From owner-molluscs@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!daresbury!not-for-mail From: fberthe@ifremer.fr (Franck Berthe, DRV RA GAP, 46.36.30.07) Newsgroups: bionet.molbio.molluscs Subject: looking for 18S primers Date: 2 Oct 1995 10:44:39 +0100 Lines: 19 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <44oca7$r16@mserv1.dl.ac.uk> X-Sender: fberthe@ronce Original-To: molluscs@dl.ac.uk Hi netters, in order to sequence a part of this gene, I am looking for PCR primers that could amplify 18srRNA gene from Marteilia sp., a Protozoa : Acetospora, flat oyster (Ostrea edulis) parasite.=20 Can anyone help me ? Lot of thanks. Dr. Franck BERTHE Unit=E9 de Recherche en Pathologie et Immunologie G=E9n=E9rales Laboratoire G=E9n=E9tique, Aquaculture, et Pathologie. IFREMER, La Tremblade BP 133, 17390, La Tremblade, FRANCE. tel : (33) 46 36 98 36 (direct line (33) 46 36 98 43) fax : (33) 46 36 37 51 From owner-molluscs@net.bio.net Sun Oct 01 23:00:00 1995 Newsgroups: bionet.molbio.molluscs Path: biosci!daresbury!nntp-trd.UNINETT.no!Norway.EU.net!EU.net!news.sprintlink.net!tank.news.pipex.net!pipex!warwick!bsmail!usenet From: Harry.Witchel@bristol.ac.uk (Harry Witchel) Subject: CTAB DNA prep: How To do it Message-ID: Sender: usenet@uns.bris.ac.uk (Usenet news owner) Nntp-Posting-Host: rm2.pys.bris.ac.uk Reply-To: harry.witchel@bristol.ac.uk Organization: Physiology / Med. School / Bristol X-Newsreader: WinVN 0.91.4 Date: Mon, 2 Oct 1995 09:19:19 GMT Lines: 175 Hello snail lovers! I have recently received an e-mail asking for an explanation of what a CTAB DNA prep is, so I assume that there may be more people out there who would like to know. CTAB is formally known as "hexadecyl trimethyl ammonium bromide" (Sigma H-6269), and it is a detergent with the property that DNA will precipitate out of a CTAB buffer unless there is high salt (typically NaCl > .7 M). There are many different kinds of CTAB preps (surprise! surprise!). There is one in Current Protocols in Molecular Biology (Ausubel -- The Red Book), although I do not see one in Maniatis 1989. If you have a choice between Maniatis or the Red Book, buy the Red Book because it has a much better index which makes it much more useful. I also found a CTAB prep on a giant document Elizabeth sent me on mollusc DNA preparation (the document is especialy good for mtDNA). If that document is generally available on a website, I do not know the address of that site; it may be useful to occasionally mail that document to this newsgroup as a kind of FAQ. Is that a useful idea Elizabeth? The protocol I present below is adapted from Winnepenninckx (No I can't pronounce it either), 1993, Trends in Genetics, vol. 9, p.407. Essentially the prep is 1) Freeze and grind tissue 2) Solubilize tissue in CTAB buffer 3) Add Proteinase K and digest homogenate 4) Chloroform extract proteins 5) Alcohol precipitate DNA I tend to get full length DNA for Southerns, but I also get a bit of RNA contamination, which I now deal with by doping the final product with a bit of RNase and just letting the RNase do its stuff indefinitely. Here it is: CTAB Buffer ----------- 2% w/v CTAB 1.4 M NaCl 20 mM EDTA 100 mM Tris-HCl pH 8 .2% v/v B-mercaptoethanol (optional) - DISSECT TISSUE, FREEZE IN LIQUID NITROGEN, AND GRIND IT I tend to use rather a lot of tissue, such as 10 cerebral ganglia or 10 buccal glands from a common garden snail. A buccal gland is 9 cubic mm. I pour liquid nitrogen in my mortar, put the tissue in to freeze it, and then grind. The mortar and pestle are sturdy ceramic unglazed ones which I got from a kitchen store; they can be autoclaved, but you should probably use a a different set for each species. If you are doing many species, you may be better off grinding them in disposible plactic. - SLOWLY ADD TISSUE POWDER TO CTAB BUFFER I use 5-10 ml of preheated (55C) CTAB buffer in a 20 ml disposible plastic universal tube. I add the powder a little at a time and mix the homogenate repeatedly while doing this by inverting the tube; this prevents undigestible clumps of tissue from forming. Remember that genomic DNA is very easily sheared, so you should mix the solution by inverting a capped tube (rather than by shaking), and you should not even dream of using a dounce homogeniser. - ADD 75 ul OF PROTEINASE K, MIX HOMOGENATE, DIGEST AT 55C Proteinase K stock (20 mg/ml in water) is as per Maniatis. As usual, mix by inversion. The incubation takes as long as necessary for all the tissue to solubilize. At a minimum, you should leave it for 30 minutes. I tend to go for 1-3 hours, but if the tissue is recalcitrant and will not solubilize, I might add another 75 ul of Proteinase K and let the reaction run overnight at 55C; the DNA still seems full length. If you have clumps of tissue which will not disperse, or if your tissue contains a lot of elastic fibers, it will never fully solubilize, in which case you should decant the fluid and discard the solids. - CHLOROFORM EXTRACT TWICE This eliminates the Proteinase K as well as the tissue protein. Chloroform as usual contains 1:24 of isoamyl alcohol. Most protocols with CTAB use only chloroform and not phenol, and I assume there is a good reason for that. I do my chloroform extractions in corex tubes -- I prefer the 30 ml tubes, but most people only have 15 ml tubes. I gently pour my homogenate into the corex tube. I mix the chloroform with the homogenate by squirting the chloroform (but not the DNA homogenate) through a 10 ml glass pipet; using a pipet bulb I squirt the chloroform into the homogenate, then I suck the unmixed chloroform from the bottom of the tube back into the pipet, and I squirt that chloroform back into the homogenate. I think squirting is safer than covering the tube with two layers and parafilm and shaking because chloroform has a habit of blowing holes through parafilm. I then centrifuge the mixture fairly hard: SS-34 rotor, 10-12,000 rpm, 10C, 20-30 minutes. The first extraction will have a lot of junk at the interface, and you may be forced to leave over between .5-1 ml of homogenate to avoid getting the interface. Sometimes the homogenate will be brown, but in nice tissues like buccal gland, the homogenate ends up clear. To remove the upper homogenate layer to a new tube, I use a 1 ml pipetman and commercially available "wide bore pipet tips". You can make your own wide bore pipet tips by sterilely cutting the end off some blue tips. If you use a glass centrifuge tube, it will be much easier to see the interface, and you will know whether you have gunk at the interface. If you have much gunk at the interface after 2 chloroform extractions, try a third extraction. - ADD 2/3 VOLUMES OF ISOPROPYL ALCOHOL AND SPOOL DNA At this point I have put my cleared homogenate into a 20 ml plastic universal tube again, and I gently add the isopropanol and mix by inversion. I then leave the DNA to precipitate for a few minutes at room temperature while I make a "spool". A spool is just a glass rod with a tiny bend at the end. To make a spool, use a long pasteur pipet, and close off the skinny end using a bunsen burner. Then make a tiny bend (I use a 45 degree angle, but I don't think it matters) about 2-3 mm from the end of the pipet. Let the glass cool for a minute, then stick the thin end of the spool into your DNA solution, and capture all the strands of DNA onto the spool by moving the spool in circles around the edge of the the universal tube. The DNA ideally should be made of white threads, but it may be clear (RNA) and it may be brown (oops! Don't give up, it may still be good). Once all the DNA is on the spool, you can gently remove it from the isopropyl solution and drip the alcohol off the DNA by touching the DNA to the edge of the tube. If you do not see any strands of DNA at this point, there is probably too little to spool. You will have to let the DNA precipitate overnight at room temperature, then centrifuge the solution at 10,000 rpm. - WASH THE DNA IN 70% ETHANOL, THEN IN 100% ETHANOL 70% ethanol will get the salts off the DNA and into the water, then 100% ethanol will get the water off the DNA. I wriggle the spool in a tube of each ethanol for 30 seconds or so. If you had to centrifuge the DNA, the washes are done on the pellet in the centrifuge tube, as you would with a plasmid prep. - SCRAPE THE DNA INTO AN EPPENDORF TUBE AND DRY THE DNA Get as much alcohol off the DNA as possible, then scrape the DNA off the glass spool using the edge of the eppendorf tube or a clean yellow tip. If there is any fluid at the bottom of the tube, pipet it out. The reason you dry the DNA is that ethanol interferes with gel electrophoresis of high MW DNA, so if you are going to continue to purify the DNA using some extra steps, it is unnecessary to dry it at this point. I dry my DNA for 15 minutes under a low vacuum by covering my eppendorf tube with parafilm, poking holes in the parafilm using a syringe needle, and putting it in the aspirator. If there is ethanol (from the side of an undried tube) in your final DNA sample, it may not run correctly in the gel. - REDISSOLVE DNA IN TE OR T.1E T.1E is 10 mM Tris pH 8/.1 mM EDTA, which is ultimately better for doing reactions later. If I have to centrifuge the DNA, I may try as little as 20 ul of T.1E, but if I have a huge wadge of DNA floating on the spool, then I might use 100-200 ul. At the very least the DNA should be left to redissolve at 4C for an hour, but I like to give my DNA overnight to digest. Then I electrophorese the DNA to see if it is any good. Problems with electrophoresis of high MW DNA include: the DNA may float out of the well (ethanol or organics present in sample), it may refuse to leave the wells (voltage too high, high salt, or DNA is not redissolved), it may have a shooting star effect (too much DNA in a small lane), and there may be a streak running down the center of the lane (RNA or smaller DNAs dragging through the high MW DNA). To reduce electrophoresis problems, you may try any of the following: dry DNA (no more than 15 minutes in a vacuum), run low percentage (.6%) agarose gels, and run the gel at a lower voltage (especially during the first 15 minutes as the DNA attempts to enter the gel matrix). Harry Witchel Robert Meech's Laboratory Dept. Physiology Medical School Bristol From owner-molluscs@net.bio.net Sun Oct 01 23:00:00 1995 Path: biosci!MAILBOX.UQ.OZ.AU!S.Degnan From: S.Degnan@MAILBOX.UQ.OZ.AU (Sandie Degnan) Newsgroups: bionet.molbio.molluscs Subject: molluscan microsatellites Date: 2 Oct 1995 12:23:44 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net To Elizabeth Boulding re your message of 23.9.95 - We currently are developing microsatellite loci for abalone (Haliotis spp.), and it looks as though we'll end up with a dozen or so. Once we have them up and running, we'd be happy to try them out on other molluscs, or send them to others or may want to try them on their study species. Please contact me if you're interested. A couple of people in our lab have put in a good effort to get the automated fluorescent screening underway, but to no avail. It seems fickle. Nonetheless, the laboratory of Dr. Steve Moore, CSIRO Division of Tropical Animal Production (Molecular Animal Genetics Centre), Gehrmann Labs, University of Queensland routinely screens large numbers of individuals via automated methods, with great success. It would be worth your while to contact them. I could probably get hold of an email address for you if you're interested. Cheers, Sandie Degnan Dr. Sandie Degnan Molecular Zoology Lab Department of Zoology University of Queensland Brisbane 4072 Qld Australia ph (07) 365 2966 fax (07) 365 1655 From owner-molluscs@net.bio.net Mon Oct 02 23:00:00 1995 Newsgroups: bionet.molbio.molluscs Path: biosci!bcm.tmc.edu!cs.utexas.edu!news.sprintlink.net!dispatch.news.demon.net!demon!sunsite.doc.ic.ac.uk!warwick!bsmail!usenet From: Michael Rickard Subject: Another CTAB DNA prep Message-ID: Sender: usenet@uns.bris.ac.uk (Usenet news owner) Nntp-Posting-Host: rm2.pys.bris.ac.uk Organization: University of Bristol, England Date: Tue, 3 Oct 1995 08:38:50 GMT Lines: 68 Hi -- I have found another protocol if mine does not work. I have not tried this one. Harry Here it is: From: Andrew McArthur Subject: CTAB DNA Extraction Protocol To: mollusc-molecular-news@sfu.ca Status: RO I recieved several requests for this protocol, so I'm posting it to the MMN. It has consistantly worked where others have not (note that my work is mainly with gastropods). As mentioned before, it has only failed in Solenogastres. Contact Mike Black (below) if you have questions about that. DNA ISOLATION WITH CTAB MODIFIED FROM DOYLE & DOYLE (1987) PHYTOCHEMICAL BULLETIN 19: 11-15 BY MIKE BLACK AT RUTGERS (MBLACK@ZODIAC.RUTGERS.EDU). - USE 1 TO 2 CUBIC MILLIMETERS OF TISSUE - IN A 1.5 ML EPPENDORF TUBE, GRIND TISSUE IN 300 UL OF 2X CTAB @ 60C - INCUBATE IN WATER BATH @ 60C FOR 30 MIN. - ADD 300 UL CHLOROFORM-ISOAMYLALCOHOL AND SHAKE FOR 2 MIN. - CENTRIFUGE @ 5000G FOR 2 MIN. - LABEL FRESH EPPENDORFS AND ADD 600 UL 70% ETOH AND 25 UL 3M NAOAC - ADD AQUEOUS (TOP) PHASE FROM SAMPLE TUBES TO ABOVE - CENTRIFUGE @ 5000G FOR 2 MIN. - DECANT FLUID AND DRAW OFF EXCESS ETOH FROM PELLET (CAREFUL!) - ADD 200 UL 70% ETOH AND WASH PELLET BY INVERTING SEVERAL TIMES - CENTRIFUGE @ 5000G FOR 2 MIN. - POUR OFF ETOH AND DRAW OFF EXCESS ETOH FROM PELLET (CAREFUL!) - DRY IN OVEN (60-70C) FOR 2-5 MIN. (ONLY TO EVAPORATE EXCESS ETOH, NOT TO DRY PELLET) - ADD 25-100 UL 0.1X TBE BUFFER TO RE-DISSOLVE DNA PELLET - PLACE IN OVEN (60-70C) FOR 30 MIN. (OR OVERNIGHT) - CHECK EXTRACTION BY AGAROSE GEL ELECTROPHORESIS I'VE MADE SOME MODIFICATONS OF MY OWN. SINCE I USE TOUGH GASTROPOD FOOT, I ADD SOME SIGMA WHITE QUARTZ SAND (-50 TO 70 MESH) TO HELP IN THE GRIDING (THANKS CLARK!). 1.5 ML EPPINDORF GRINDERS ARE AVAILABLE FROM THE NORMAL EQUIPMENT SUPPLIERS FOR A MOLECULAR BIOLOGY LAB OR YOU CAN MAKE THEM OUT OF SURF BOARD RESIN (THANKS FOOL'S GUIDE). I USE 96:4 CHLOROFORM:ISSA AND THE FIRST SPIN IS AT 13K FOR 10 MIN. I ALSO USE 1 ML OF 95% ETOH INSTEAD OF 600 UL OF 70% ETOH IN THE PRECIPITATION. CTAB ISOLATION BUFFER (I IGNORE THE PH AND USE SOLIDS ONLY): 100 MM TRIS-HCL, PH 8.0 1.4 M NACL 20 MM EDTA 2% HAXADECYLTRIMETHYLAMMONIUM (CTAB) 0.2% 2-MERCAPTOETHANOL BEST OF LUCK! ANDREW MCARTHUR AMCARTHU@UVVM.UVIC.CA From owner-molluscs@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!OSF1.GMU.EDU!ladamkew From: ladamkew@OSF1.GMU.EDU ("S. LAURA ADAMKEWICZ") Newsgroups: bionet.molbio.molluscs Subject: Re: Another CTAB DNA prep Date: 3 Oct 1995 18:45:22 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net Andrew McArthur's protocol has always worked well for me. If you have really slimey molluscs I recommend adding 2% polyvinyl pyrrolidine to it. ==================================================================== = Laura Adamkewicz e-mail ladamkew@gmu.edu = = Dept. of Biology tel 703-993-1047 = = George Mason University fax 703-993-1046 = = Fairfax, VA 22030 USA = ==================================================================== From owner-molluscs@net.bio.net Tue Oct 03 23:00:00 1995 Path: biosci!UVAIX3E1.COMP.UVIC.CA!amcarthu From: amcarthu@UVAIX3E1.COMP.UVIC.CA (Andrew G. McArthur) Newsgroups: bionet.molbio.molluscs Subject: Updated CTAB Extraction Date: 4 Oct 1995 14:15:19 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 122 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Here is a more recent version of the CTAB DNA extraction protocol recently posted to the MMN. DNA Isolation with CTAB AMCARTHU@UVAIX.UVIC.CA This protocol is from Dr. Robert Vrijenhoek's molecular biology group at Rutgers University in New Brunswick, New Jersey, U.S.A. It is based on Doyle & Doyle (1987) and a variety of other papers, some of which are listed at the bottom. I include some of our modifications (we developed our own protocol independently but merged it with the Rutger's protocol). If this does not work for your creatures and you have tried most traditional methods, give Hempstead et al. (1990) a try (we have never had the need to try it). We use this method on invertebrates, vertebrates, and bacculovirus pips. It works on invertebrates frozen or preserved in alcohol or DNE solution. We even have good success for formalin preserved material. It works on vertebrates frozen, preserved in alcohol, or dried skin samples. It has worked well for <1 mm long gastropods (half shell at that) followed by PCR, but we suggest you examine alternate techniques for extractions from blood or single hairs (we have some ideas on this). Note: A recent paper has a method very similar to this. You might want to look it up: Winnepenninckx, Backeljau & de Wachter. 1993. Extraction of high molecular weight DNA from molluscs. Trends in Genetics 9: 407. 1. Grind 1-2 cubic millimetres of tissue in a 1.5 ml eppendorf tube with 300 ul of 2X CTAB buffer @ 60C (we often add a pinch of white quartz sand, -50 to 70 mesh, from Sigma to help grind snail foot tissue, tough fish parts, or mammal skin samples) 2. Incubate in water bath @ 60C for 30 min. 3. Add 300 ul of chloroform-isoamyl alcohol (96:4) and shake for 2 min. 4. Centrifuge @ 15,000g for 10 min. 5. Label fresh eppendorfs and add 600 ul cold 70% EtOH, 25 ul 3M NaOAc, and the aqueous from the chloroform extraction. Mix by inversion. 6. Centrifuge @ 15,000g for 10 min. 7. Pour off EtOH and use pipette (or kimwipe) to remove excess EtOH. Dry pellet in oven (60-70C) for 2-5 min. (not too long or pellet will dry out). (we have used a speedvac instead of an oven with no problems) 8. Re-dissolve the pellet in 25 ul of 0.1X TE and place in oven (60-70C) for 30 min. or overnight to get all DNA into solution. (resuspend in more 0.1X TE if a large pellet but remember that is always easier to dilute later than concentrate) 9. Verify quality and quantity using agarose gels, spectrophotometers, or fluorometers. (CTAB buffer does cause some shearing of DNA but we PCR from the extract all the time. The CTAB extraction also yields large quantities of good quality RNA. We have not found that the RNA interferes with PCR as reported in the literature. We have not tried Southern or Northern blots on CTAB extracted DNA or RNA.) 2X CTAB isolation buffer: 100 mM Tris-HCl, pH 8.0 1.4 M NaCl 20 mM EDTA 2% Hexadecyltrimethylammonium bromide (CTAB) 0.2% 2-mercaptoethanol (We actually make the buffer from solids and do not adjust pH. For a 100 ml batch this takes 1.21 gm Tris, 8.182 gm NaCl, 0.74 gm EDTA, 2.0 gm CTAB, 0.2 ml 2-mercaptoethanol, and water to volume. Take proper precautions when handling 2-mercaptoethanol. We have not examined the shelf-life of the CTAB buffer but have used a single batch for 2-3 months without problems.) Literature: Del Sal et al. (1989) The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. BioTechniques 7: 514-521. Doyle, J.J. & Doyle, J.L. (1987) A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochemical Bulletin 19: 11-15 Gustincich et al. (1991) A fast method for high-quality genomic DNA extraction from whole human blood. BioTechniques 11: 298-302. Hempstead et al. (1990) A method for the preparation of high-molecular-weight DNA from marine and freshwater triclads (Platyhelminthes, Turbellaria). DNA and Cell Biology 9: 57-61. Maki et al (1991) A cationic detergent, cetyltrimethylammonium bromide (CTAB), selectively dissociates the intermediate filament of the fibroblast. Biochemical and Biophysical Research Communications 175: 768-774. Rogstad, S.H. (1992) Saturated NaCl-CTAB solution as a means of field preservation of leaves for DNA analyses. Taxon 41: 701-708. --------------------------------------------------------------- Please note that my email has changed: amcarthu@uvaix.uvic.ca But don't worry, email sent to my old address will still get to me (for now). --------------------------------------------------------------- --------------------------------------------------------------- Andrew G. McArthur Department of Biology University of Victoria P.O. Box 1700 Victoria, British Columbia Canada, V8W 2Y2 amcarthu@uvaix.uvic.ca http://darwin.ceh.uvic.ca/people/mcarthur.html --------------------------------------------------------------- --------------------------------------------------------------- Check out the DEEPSEA mailing list! (deepsea@uvvm.uvic.ca) http://darwin.ceh.uvic.ca/deepsea/deepsea.html --------------------------------------------------------------- From owner-molluscs@net.bio.net Thu Oct 05 23:00:00 1995 Path: biosci!ARMS.GPS.CALTECH.EDU!dschida From: dschida@ARMS.GPS.CALTECH.EDU (Bill Dschida) Newsgroups: bionet.molbio.molluscs Subject: chiton codon usage table Date: 6 Oct 1995 16:42:21 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <9510062347.AA02329@arms.gps.caltech.edu> NNTP-Posting-Host: net.bio.net Hello Newsgroup, I'm in search of a list of codon usage/preferences for chiton (or more generally, for molluscs). Thank you, Bill Dschida Caltech, Division of Geological and Planetary Sciences dschida@arms.gps.caltech.edu From owner-molluscs@net.bio.net Tue Oct 10 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!chi-news.cic.net!simtel!zombie.ncsc.mil!cs.umd.edu!haven.umd.edu!purdue!news.bu.edu!transfer.stratus.com!news3.near.net!yale!news.ycc.yale.edu!news From: Greg Fitzgerald Newsgroups: bionet.molbio.hiv,bionet.molbio.methds-reagnts,bionet.molbio.molluscs,bionet.molbio.proteins,bionet.molbio.rapd Subject: Re: Make a $1,000,000 in 90 Days! Date: 11 Oct 1995 16:58:50 GMT Organization: Yale University Biology Department Lines: 3 Message-ID: <45gt4a$flf@news.ycc.yale.edu> References: <45ge3g$o1j$5@mhafn.production.compuserve.com> NNTP-Posting-Host: 130.132.107.28 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: 102771.3337@CompuServe.COM X-URL: news:45ge3g$o1j$5@mhafn.production.compuserve.com Xref: biosci bionet.molbio.hiv:1675 bionet.molbio.methds-reagnts:34741 bionet.molbio.molluscs:172 bionet.molbio.proteins:5905 bionet.molbio.rapd:1324 Get out of this site you fool! From owner-molluscs@net.bio.net Tue Oct 10 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!chi-news.cic.net!simtel!zombie.ncsc.mil!cs.umd.edu!haven.umd.edu!purdue!news.bu.edu!transfer.stratus.com!news3.near.net!yale!news.ycc.yale.edu!news From: Greg Fitzgerald Newsgroups: bionet.molbio.hiv,bionet.molbio.methds-reagnts,bionet.molbio.molluscs,bionet.molbio.proteins,bionet.molbio.rapd Subject: Re: Make a $1,000,000 in 90 Days! Date: 11 Oct 1995 16:58:39 GMT Organization: Yale University Biology Department Lines: 3 Message-ID: <45gt3v$flf@news.ycc.yale.edu> References: <45ge3g$o1j$5@mhafn.production.compuserve.com> NNTP-Posting-Host: 130.132.107.28 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: 102771.3337@CompuServe.COM X-URL: news:45ge3g$o1j$5@mhafn.production.compuserve.com Xref: biosci bionet.molbio.hiv:1674 bionet.molbio.methds-reagnts:34740 bionet.molbio.molluscs:171 bionet.molbio.proteins:5904 bionet.molbio.rapd:1323 Get out of this site you fool! From owner-molluscs@net.bio.net Wed Oct 11 23:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!swrinde!emory!news.cc.emory.edu!usenet From: Brad Thomas Newsgroups: bionet.molbio.hiv,bionet.molbio.methds-reagnts,bionet.molbio.molluscs,bionet.molbio.proteins,bionet.molbio.rapd Subject: Re: Make a $1,000,000 in 90 Days! Date: 11 Oct 1995 23:51:55 GMT Organization: Emory University Lines: 4 Message-ID: <45hlar$894@moe.cc.emory.edu> References: <45ge3g$o1j$5@mhafn.production.compuserve.com> NNTP-Posting-Host: alvin.gen.emory.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:45ge3g$o1j$5@mhafn.production.compuserve.com Xref: biosci bionet.molbio.hiv:1676 bionet.molbio.methds-reagnts:34766 bionet.molbio.molluscs:173 bionet.molbio.proteins:5915 bionet.molbio.rapd:1326 Does this have something to do with smuggling aliens ? (Sorry for the bandwidth) From owner-molluscs@net.bio.net Mon Oct 16 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!dispatch.news.demon.net!demon!halmarax.halmarax.demon.co.uk!not-for-mail From: Tony@halmarax.demon.co.uk (Tony Halmarack) Newsgroups: bionet.molbio.molluscs Subject: eyestalk of a snail Date: Tue, 17 Oct 1995 11:47:23 +0100 Organization: Detectox Lines: 4 Distribution: world Message-ID: <19951017.114723.89@halmarax.halmarax.demon.co.uk> Reply-To: Tony@halmarax.demon.co.uk NNTP-Posting-Host: halmarax.demon.co.uk X-NNTP-Posting-Host: halmarax.demon.co.uk X-Newsreader: Archimedes TTFN Version 0.36 Please can anyone tell me the name of the eyestalk of a snail? -- Tony Halmarack From owner-molluscs@net.bio.net Tue Oct 17 23:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!btnet!zetnet.co.uk!dispatch.news.demon.net!demon!halmarax.halmarax.demon.co.uk!not-for-mail From: Tony@halmarax.demon.co.uk (Tony Halmarack) Newsgroups: bionet.molbio.molluscs Subject: eyestalk of snail Date: Tue, 17 Oct 1995 21:19:44 +0100 Organization: Detectox Lines: 4 Distribution: world Message-ID: <19951017.211944.79@halmarax.halmarax.demon.co.uk> Reply-To: Tony@halmarax.demon.co.uk NNTP-Posting-Host: halmarax.demon.co.uk X-NNTP-Posting-Host: halmarax.demon.co.uk X-Newsreader: Archimedes TTFN Version 0.36 Please can someone tell me the name of the eyestalk of a snail? -- Tony Halmarack From owner-molluscs@net.bio.net Tue Oct 24 22:00:00 1995 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!tank.news.pipex.net!pipex!oleane!jussieu.fr!univ-lyon1.fr!news From: Erwan.LeRoy@cipcinsa.insa-lyon.fr (Erwan Le Roy) Newsgroups: bionet.molbio.molluscs Subject: ALOPECY VIVISCAL Date: 25 Oct 1995 12:01:50 GMT Organization: INSA Centre Informatique du 1er Cycle. Lines: 5 Message-ID: <46l8ve$jko@cismsun.univ-lyon1.fr> NNTP-Posting-Host: pc110-121.insa-lyon.fr Mime-Version: 1.0 X-Newsreader: WinVN 0.99.2 Do you know this new treatment for alopecy made with molluscs. Tell me what you know about it and where can i find it. From owner-molluscs@net.bio.net Mon Oct 30 22:00:00 1995 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!howland.reston.ans.net!newsfeed.internetmci.com!in2.uu.net!news.compuserve.com!news.production.compuserve.com!news From: Brian B. Jiang <73344.437@CompuServe.COM> Newsgroups: bionet.molbio.gene-linkage,bionet.molbio.genome-program,bionet.molbio.hiv,bionet.molbio.methds-reagnts,bionet.molbio.molluscs Subject: Anti-Immigration Law Status/Seminar Date: 31 Oct 1995 08:33:21 GMT Organization: The Law Office of Brian B. Jiang Lines: 105 Message-ID: <474n0h$9r9$6@mhafm.production.compuserve.com> Xref: biosci bionet.molbio.gene-linkage:880 bionet.molbio.genome-program:1562 bionet.molbio.hiv:1692 bionet.molbio.methds-reagnts:35666 bionet.molbio.molluscs:179 RE: New Development On the Anti-Immigration Bill(HR 2202) and Upcoming Seminar Hi folks: As of today, the House Judiciary Committee has completed its markup on the Lamar Bill after 9 days of debate over several weeks. Despite all the bad news, such as reduction of legal immigration and elimination of some family categories, there are a few signs of relief. Both outstanding researchers/professors and national interest waivers were restored to the Bill. So professionals( scientists, post-docs, engineers etc.) can still apply for permanent residence without labor certification and sponsorship of their employers. On the other hand, for those who have to apply through labor cert, they are, as usual, still at the mercy of the Labor Dept. Any positive change is yet to be seen. Although waiver of labor certification and employer sponsorship is still obtainable for those who qualify, difficulties have been on the rise and more are expected. Nearly all four Service Centers have implemented some newly proposed requirements that make it very difficult to obtain an approval. According to administrative rules no law should be carried out until it is final. Despite the rules, INS is already treating the proposals like enforceable law. In the real world, things are not always the way they are supposed to be. This is even more so in immigration practice with today's anti-immigration fervor. I am an immigration attorney specializing in employment based immigration(1st and 2nd preference). I will hold seminars on how to apply for a green card without labor certification and sponsorship of the employer. The intended audiences of my seminar are professionals with advanced degrees or a bachelor degree plus five years of work experience. I will also cover the impact of HR 2202 on future immigration. The locations of the seminar will include: 1. University of Florida at Gainesville University Center Hotel (President's Ballroom) 1535 SW Archer Road Gainesville, FL Tel: (904)371-3333 Time: 11/12/95 Sunday 2:00-4:00pm 2. Columbia University at New York City Columbia University 517 Hamilton Hall (close to W. 116th St. & Broadway) Time: 11/13/95 Monday 7:30-9:30pm 3. Princeton University Location to be decided Time 11/14/95 Tuesday 4. Baltimore near University of Maryland Holiday Inn Baltimore Inner Harbor (Chesateake Ballroom #1) 301 W. Lombard Street Baltimore, MD Tel: (410)685-3500 Time: 11/15/95 Wed. 7:30-9:30pm 5. Pittsburgh near Univ. of Pittsburgh Pittsburgh Green Tree Marriott (Near University of Pittsburgh) 101 Marriott Drive Pittsburgh, PA Tel: (412)922-8400 Time: 11/16/95 Thursday 7:30-9:30pm 6. Boston at Cambridge Hyatt Regency Cambridge 575 Memorial Drive Cambridge, MA Tel: (617)492-1234 Time 11/17/95 Friday 7:30-9:30pm 7. University of Connecticut Location to be decided Time 11/18/95 Saturday 2:30-4:30pm 8. Palo Alto near Stanford University Holiday Inn Palo Alto (Near Stanford University) 625 El Camino Real Palo Alto, CA Tel: (415)328-2800 Time 11/19/95 Sunday 7:30-9:30pm I will conduct these seminars during these time. All questions, comment, and suggestions are welcome. You can also reach me at tel. (619)278-5480 or (619)278-5492. Brian B. Jiang Esq. Email: 73344.437@compuserve.com From owner-molluscs@net.bio.net Tue Oct 31 22:00:00 1995 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.molluscs Subject: IMPORTANT: BIOSCI miniFAQ Date: 1 Nov 1995 02:03:30 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 196 Sender: daemon@net.bio.net Distribution: world Message-ID: <199511011000.CAA15137@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. 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. 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. E-mail users can search the BIOSCI archives by using our waismail e-mail server. For instructions send the message help to waismail@net.bio.net. Leave the Subject: line blank (anything entered on the Subject: line is ignored). 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