net.bio.net Wed Oct 11 23:00:00 1995 Path: biosci!biosci!not-for-mail From: MMCCARTHY@biotechnet.com Newsgroups: bionet.journals.letters.biotechniques Subject: Citation Information for BioTechniques Articles Date: 12 Oct 1995 12:55:31 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Approved: MMCCARTHY@BIOTECHNET.COM Distribution: world Message-ID: <01HWCWOSYI5E8X0JI8@delphi.com> NNTP-Posting-Host: net.bio.net When you receive your October issue of BioTechniques, check beneath the title of each article for the complete citation information (volume number, inclusive page numbers, month and year of publication) for that article. This information has been incorporated into the BioTechniques page format as the result of a suggestion posted to this newsgroup by Paul Hengen (pnh@ncifcrf.gov), who pointed out that determining the correct citation information for BioTechniques articles (especially from photocopies) can be difficult because all pages in the journal are not numbered. One purpose of the BIOTECHNIQUES newsgroup is to facilitate rapid communication between readers and the BioTechniques editorial staff - kind of an electronic "Letters to the Editor". (The *primary purpose* of the newsgroup is to enable readers, authors and all interested individuals to discuss and troubleshoot the techniques, including general approaches and specific protocols, that appear monthly in the journal, BioTechniques.) So if you have an idea that you think would make BioTechniques more valuable, interesting or easier to use, post your suggestions here - we'd like to hear your opinions! Mary McCarthy (Moderator) Associate Editor, BioTechniques From owner-biotechniques@net.bio.net Tue Oct 17 23:00:00 1995 Path: biosci!biosci!not-for-mail From: Robert Horton Newsgroups: bionet.journals.letters.biotechniques Subject: AmpliGrease and PCR Dye FAQ Date: 18 Oct 1995 09:12:21 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 207 Sender: daemon@net.bio.net Approved: MMCCARTHY@BIOTECHNET.COM Distribution: world Message-ID: <199510172144.QAA18694@biosci.cbs.umn.edu> NNTP-Posting-Host: net.bio.net These questions refer to two publications in BioTechniques: References: Hoppe, B.L., Conti-Tronconi, B.M. and Horton, R.M. "Gel-loading dyes compatible with PCR". BioTechniques, 12(5):679-80, 1992. Horton, R.M., Hoppe, B.L., and Conti-Tronconi, B.M. AmpliGrease: "Hot-start" PCR using petroleum jelly. BioTechniques 16(1):42-43, 1994. The first paper describes gel-loading dyes that can be added to a PCR without interfering with the reaction, and the second describes the use of ordinary petroleum jelly (Vaseline TM) as a meltable barrier for hot-start PCR. The hot-start protocol makes use of the PCR-compatible dye. No, there is no real commercial product called "AmpliGrease". : # Since publication, have you made any improvements? A few small things: 1) Heat the sample block up to denaturation temperature before you put the samples in. The grease melts at about 50degrees, and you can get some "cold-start" effect from this first cycle. We have seen actual examples where reactions came out noticeably cleaner when starting in a hot block. 2) Don't cycle the reactions too fast. I did some reactions in a home-made robo-cycler, and it took about 40 cycles to reach the same yield as 30 cycles in a conventional block-type machine. The robot cycled the tubes so fast that there was not enough time for mixing by diffusion. I suspect that if you included a long initial soak (maybe even a long 72degree soak) in the first cycle, you should be able to cycle fast afterwards, but I never tried it (they made me take my robot apart...) :( 3) an oven is more convenient for melting the grease than a hot plate (see below). : # There is a hot bonnet on the PCR-machine that I use; do I : still overlay with oil? I would overlay with oil, for two reasons. First, the oil protects the itty bitty droplet of top mix from evaporation, which can be significant even before you put the tubes in the cycler if you leave the lid open for too long. Remember, we recommend 10ul total reaction volume, because this protocol depends on diffusion to mix top and bottom mixes. The Perkin-Elmer Cetus protocol recommends 100ul volumes because they use convective mixing. In our protocol, the densities of the top and bottom mixes are so different it would presumably be difficult to get good convective mixing. 10ul can evaporate while you're not looking. Second, the oil mixes with the Vaseline during cycling, to make a mixture that stays liquid afterwards. You need to add oil to 2.5 to 3 times the volume of grease to prevent resolidification. If the grease resolidifies, you have a real mess to deal with in trying to pipette out your sample. It will really plug up a pipette tip. : # Where do you heat the tubes to melt the AmpliGrease? Most importantly, you do NOT want to use your PCR machine, because of the potential for contamination. We originally used a hot plate (just watch the samples to be sure they don't boil!). Now I use a little oven set at about 70 degrees. I bet a hairdryer would work, if you can find a real hot one. : # Can I overlay the samples with heated, fluid AmpliGrease, : rather than heating it afterwards? I haven't tried it. If you use warm Vaseline and a large gauge needle, it is pretty easy to dispense in the "gel" state. If the room is cold and/or your needle is too small, its difficult. I did try dispensing liquid wax some time back - boy, that's a pain (hard to keep melted). : # Can you please send me a protocol for the production of your : Cresol Red and Sucrose loading dye, and also where to buy : them? We have used Cresol Red from Aldrich and from Sigma, but we've only tried the free acid (Sigma cat #C-7524). There is also a salt of cresol red which is more soluble, but I haven't tried it. With the free acid, we make essentially a saturated solution of Cresol Red in 60% sucrose. In a 50ml tube, put 30g sucrose and ~20 mg cresol red. Mix well (preferably, let it tumble for a while.). Sterile filter, aliquot, and freeze for long-term storage (we aliquot all PCR reagents for purposes of contamination control). The solution is actually so hypertonic that we've never seen anything grow in it, even at room temp for several months, but its probably best to keep the working aliquot in the refrigerator, and stocks in the freezer. In my most recent batch of loading dye, I used Sigma Ultra-grade sucrose (cat #S-7903). I suspect that if one were studying plant genes, especially sugar beets or cane, one might want to be more careful in the choice of sucrose, but we have never been able to attribute a band to the sucrose. (I have, however, cloned E. coli genes from "mammalian cDNA". Presumably this is because the enzymes used, RT, Taq, etc., are recombinant and are generally contaminated with some amount of bug DNA.) : # Where at the supermarket do I ask for Regular or Molecular : Grade Vaseline? Unfortunately, you still need to do your own quality control on Vaseline at this point in time. Practically, this means you just need to try it and see if it works. Just be aware that not all batches of Vaseline are suitable for use in PCR. As mentioned in the paper, we found one jar which gave sporadic results, as if an inhibitor were present, but not mixed in very well. Four or five other jars have worked flawlessly. If you could get Molecular Grade Vaseline, I'm sure it would cost 100 times as much. :) Other points: It has recently come to my attention that another group published the use of tartrazine (yellow food coloring, FD&C #5) as a PCR-compatible loading dye before we did (Wittwer, C.T., and Garling, D.J, Rapid cycle DNA amplification: time and temperature optimization. BioTechniques 10(1):76-83, 1991). It was a real twilight-zone experience for me to see this for several reasons: 1) they used one of the same dyes 2) they published in the same journal 3) Dr. Wittwer's works on rapid-cycle PCR are among my all-time favorite PCR papers. There is no doubt I read Dr. Wittwer's paper when it first appeared. Perhaps it started me thinking about coloring PCRs. When Bobbi Hoppe approached me about doing an undergraduate research project, I had this project in the back of my mind. I went to the store and bought a pack of food colors. Only yellow worked. I'm sure that's what Dr. Wittwer's group did, too. Before we submitted this paper, we looked high and low for earlier work, and did not find anything. I had by then completely forgotten that Wittwer's group had mentioned it in their rapid-cycling paper, and it did not occur to me to look there. There is no doubt we should have referenced them; as far as I know, theirs is the first report of using dyes to color PCRs (but then, I've been known to miss such things). If anyone knows of other works in this area that I have missed, I would appreciate having them brought to my attention (normal Medline searches don't bring up much). Idaho Technologies, the manufacturers of the commercial version of the hot-air rapid PCR cycler, now recommend the use of sucrose and cresol red (see the e-journal "The Rapid Cyclist" at http://128.110.195.115/ for protocols). There is a PCR-compatible red dye in Redivue radiolabeled nucleotides (Amersham). It is very intense, and is definitely not cresol red, as judged by differences in migration and the fact that it doesn't turn yellow at pH 7. It is PCR-compatible. Amersham won't say what it is, though (I called 'em). There is a commercially available PCR-compatible gel-loading dye called RediLoad, advertised (where else?) in BioTechniques. The supplier is Research Genetics, Inc., Huntsville, AL, home page: http://www.resgen.com/, FAX: 205-536-9016. They charge $77 for enough to do 1000 one hundred microliter reactions. I don't know what the dye is, but I'd guess it might be the same as Amersham's Redivue dye (judging from the color in the photo and its gel migration). You can get good at pipetting a small sample from beneath oil (even the thicker grease/oil mix that results from AmpliGrease hot starts), but it takes practice. A few years back, it seemed like you couldn't open an issue of BioTechniques without seeing yet another method for removing the blasted oil from a PCR - I think it has frustrated quite a few people. A few hints: - Watch out for bubbles in which your sample is enveloped in a skin of oil. They can float out of the well and burst on the surface, spreading your sample far and wide. With practice, you can pop these bubbles against the side of the well as they start to form. Oil floating to the top without your sample is no big deal. Smooth, fairly rapid sample application will help prevent more bubbles from forming as you load. - When you're trying to fish out that itty bitty droplet of reaction volume from underneath the oil, rest your hands on some solid support. Use spare fingers from the hand holding the tube to steady the pipette tip. This is a delicate operation; steadying the tip with your other hand is not cheating! - Practice a few times before you give up and resort to trying to remove the oil! You'll probably get the hang of it. I thank from Dr. Steffano Loffrede (Division of Gastroenterology, Johns Hopkins University School of Medicine) for stimulating questions. Comments or questions regarding this posting should be directed to: horton@biosci.cbs.umn.edu Robert M. Horton (PhD!) /\ "Crash programs fail because of the theory that U of M Dermatology Dept || with nine women pregnant you get a baby a month" Box 98 UMHC, 4-154 PWB /||\ -W. von Braun. Disclaimer: "Bob who?" Minneapolis, MN 55455 ^^ horton@biosci.cbs.umn.edu (612)625-8941 From owner-biotechniques@net.bio.net Thu Oct 19 23:00:00 1995 Path: biosci!biosci!not-for-mail From: BIOSCI Administrator Newsgroups: bionet.journals.letters.biotechniques Subject: IMPORTANT: BIOSCI miniFAQ Date: 20 Oct 1995 06:20:27 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 196 Sender: daemon@net.bio.net Approved: MMCCARTHY@BIOTECHNET.COM Distribution: world Message-ID: <199510200900.CAA29679@net.bio.net> NNTP-Posting-Host: net.bio.net This is a new "miniFAQ" 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/. Contents: -------- 1) What to do about "spams," i.e., junk mail, ads, etc. 2) Examples of subscribing and unsubscribing to the mailing lists. 3) How to access BIOSCI/bionet newsgroup archives. 4) The BIOSCI user address and research interest directory. 1) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups) and mailing lists. The same postings are distributed on both 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 newsgroups from about 95% of the spams that are being sent to date. This means 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. Unfortunately there are easy ways for determined spammers to override the moderation mechanism. We are working on new systems to provide access to our newsgroups over the WWW. These should be available soon, probably November 1995, and will allow you to use your Web browser to look at the news postings. While this 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. 2) 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. 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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. 3) How to access BIOSCI/bionet newsgroup archives. -------------------------------------------------- Back postings of all BIOSCI/bionet newsgroups can be found on the World Wide Web at URL http://www.bio.net/. There are several searchable newsgroup indices at this site. 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