From owner-diagnostics@net.bio.net Tue Mar 14 22:00:00 1995 Path: biosci!biosci!not-for-mail From: kristoff@net.bio.net (David Kristofferson) Newsgroups: bionet.diagnostics Subject: another test of bionet.diagnostics Date: 14 Mar 1995 16:33:56 -0800 Organization: BIOSCI International Newsgroups for Biology Lines: 13 Distribution: world Message-ID: <3k5clk$ni2@net.bio.net> NNTP-Posting-Host: net.bio.net Test of the USENET newsgroup and the news-to-mail gateway. Please ignore. Please await the posting of the official opening announcement before posting into this USENET newsgroup. Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-diagnostics@net.bio.net Tue Mar 14 22:00:00 1995 Path: biosci!BIONET.IG.COM!kristoff From: kristoff@BIONET.IG.COM (David Kristofferson) Newsgroups: bionet.diagnostics Subject: test of diagnost@net.bio.net Date: 14 Mar 1995 16:31:04 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net test of the mail-to-news gateway. Please ignore. From owner-diagnostics@net.bio.net Tue Mar 14 22:00:00 1995 Path: biosci!ADMIN3.USASK.CA!CHELACK From: CHELACK@ADMIN3.USASK.CA Newsgroups: bionet.diagnostics Subject: Immunohisto marker for macrophages needed Date: 15 Mar 1995 06:47:57 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HO5PWDPZAQ8WZS0M@ADMIN3.USASK.CA> NNTP-Posting-Host: net.bio.net Hi, I keep getting requests for a staining paraffin sections for macrophages in particular cases of suspected histiocytic tumors in dogs. What we are looking for is a marker for macrophages/histiocytes in paraffin sections which could be used in any species. Is there anyone out there in netland who is already doing this? My initial thoughts are that an antibody to myeloperoxidase or alpha 1 anti tryosin might be the way to go but I am short on time and wonder if anyone has validated such an antibody on paraffin sections, or for that matter in other species. Most of the commercially available antibodies such as Mac-1 or Mac-3 do not seem to work on paraffin sections of canine tissues so I am looking for a more generic marker. Thanks for any advise. Regards BJC - Brian Short people... last to know its raining, first to know its a flood. From owner-diagnostics@net.bio.net Tue Mar 14 22:00:00 1995 Path: biosci!a1.kids.wustl.edu!BULLER From: BULLER@a1.kids.wustl.edu (Richard Buller 454 6039) Newsgroups: bionet.diagnostics Subject: Re: Low Boiling Point Date: 15 Mar 1995 05:10:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HO5N1WSQ9I8WXC92@mr.kids.wustl.edu> NNTP-Posting-Host: net.bio.net ICAgICAgICAgICAgICAgVG8gdGhlIGluZGl2aWR1YWwgY29uY2VybmVkIGFib3V0 IHRoZSBsb3cgcG9pbnQgb2Ygd2F0ZXIgaW4gUXVpdG8sIHRoZSANCiAgICAgICAg ICBwdXJwb3NlIG9mIGdvaW5nIHRvIDk1sEMgaXMgdG8gYXNzdXJlIGRlbmF0dXJh dGlvbiBvZiB0ZW1wbGF0ZSBETkEgc3RyYW5kcy4gIA0KICAgICAgICAgIEFzIHlv dSBjYW5ub3QgYWNoaWV2ZSB0aGlzLCBhdCBsZWFzdCB3aXRob3V0IHRoZSB1c2Ug b2YgYSBwcmVzc3VyZSBjb29rZXIsIHlvdSANCiAgICAgICAgICBtYXkgd2FudCB0 byBpbnZlc3RpZ2F0ZSB0aGUgdXNlIG9mIGNvLXNvbHZlbnRzIHN1Y2ggYXMgZ2x5 Y2Vyb2wgb3IgRE1TTyBpbiB0aGUgDQogICAgICAgICAgUENSIHJlYWN0aW9uIG1p eHR1cmUuICBUaGVzZSBzdWJzdGFuY2VzIGhhdmUgYmVlbiB1c2VkIGluIFBDUiBy ZWFjdGlvbnMgdG8gDQogICAgICAgICAgYWNoaWV2ZSBjb21wbGV0ZSBkZW5hdHVy YXRpb24gb2YgaGlnaCBHQyBjb250ZW50IEROQSBzdWNoIGFzIGZvdW5kIGluIHNv bWUgDQogICAgICAgICAgaGVycGVzIHZpcnVzZXMuICBJIHdvdWxkIHRyeSB5b3Vy IHJlYWN0aW9uIGZpcnN0IHdpdGhvdXQgYW55IHNwZWNpYWwgDQogICAgICAgICAg cHJvY2VkdXJlcywgODkgQyBtYXkgYmUgaGlnaCBlbm91Z2ggZm9yIHlvdXIgdGVt cGxhdGUuDQogICAgICAgICAgDQogICAgICAgICAgUmljaGFyZCBCdWxsZXINCiAg ICAgICAgICBXYXNoaW5ndG9uIFVuaXZlcnNpdHkNCiAgICAgICAgICBTdC4gTG91 aXMsIE1PICANCg0KDQoNCg== From owner-diagnostics@net.bio.net Tue Mar 14 22:00:00 1995 Path: biosci!daresbury!not-for-mail From: David Kristofferson Newsgroups: bionet.diagnostics Subject: test of diagnost@daresbury.ac.uk Date: 15 Mar 1995 00:39:17 -0000 Lines: 2 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3k5cvl$g7e@mserv1.dl.ac.uk> Original-To: diagnost@dl.ac.uk test of diagnost@daresbury.ac.uk; please ignore From owner-diagnostics@net.bio.net Tue Mar 14 22:00:00 1995 Path: biosci!biosci!not-for-mail From: kristoff@net.bio.net (David Kristofferson) Newsgroups: bionet.diagnostics Subject: test of bionet.diagnostics Date: 14 Mar 1995 16:16:09 -0800 Organization: BIOSCI International Newsgroups for Biology Lines: 11 Distribution: world Message-ID: <3k5bk9$m8h@net.bio.net> NNTP-Posting-Host: net.bio.net This is a test of the bionet.diagnostics USENET newsgroup and the news-to-mail gateway. Please do not post into this USENET newsgroup until our tests are complete and an official opening announcement in issued here. Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-diagnostics@net.bio.net Wed Mar 15 22:00:00 1995 Path: biosci!sasa.gov.uk!burns From: burns@sasa.gov.uk (Robert Burns) Newsgroups: bionet.diagnostics Subject: Mabs from PAGE bands Date: 16 Mar 1995 07:21:07 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 29 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Hi all, my first posting to the "now permanent" diagnost list My question: Has anybody raised Mabs using protein bands excised from PAGE gels as the immunogen? If so can you use the gel band itself as an implant without having to elute the protein? Second question ( I have asked this before but for the benefit of newcomers) Is anybody interested in Fungal diagnostics by serological methods? Robert Robert Burns Monoclonal Antibody Unit Scottish Agricultural Science Agency East Craigs Edinburgh Scotland burns@sasa.gov.uk From owner-diagnostics@net.bio.net Wed Mar 15 22:00:00 1995 Path: biosci!biosci!not-for-mail From: biohelp@net.bio.net (BIOSCI Administrator) Newsgroups: bionet.diagnostics Subject: DIAGNOSTICS/bionet.diagnostics is ready! Date: 16 Mar 1995 13:25:09 -0800 Organization: BIOSCI International Newsgroups for Biology Lines: 201 Distribution: world Message-ID: <3kaabl$e82@net.bio.net> NNTP-Posting-Host: net.bio.net The DIAGNOSTICS/bionet.diagnostics newsgroup is ready for operation. PLEASE NOTE that many USENET sites do not allow automatic creation of new USENET groups!!! If you do not see bionet.diagnostics in your newsreader within another day or two, ask your news system administrator to act on our "newgroup" message to enable the group at your site. We have already done several tests and are certain that the group is currently propagating around the network. 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Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-diagnostics@net.bio.net Fri Mar 17 22:00:00 1995 Path: biosci!sasa.gov.uk!browning From: browning@sasa.gov.uk (Isla Browning) Newsgroups: bionet.diagnostics Subject: Medical database development Date: 18 Mar 1995 01:40:02 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net 4D programmer wanted to assist in developing industry standard medical database for UK market From owner-diagnostics@net.bio.net Fri Mar 17 22:00:00 1995 Path: biosci!aol.com!PandaGroup From: PandaGroup@aol.com Newsgroups: bionet.diagnostics Subject: Chlamydia Testing Date: 18 Mar 1995 10:45:43 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <950318134234_53459775@aol.com> NNTP-Posting-Host: net.bio.net I am writing an article on current clinical testing practices and products for the diagnosis of chlamydial infection, and would like professional opinions as to the current state of the art, how useful current test procedures are to clinicians, problems experienced, etc. The article will be published by the College of American Pathologists. If you would like to share your opinions on this subject with me, please e-mail a phone number and convenient time for me to call. For point of reference in picking a time, I'm located in California. Thank you. Dr. Ken Krul From owner-diagnostics@net.bio.net Sun Mar 19 22:00:00 1995 Path: biosci!sasa.gov.uk!browning From: browning@sasa.gov.uk (Isla Browning) Newsgroups: bionet.diagnostics Subject: Use of PVP in ELISA Date: 20 Mar 1995 03:29:54 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net As suggested by Bioreba we use polyvinylpyrrolidone in extraction and = conjugate buffers when carrying out ELISA for detection of virus in plant sap. I would be grateful if someone could enlighten me as to why = it is used . Is its action as a blocker or as an antioxidant or does = it have some other function? Isla Browning SASA Edinburgh 8/3/95 =01=01=01=01 From owner-diagnostics@net.bio.net Sun Mar 19 22:00:00 1995 Path: biosci!INTERNETMAIL.PR.CYANAMID.COM!Bart_Corsaro_at_USLRMG01 From: Bart_Corsaro_at_USLRMG01@INTERNETMAIL.PR.CYANAMID.COM Newsgroups: bionet.diagnostics Subject: Re: Mabs from PAGE bands Date: 20 Mar 1995 10:22:11 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 55 Sender: daemon@net.bio.net Distribution: world Message-ID: <9502207957.AA795734383@internetmail.pr.cyanamid.com> NNTP-Posting-Host: net.bio.net Robert, I haven't made MABs with PAGE eluted proteins but I have made polyclonal antibodies with them and the antisera I generated had an excellent specific titer. I used it at a dilution of > 1 : 5000 for Western blots. The antisera however, could not be used for immunopercipitation. At the time we hypothesized that the sera was reacting to the linearized protein and that little if any secondary protein structure had reformed after elution from the gel. While doing this work we considered using the whole gel, but we didn't think we could get the protocol passed the Animal Safety Committee so we didn't bother trying. From what I remember, the thought was that the SDS and the acrylamide would cause severe lesion at the point of injections and cause undue suffering for the animal. If you want our protocol let me know and I'll try to dig it up. Bart Corsaro Lederle-Praxis Biologicals West Henrietta, New York EMail: Bart_Corsaro_at_USLRMG01@internetmail.pr.cyanamid.com _______________________________________________________________________________ My question: Has anybody raised Mabs using protein bands excised from PAGE gels as the immunogen? If so can you use the gel band itself as an implant without having to elute the protein? Second question ( I have asked this before but for the benefit of newcomers) Is anybody interested in Fungal diagnostics by serological methods? Robert Robert Burns Monoclonal Antibody Unit Scottish Agricultural Science Agency East Craigs Edinburgh Scotland burns@sasa.gov.uk From owner-diagnostics@net.bio.net Sun Mar 19 22:00:00 1995 Path: biosci!VTH1.VTH.COLOSTATE.EDU!dgetzy From: dgetzy@VTH1.VTH.COLOSTATE.EDU (David Getzy) Newsgroups: bionet.diagnostics Subject: Re: Immunohisto marker for macrophages needed Date: 20 Mar 1995 07:28:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 41 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <01HO5PWDPZAQ8WZS0M@ADMIN3.USASK.CA> NNTP-Posting-Host: net.bio.net Brian, We have recently completed a comparison study of malignant histiocytosis and anaplastic giant cell carcinoma in the canine. For the immunohistochemical studies, we used both anti-alpha 1 antitrypsin and anti-lysozyme. Both of these markers worked well, but in general, the signal from the anti alpha 1 antitrypsin was the strongest. The paper can be found in the journal: Veterinary Clinical Pathology 23:118-122, 1994. If you have any further questions on the technique, please call or e-mail. Good luck! ********************************************************************* * Dave Getzy * Director * CSU Diagnostic Laboratories * 300 West Drake, Fort Collins, Co 80523 * PHONE 303-491-1281 FAX 303-491-0320 * EMAIL dgetzy@vth1.vth.colostate.edu ******************************************************************** On 15 Mar 1995 CHELACK@admin3.usask.ca wrote: > Hi, I keep getting requests for a staining paraffin sections for macrophages > in particular cases of suspected histiocytic tumors in dogs. What we are > looking for is a marker for macrophages/histiocytes in paraffin sections > which could be used in any species. Is there anyone out there in netland > who is already doing this? My initial thoughts are that an antibody to > myeloperoxidase or alpha 1 anti tryosin might be the way to go but I am > short on time and wonder if anyone has validated such an antibody on paraffin > sections, or for that matter in other species. Most of the commercially > available antibodies such as Mac-1 or Mac-3 do not seem to work on paraffin > sections of canine tissues so I am looking for a more generic marker. > Thanks for any advise. > > Regards > BJC - Brian Short people... last to know its > raining, first to know its a flood. > > From owner-diagnostics@net.bio.net Sun Mar 19 22:00:00 1995 Path: biosci!IC.NET!cliu From: cliu@IC.NET (C. Liu) Newsgroups: bionet.diagnostics Subject: Re: Use of PVP in ELISA Date: 20 Mar 1995 06:45:35 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 20 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >As suggested by Bioreba we use polyvinylpyrrolidone in extraction and >conjugate buffers when carrying out ELISA for detection of virus in >plant sap. I would be grateful if someone could enlighten me as to why > it is used . Is its action as a blocker or as an antioxidant or does it have >some other function? > > >Isla Browning >SASA >Edinburgh >8/3/95 > I believe it's used as a blocking agent in your assay. It is a hydrophilic polymer that is a wetting agent, and probably reduces the nonspecific binding to plastic in microtiter plates. --- cliu@ic.net Internet connectivity provided by ICNET: +1 (313)998-0090 From owner-diagnostics@net.bio.net Mon Mar 20 22:00:00 1995 Path: biosci!ZEUS.DATASRV.CO.IL!ila2027 From: ila2027@ZEUS.DATASRV.CO.IL (Falk Fish) Newsgroups: bionet.diagnostics Subject: Re: RHEUMATOID FACTOR Date: 20 Mar 1995 20:08:57 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net We indeed discussed RF a couple of months ago. Additional ways to reduce RF interference is by diluting the specimen (RF, being a low affinity antibody, will be less active) and conducting reations at higher temperature. I missed the initial message, so I have no idea of the system involved. There are additional ways to counteract RF, but they depend on what one is trying to measure. Falk Fish, Tel-Aviv, Israel From owner-diagnostics@net.bio.net Mon Mar 20 22:00:00 1995 Path: biosci!CMU.CHIANGMAI.AC.TH!mdbci001 From: mdbci001@CMU.CHIANGMAI.AC.TH (Prachya Kongtawelert) Newsgroups: bionet.diagnostics Subject: Re: Use of PVP in ELISA Date: 20 Mar 1995 17:16:47 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 36 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net As I have recalled, there was a paper concerning about the use of polyvinylpyrrolidone as a blocker in ELISA in Analytical Biochemistry, 1994. ************* Prachya Kongtawelert, Ph.D. Res Lab for Joint Dis., Dept of Biochem., Fac of Med., Chiangmai University, Chiangmai, Thailand 50200 Tel No. 66-53-894188 Fax No. 66-53-894188, 217144 email:mdbci001@chiangmai.ac.th On 20 Mar 1995, Isla Browning wrote: > As suggested by Bioreba we use polyvinylpyrrolidone in extraction and = > > conjugate buffers when carrying out ELISA for detection of virus in > plant sap. I would be grateful if someone could enlighten me as to why = > > it is used . Is its action as a blocker or as an antioxidant or does = > it have > some other function? > > > Isla Browning > SASA > Edinburgh > 8/3/95 > =01=01=01=01 > > > > From owner-diagnostics@net.bio.net Mon Mar 20 22:00:00 1995 Path: biosci!CORNELL.EDU!gsr1 From: gsr1@CORNELL.EDU (Geoffrey Rule) Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 21 Mar 1995 07:38:53 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 32 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net At 8:57 AM 3/13/95, forbes@cip.org.ec wrote: >We would like to initiate some PCR based methods near Quito, >Ecuador at an altitude of 3050 m above sea level. Water boils >here at 89 C which will not allow us to bring the vials up above >this temperature. Our PCR machine has not yet arrived but we >would like to have some ideas on how this problem might be solved >before it does. >Many thanks >Greg Forbes > Hi Greg, I haven't seen any replies to your message. Have you received any? Your question is somewhat intruiging. I wonder, first of all, would DNA and the enzymes behave as you would like, at the lower temperature, since they are at lower pressure at the same time? Another thought; if the tubes are tightly capped the reactions may not bump, or boil, so long as the pressure within each tube is contained. Essentially, the pressure within each tube will actually be mucher higher than your ambientt air pressure anyway and so you will not have a problem. Failing that, I can only imagine that you might build some sort of pressure controlled box to contain the thermal cycler while performing PCR. Please let me know what you come up with, and best of luck! Geoff Rule Geoffrey S. Rule Analytical Chemistry Labs NYSAES/ Cornell University Geneva, NY 14456 1-315-787-2435 From owner-diagnostics@net.bio.net Mon Mar 20 22:00:00 1995 Path: biosci!ZEUS.DATASRV.CO.IL!ila2027 From: ila2027@ZEUS.DATASRV.CO.IL (Falk Fish) Newsgroups: bionet.diagnostics Subject: Re: Use of PVP in ELISA Date: 20 Mar 1995 20:22:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net On 20 Mar 1995, Isla Browning wrote: > As suggested by Bioreba we use polyvinylpyrrolidone in extraction and = > > conjugate buffers when carrying out ELISA for detection of virus in > plant sap. I would be grateful if someone could enlighten me as to why = > > it is used . Is its action as a blocker or as an antioxidant or does = > it have > some other function? > > > Isla Browning > SASA > Edinburgh > 8/3/95 > =01=01=01=01 > > > PVP may be am absorbent for all kins of noxious compounds (Phenols, etc) which are abundanr in plant tissue. Just a wild guess. Falk. From owner-diagnostics@net.bio.net Mon Mar 20 22:00:00 1995 Path: biosci!ZEUS.DATASRV.CO.IL!ila2027 From: ila2027@ZEUS.DATASRV.CO.IL (Falk Fish) Newsgroups: bionet.diagnostics Subject: Re: Mabs from PAGE bands Date: 20 Mar 1995 20:20:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 41 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net On 16 Mar 1995, Robert Burns wrote: > Hi all, > my first posting to the "now permanent" diagnost list > > My question: Has anybody raised Mabs using protein bands excised > from PAGE gels as the immunogen? If so can you use the gel band > itself as an implant without having to elute the protein? > > Second question ( I have asked this before but for the benefit of > newcomers) Is anybody interested in Fungal diagnostics by serological > methods? > > > Robert > > > > > > Robert Burns > > Monoclonal Antibody Unit > Scottish Agricultural Science Agency > East Craigs > Edinburgh > Scotland > > burns@sasa.gov.uk > > > > Yes, you could immunize with the gel without elution, but macerate (grind) it first to smaller particles. Falk Fish. From owner-diagnostics@net.bio.net Mon Mar 20 22:00:00 1995 Path: biosci!agresearch.cri.nz!COLLINR From: COLLINR@agresearch.cri.nz ("Collin, Roger") Newsgroups: bionet.diagnostics Subject: Use of PVP in ELISA Date: 21 Mar 1995 14:10:29 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HOFLD3V57UHSKHCA@INVERMAY.CRI.NZ> NNTP-Posting-Host: net.bio.net With regard to using PVP there is a paper in Analytical Biochemistry 208:397-99, 1993 on "PVP as a blocking agent in immunochemical studies" byJW Haycock. This paper recommends the use of PVP in conjunction with Tween 20 in Western Blots as it gives a high signal to background ratio. I use this combination in an indirect competitive ELISA for sporidesmin (a mycotoxin) and have found that it works very well. Regards Dr Roger Collin AgResearch, Hamilton, New Zealand From owner-diagnostics@net.bio.net Mon Mar 20 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!uunet!newsflash.concordia.ca!canopus.cc.umanitoba.ca!zeno.ibd.nrc.ca!mansfield From: mansfield@zeno.ibd.nrc.ca (Jim Mansfield) Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 21 Mar 1995 18:12:55 GMT Organization: NRC Institute for Biodiagnostics Lines: 39 Message-ID: <3kn4v7$i25@canopus.cc.umanitoba.ca> References: NNTP-Posting-Host: zeno.ibd.nrc.ca In article odonnell@sasa.gov.uk ("Kevin O'Donnell") writes: ><> > > >We would like to initiate some PCR based methods near Quito, > >Ecuador at an altitude of 3050 m above sea level. Water boils > >here at 89 C which will not allow us to bring the vials up above > >this temperature. Our PCR machine has not yet arrived but we > >would like to have some ideas on how this problem might be solved > >before it does. > >Many thanks > >Greg Forbes ><> > >What an interesting problem! I somehow missed this when it was first posted. >My immediate thought is: would this still be a problem if you had one of those >thermal cyclers with a screw down heated lid? Also, if water boils at a lower >temperature, will the Tm of your DNA primers be lower as well? Hi, I don't know much about what a PCR machine is, but here are some solutions to the low boiling point problem: 1) use water/ethylene glycol mixture 2) use a saturated salt solution of water 3) heat it inside a pressure cooker I would think #1 would be your best choise - assuming the water you are heating the vials in does not come in contact with your sample :-) Hope that helps, -Jim -- --------------------------------------------------------------------- Jim Mansfield internet: mansfield@ibd.nrc.ca Institute for Biodiagnostics, NRCC phone: (204) 984-5191 http://www.ibd.nrc.ca/~mansfield/ fax: (204) 984-5472 From owner-diagnostics@net.bio.net Mon Mar 20 22:00:00 1995 Path: biosci!sasa.gov.uk!odonnell From: odonnell@sasa.gov.uk ("Kevin O'Donnell") Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 21 Mar 1995 08:27:16 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 59 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net <> >We would like to initiate some PCR based methods near Quito, >Ecuador at an altitude of 3050 m above sea level. Water boils >here at 89 C which will not allow us to bring the vials up above >this temperature. Our PCR machine has not yet arrived but we >would like to have some ideas on how this problem might be solved >before it does. >Many thanks >Greg Forbes <> What an interesting problem! I somehow missed this when it was first posted. My immediate thought is: would this still be a problem if you had one of those thermal cyclers with a screw down heated lid? Also, if water boils at a lower temperature, will the Tm of your DNA primers be lower as well? Wish I knew more physics. Kevin O'Donnell Diagnostics and Molecular Biology Scottish Agricultural Science Agency Edinburgh odonnell@sasa.gov.uk Kevin O'Donnell Diagnostics and Molecular Biology Scottish Agricultural Science Agency Edinburgh odonnell@sasa.gov.uk From owner-diagnostics@net.bio.net Tue Mar 21 22:00:00 1995 Path: biosci!ACADEM01.MTY.ITESM.MX!dsleal From: dsleal@ACADEM01.MTY.ITESM.MX ("M.C. Diana Sara Leal Klevezas") Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 22 Mar 1995 07:17:28 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <01HOEVO20DSI001171@GW.AGR.CA> NNTP-Posting-Host: net.bio.net On 21 Mar 1995 WHEATCROFTR@ncccot.agr.ca wrote: > Elevate the boiling point by adding DMSO Pardon me, but a think that using DMSO is not a good idea, since =BFf=3D=A7=ACN=E9{=ABE=B4=E4x=AANx<=B7=FE=D2'=D9"=B7=CC=A6LMW}2y=E3=F0=EB= =B4=EF=BE=A0 p4D=C5&=D6 =E6=B6=ACc7=A7'D&$=EADMSO brakes hidrogen bonds and it interferes with the anealing process.DMSO is used in strand separation procedures.=20 =A30=09=AA}"=C1=AF=E4V=D8=DB NNTP-Posting-Host: net.bio.net Elevate the boiling point by adding DMSO. From owner-diagnostics@net.bio.net Tue Mar 21 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!ix.netcom.com!netnews From: gl1@ix.netcom.com (Gene Levinson) Newsgroups: bionet.diagnostics Subject: mammalian telomerase Date: 22 Mar 1995 00:31:44 GMT Organization: Netcom Lines: 3 Distribution: world Message-ID: <3knr5g$kq6@ixnews3.ix.netcom.com> NNTP-Posting-Host: ix-dc6-01.ix.netcom.com Does anyone know of sequence data for mammalian telomerase, or have any clues as to why there is none available? Typically, folks are using enzyme assays for detection of telomerase activity. From owner-diagnostics@net.bio.net Tue Mar 21 22:00:00 1995 Path: biosci!UVA.PCMAIL.VIRGINIA.EDU!mgk2r From: mgk2r@UVA.PCMAIL.VIRGINIA.EDU ("Michael G. Kurilla") Newsgroups: bionet.diagnostics Subject: Re: Date: 22 Mar 1995 12:52:14 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 63 Sender: daemon@net.bio.net Distribution: world Message-ID: <199503222052.PAA26702@uva.pcmail.Virginia.EDU> NNTP-Posting-Host: net.bio.net On Mar 18, 9:19am, "Kevin O'Donnell" wrote: > Subject: Re: > <> > > . We've tried laborious/exhaustive DNA extractions, carefully > > monitoring DNA concentration, ensuring the integrity etc. of our reagen= > t > >stocks, but nothing seems to get over the reproducibility problem. = > If there is > >anyone out there who has experienced similar problems > > we would like to hear from you, believe me! > > We have just been through exactly this problem! A promising assay just = > wasn't > giving reproducible results - it was driving us mad. Eventually, we trac= > ed it to > the *water*. The water used was filtered and autoclaved but when run = > side by > side with reactions using water from a different source it was clear that= > it was > indeed the culprit. Why? We don't know. It might be fluctuations in = > the pH or > ionic concentration of the water source. > What type of PCR machine are you using? There have been reports of reproducibility difficulties between various wells of the machine. I know this was documented in the original Perkin-Elmer machine (there was an article in Biotechniques about this). This was demonstrated by preparing a master mix (4.8mls) and running 48 identical reactions in all the wells of the machine. The variation from well to well was quite impressive. With regards to the water, I would a little skeptical of pH or ionic solutes if you are using water prepared from a source that bases purity on electrical resistance of the water. We use a Millipore system that measures the ohms (pure water is something like >18Mohm). All we do after that is purify the water through a 0.22 micron filter and store in aliquots. One point to consider is that these systems don't measure organics (unless they carry a charge). The final filtration is simply to ensure elimination of bacterial contamination. > We've decided to splash out (sorry) on some bottled water in future. > > This may well not be what's causing your problems of course. For example= > : > > >Our latest ploy is to hang rosary beeds on the > > PCR machine and play Elvis Costello very loud, but this has met with = > only > > limited success. > > Your problem may well be the variation in quality of Elvis Costello album= > s. > Have you been playing 'Goodbye Cruel World' on the days it hasn't worked = > > and 'Imperial Bedroom' on the days that it has? > I've been told that a horseshoe over the machine works even you don't believe that it brings good luck. Mike Kurilla From owner-diagnostics@net.bio.net Wed Mar 22 22:00:00 1995 Path: biosci!GNV.IFAS.UFL.EDU!CRS From: CRS@GNV.IFAS.UFL.EDU ("Charles R. Semer IV") Newsgroups: bionet.diagnostics Subject: Addresses of materials and reagent suppliers Date: 22 Mar 1995 16:35:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 15 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HOG4YXK0WO90N7I5@gnv.ifas.ufl.edu> NNTP-Posting-Host: net.bio.net Dear Net, I am near finishing a Book to be published by American Phytopathological Press titled" Methods and Practice of Plant Disease Diagnosis". In the introductory chapter as an addenda I would like to include the addresses of material and reagent suppliers. I can obtain the US addresses that would be suitable for inclusion in this listing but I do not have any information about suppliers, names and addresses outside the US. Can you provide some assistance? Rather than clutter up this group with an endless listing of this nature please send the information to me at crs@gnv.ifas.ufl.edu. If this group would like a copy of the completed list I will be happy to provide one in June. Thanks in advance for your assistance in this matter. Regards, Charles (Chuck) R. Semer IV crs@gnv.ifas.ufl.edu From owner-diagnostics@net.bio.net Wed Mar 22 22:00:00 1995 Path: biosci!AARDVARK.UCS.UOKNOR.EDU!SMASOUD From: SMASOUD@AARDVARK.UCS.UOKNOR.EDU Newsgroups: bionet.diagnostics Subject: RE:low boiling point Date: 23 Mar 1995 09:20:11 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HOH1U0I1VQ007FAW@aardvark.ucs.uoknor.edu> NNTP-Posting-Host: net.bio.net In addtion of increasing the boiling temp., DMSO weaken the douple strand interactions. This should lower the annealing temp. and lower denaturing temp. I would recomend playing with both parammeters if you want to use DMSO in PCR. I have used DMSO before in PCR and was very effective. I found that you need to find the optimum concentration for any DNA/primers combination. DMSO inhibit taq activity at high concentrations. Sameer Masoud Plant Biology Division S. R. Noble Foundation 2510 Sam Noble Parkway Ardmore, OK 73402 Tel (405) 221 7309 FAX (405) 221 7380 From owner-diagnostics@net.bio.net Wed Mar 22 22:00:00 1995 Path: biosci!VM1.NODAK.EDU!DEVAULT From: DEVAULT@VM1.NODAK.EDU (James D DeVault) Newsgroups: bionet.diagnostics Subject: species 2000 Date: 23 Mar 1995 06:35:53 -0800 Organization: USDA/ARS Bioscience Laboratory, Fargo, ND Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <199503231435.GAA20102@net.bio.net> NNTP-Posting-Host: net.bio.net If anyone has a back up of a post regarding species 2000 please forward. Our system crashed during my reading, and the post was lost. If this is the wrong news group from which this post was sent, have a great day anyway. thanx jim deVault From owner-diagnostics@net.bio.net Wed Mar 22 22:00:00 1995 Path: biosci!NCCCOT.AGR.CA!WHEATCROFTR From: WHEATCROFTR@NCCCOT.AGR.CA Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 22 Mar 1995 20:10:37 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: <01HOGBWKERZ6001C7V@GW.AGR.CA> NNTP-Posting-Host: net.bio.net >On 21 Mar 1995 WHEATCROFTR@ncccot.agr.ca wrote: > Elevate the boiling point by adding DMSO >Pardon me, but a think that using DMSO is not a good idea, since >=BFf=3D=A7=ACN=E9{=ABE=B4=E4x=AANx<=B7=FE=D2'=D9"=B7=CC=A6LMW}2y=E3=F0=EB= >=B4=EF=BE=A0 p4D=C5&=D6 =E6=B6=ACc7=A7'D&$=EADMSO brakes >hidrogen bonds and it interferes with the anealing process.DMSO is used in >strand separation procedures.=20 >=A30=09=AA}"=C1=AF=E4V=D8=DBReturn-path: Dear Diana and other DMSO doubters, See Shen et al, Trends in Genetics 8:227 (1992). Title: DMSO improves PCR... I think they had 5% in their samples. This should push the BP up enough for Quito. [I believe the BP of neat DMSO is 189 C at sea level] Roger W. From owner-diagnostics@net.bio.net Wed Mar 22 22:00:00 1995 Path: biosci!PLAINS.NODAK.EDU!dwatson From: dwatson@PLAINS.NODAK.EDU (David Watson) Newsgroups: bionet.diagnostics Subject: PCR at high altitude Date: 23 Mar 1995 14:36:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 18 Sender: daemon@net.bio.net Distribution: world Message-ID: <199503232235.AA11783@plains.NoDak.edu> NNTP-Posting-Host: net.bio.net Dear Netters: A quick observation (somebody correct me if this is not accurate): Taq is an enzyme, albeit one with a very high temperature optimum. Therefore, at a temp slightly below boiling in Quito (89 C, was it?), shouldn't Taq still work, even if slightly less than optimally, such that routine amplifications could still be accomplished? Does anyone know how rapidly Taq's efficiency falls off either side of 94 C? I assume the dropoff is steeper above 94 C? |===========================================| David A. Watson, Ph.D. (dwatson@plains.nodak.edu) Asst. Prof. of Veterinary and Microbiological Sciences North Dakota State University --------------------------------------------------------------------------- "Some are wise, some otherwise." |===========================================| From owner-diagnostics@net.bio.net Wed Mar 22 22:00:00 1995 Path: biosci!ACADEM01.MTY.ITESM.MX!dsleal From: dsleal@ACADEM01.MTY.ITESM.MX ("M.C. Diana Sara Leal Klevezas") Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 23 Mar 1995 13:34:46 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 30 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <01HOGBWKERZ6001C7V@GW.AGR.CA> NNTP-Posting-Host: net.bio.net Dear Roger W. I didn't realize that such a little DMSO could have that effect on temperature. Pardon my ignorance, I've got your point and thank you for the lecture. Dear netters: Sorry my last message did't get thourgh complete, I have some other point regarding Greg's question. Fist of all I agreed with Geoff's comments. The presure inside the tubes must be much higher than enviromental pressure, Presure builds up when liquids and air within the tubes expand, therefore pressure in warmer-than-enviroment tubes must increase with temperature. Just make sure to use a good quality tube with a well-fit cap. The only sure way to know the answers to Greg's questions is trying it, and I'm sure everyone would like to know how this goes for Greg. If I'm wrong regarding the pressure building up in warm tubes, the possibilities are even more exciting, here is the idea: The water boils because the molecules get excited enough to do so. If less energy is needed to boil the water, less energy might be needed to break the hidrogen bonds between DNA strands, which means that under those circumstances, one might need lower temperatures to reach DNA melting point. If this is true, the consecuences are that your Taq will last loger!!!. Regards to everyone, Diana Sara Leal-Klevezas Centro de Investigacion Biomedica del Noreste Instituto Mexicano del Seguro Social Monterrey, Mexico. From owner-diagnostics@net.bio.net Wed Mar 22 22:00:00 1995 Path: biosci!ZEUS.DATASRV.CO.IL!ila2027 From: ila2027@ZEUS.DATASRV.CO.IL (Falk Fish) Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 23 Mar 1995 12:52:11 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 19 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net > At 8:57 AM 3/13/95, forbes@cip.org.ec wrote: > >We would like to initiate some PCR based methods near Quito, > >Ecuador at an altitude of 3050 m above sea level.Water boils > >here at 89 C which will not allow us to bring the vials up above > >this temperature.Our PCR machine has not yet arrived but we > >would like to have some ideas on how this problem might be solved > >before it does. > >Many thanks > >Greg Forbes > > Why don't you add salt or sugar to the water in the bath, to increase its boiling point and tightly cap your test tubes? Falk. From owner-diagnostics@net.bio.net Wed Mar 22 22:00:00 1995 Path: biosci!CTS.COM!b3748 From: b3748@CTS.COM (Bryan Kiehl) Newsgroups: bionet.diagnostics Subject: Allergen Sources Date: 23 Mar 1995 15:08:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I am trying to make several assays for specific IgE. Does anyone know sources for purified allergens? Bryan Kiehl GenBio San Diego, CA 92128 (619) 592-9300, ext 309 (619) 592-9400 (FAX) b3748@cts.com From owner-diagnostics@net.bio.net Thu Mar 23 22:00:00 1995 Path: biosci!SVPAL.ORG!hsrock From: hsrock@SVPAL.ORG (Helene Sue Rock) Newsgroups: bionet.diagnostics Subject: Re: Allergen Sources Date: 23 Mar 1995 19:42:25 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 33 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net Dear Bryan, Please tell me what particular allergens you're interested in and what types of assays you're doing. Prior to my current career as full-time Homeschooling Mom, I was one of the original scientists at Allergenetics. Our original mission was to develop non-isotopic (ELISA) in-vitro allergy diagnostics. Allergenetics was sold to 3M a year before I left and has since be resold to BioWhittaker. I don't know what happened to the original product lines. But they did work pretty well and corresponded very well to the "gold standard" RAST test. Let me know what you're doing more specifically and perhaps I can be of some assistance. I am no longer in the paid job market, but I try to keep up as best I can via the Internet and my literature and friends. Good luck. Sincerely, Helene Sue Rock On 23 Mar 1995, Bryan Kiehl wrote: > I am trying to make several assays for specific IgE. > > Does anyone know sources for purified allergens? > Bryan Kiehl > GenBio > San Diego, CA 92128 > (619) 592-9300, ext 309 > (619) 592-9400 (FAX) > b3748@cts.com > > > From owner-diagnostics@net.bio.net Thu Mar 23 22:00:00 1995 Path: biosci!biotech.lan.nrc.ca!juck From: juck@biotech.lan.nrc.ca ("Juck, David") Newsgroups: bionet.diagnostics Subject: PCR at high altitude Date: 24 Mar 1995 06:45:44 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: <2F72DAD6@coursmtp.nrc.ca> NNTP-Posting-Host: net.bio.net Just a couple quick comments regarding PCR at higher altitudes and enzyme stability. Taq polymerase has a temperature optimum, according to Perkin- Elmer, of 75-80?C. The half-life of the enzyme though drops off sharply above 94?C (92.5?C/>130 min, 95?C/40 min, 97.5?C/10 min). The half-life of Taq can be extended at 95?C with the addition of 10% glycerol to the reaction mixture, but this may or may not affect the PCR. The real concern with PCR at high altitudes and not being able to reach temperatures higher than 89?C is the denaturing of the DNA strands which is necessary for further cycles of PCR. I think the addition of DMSO, as already mentioned by several others, is a good idea. After the initial denaturation of your template DNA (boiling in well sealed tubes?), would the denaturation of the PCR products, which would be much smaller in length relative to the initial template DNA, be easier to accomplish, ie. sufficient denaturation of PCR products at lower temperatures? I'm curious to find out what works. David Juck McGill University, Macdonald College Montreal, Canada ------------------------------------------------------------------------------ ------------------------------------------------------------------------------ Dear Netters: A quick observation (somebody correct me if this is not accurate): Taq is an enzyme, albeit one with a very high temperature optimum. Therefore, at a temp slightly below boiling in Quito (89 C, was it?), shouldn't Taq still work, even if slightly less than optimally, such that routine amplifications could still be accomplished? Does anyone know how rapidly Taq's efficiency falls off either side of 94 C? I assume the dropoff is steeper above 94 C? |===========================================| David A. Watson, Ph.D. (dwatson@plains.nodak.edu) Asst. Prof. of Veterinary and Microbiological Sciences North Dakota State University --------------------------------------------------------------------------- "Some are wise, some otherwise." |===========================================| From owner-diagnostics@net.bio.net Thu Mar 23 22:00:00 1995 Path: biosci!daresbury!not-for-mail From: "Juerg E. Frey" Newsgroups: bionet.diagnostics Subject: reply Date: 24 Mar 1995 16:22:53 -0000 Lines: 28 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <3kurkt$rr2@mserv1.dl.ac.uk> Original-To: "diagnost@daresbury.ac.uk" Peter Thoren wrote: >hi netters! >I am going to test spermidine in order to improve my microsatellite PCR. >I just want to know what final concentration is used by others. >Thanks in advance, >Peter Thoren >Dept.of genetics, Uppsala University >Sweden >peter.thoren@genetik.uu.se The optimal range cited in a paper on this topic (Ching-Yi Wan and Thea A. Wilkins (1993) Spermidine facilitates PCR amplification of target DNA. PCR Methods and Applications 3: 208-210) is between 0.2 to 1.0 mM. Regards - Juerg Juerg E. Frey Swiss Federal Research Station for Fruit-Growing, Viticulture and Horticulture CH-8820 Waedenswil SWITZERLAND PH: xxx411-783-6111 FAX:xxx411-780-6341 e-mail: juerg.frey@wae.faw.admin.ch From owner-diagnostics@net.bio.net Thu Mar 23 22:00:00 1995 Path: biosci!PLAINS.NODAK.EDU!dwatson From: dwatson@PLAINS.NODAK.EDU (David Watson) Newsgroups: bionet.diagnostics Subject: High-altitude PCR Date: 24 Mar 1995 08:21:16 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <199503241620.AA10807@plains.NoDak.edu> NNTP-Posting-Host: net.bio.net One more bit re: the PCR question. Tm's for most DNA's of approx. 40 % G+C are 85-90 C (in, e.g. TE buffer), such that with even 40-50% of the template melted, it might still be possible to routinely amplify targets by melting at say 88 C. I think David Juck makes a good point when he says that subsequent denaturation of the much smaller amplicon could be much easier (but only if it doesn't have a high G+C content). Before adding DMSO or glycerol or whatever, I'd be inclined to just give it one shot at 88 C. You might accomplish your objective without further fine-tuning. There might also be the added benefit of less temperature-related damage to the DNA. Dave Watson From owner-diagnostics@net.bio.net Thu Mar 23 22:00:00 1995 Path: biosci!bcm!cs.utexas.edu!news.sprintlink.net!uunet!newsflash.concordia.ca!canopus.cc.umanitoba.ca!zeno.ibd.nrc.ca!mansfield From: mansfield@zeno.ibd.nrc.ca (Jim Mansfield) Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 24 Mar 1995 17:38:34 GMT Organization: NRC Institute for Biodiagnostics Lines: 28 Message-ID: <3kv02q$fon@canopus.cc.umanitoba.ca> References: <01HOGBWKERZ6001C7V@GW.AGR.CA> NNTP-Posting-Host: zeno.ibd.nrc.ca In article dsleal@ACADEM01.MTY.ITESM.MX ("M.C. Diana Sara Leal Klevezas") writes: > >If I'm wrong regarding the pressure building up in warm tubes, the >possibilities are even more exciting, here is the idea: The water boils >because the molecules get excited enough to do so. If less energy is >needed to boil the water, less energy might be needed to break the >hidrogen bonds between DNA strands, which means that under those >circumstances, one might need lower temperatures to reach DNA melting >point. If this is true, the consecuences are that your Taq will last >loger!!!. Thankfully, hydrogen bonding and similar prcesses are not affected by pressure changes of the magnitute we are discussing - otherwise life as we know it would not be possible in Peru at all! The reason less energy and, hence lower temperatures, are needed at high altitudes is the vapour pressure of the liquid does not need to be as high before it matches atmospheric pressure. As the temp. of a liquid rises, so does its vapour pressure. Once it reaches atmospheric pressure, voila, boiling. Putting a lid on the vesselwill raise the pressure of the gas over the liquid, raising its boiling point. -Jim -- --------------------------------------------------------------------- Jim Mansfield internet: mansfield@ibd.nrc.ca Institute for Biodiagnostics, NRCC phone: (204) 984-5191 http://www.ibd.nrc.ca/~mansfield/ fax: (204) 984-5472 From owner-diagnostics@net.bio.net Fri Mar 24 22:00:00 1995 Path: biosci!ZEUS.DATASRV.CO.IL!ila2027 From: ila2027@ZEUS.DATASRV.CO.IL (Falk Fish) Newsgroups: bionet.diagnostics Subject: Re: Allergen Sources Date: 25 Mar 1995 01:03:02 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 39 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net On 23 Mar 1995, Bryan Kiehl wrote: > I am trying to make several assays for specific IgE. > > Does anyone know sources for purified allergens? > Bryan Kiehl > GenBio > Some source for allergenic extracts: ALK Laboratories, 800-325-7354, 203-949-2727, Fax 203-949-2718 This is a subsidiary of a Danish company Nelco Labs, Inc., 1-800-541-0790, 516-242-3662, Fax 516-242-3290 Allermed Labs, Inc. 800-221-2748, 619-292-1060, Fax 619-292-5934 Miles, Inc, 800-992-1120, 203-937-2000 Alo Labs, 800-654-5439, Fax 614-291-2329 Bio Whittaker, 301-898-7025 Greer Labs, Inc., 800-438-0088, 704-754-5327, Fax 704-754-5320 There are plenty other companies supplying allergenic extracts for tests and treatment, if one has the time to look for them. Europe as well has some companies but their names are stuck somewhere in unreachable neurons. Falk Fish, Tel-Aviv, Israel From owner-diagnostics@net.bio.net Sun Mar 26 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!pipex!news.pavilion.co.uk!line02.kemp-du.pavilion.co.uk!user From: jpwscyto@pavilion.co.uk (Jon Waterman-Smith) Newsgroups: bionet.diagnostics Subject: LAUNCH OF FREE MAGS/WWW PAGES FOR BIONET... Date: 27 Mar 1995 17:06:05 GMT Organization: Pavilion Internet plc, Brighton, England Lines: 29 Message-ID: NNTP-Posting-Host: line02.kemp-du.pavilion.co.uk Within the next 6 weeks a new series of magazines will be launched, which will have a hard copy available to interested parties, as well as a major publication series available on the web. The magazines will deal with many subject areas, and will have hypertext points imprinted into features, so the reader can access the WWW, and simply go to the relevant pages, following the same hypertext links as shown in the magazines. The future....... Our aim is to promote science, and scientific issues mainly in the field of bioscience. Both the magazines and the WWW pages will eventually have articles and links to publications, posters, and as many products and suppliers as we can get interested in publishing with us. So, if you know of anyone who would be interested in providing articles, publishing information to assist other scientists, or you work for a company who wants to advertise , and set up pages on the WWW, we aim to become the one stop point to leap off into the web, and other internet areas. Send me an email, and I'll keep you up to date on our progress. Also let me know if you want to register for the first issue of the first magazine to be published early May '95. Diagnostics, Biotechnology and Life-sciences will be the areas first targeted for publication. TO ALL INTERESTED ADVERTISERS - the deadline for the first issue will be in 3-4 weeks time. Please contact me urgently for more information - your advert will also secure you web space and hypertext links!!!!! I look forward to hearing from you all... Best wishes, Jon Waterman-Smith E-mail address..... jpwscyto@pavilion.co.uk From owner-diagnostics@net.bio.net Sun Mar 26 23:00:00 1995 Path: biosci!CORNELL.EDU!gsr1 From: gsr1@CORNELL.EDU (Geoffrey Rule) Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 27 Mar 1995 14:02:13 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I can't believe the amount of discussion that has been generated by this low boiling point question. Surely, with all of our collective knowledge we have all the right answers. My only question now is what happened to Greg Forbes? Are you taking all of this in Greg? Did you get your PCR machine yet? Do you have any results yet? And... does coffee taste as good if the water used in preparation only gets to 89 C ? Please, please, please, let us know how you're doing and how PCR works there in Quito. I'm on the edge of my seat. #8-) All the best, Geoff Rule From owner-diagnostics@net.bio.net Sun Mar 26 23:00:00 1995 Path: biosci!ACADEM01.MTY.ITESM.MX!dsleal From: dsleal@ACADEM01.MTY.ITESM.MX ("M.C. Diana Sara Leal Klevezas") Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: 27 Mar 1995 12:13:54 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 43 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <3kv02q$fon@canopus.cc.umanitoba.ca> NNTP-Posting-Host: net.bio.net On 24 Mar 1995, Jim Mansfield wrote: > In article dsleal@ACADEM01.MTY.ITESM.MX ("M.C. Diana Sara Leal Klevezas") writes: > > > >If I'm wrong regarding the pressure building up in warm tubes, the > >possibilities are even more exciting, here is the idea: The water boils > >because the molecules get excited enough to do so. If less energy is > >needed to boil the water, less energy might be needed to break the > >hidrogen bonds between DNA strands, which means that under those > >circumstances, one might need lower temperatures to reach DNA melting > >point. If this is true, the consecuences are that your Taq will last > >loger!!!. > > Thankfully, hydrogen bonding and similar prcesses are not affected by > pressure changes of the magnitute we are discussing - otherwise life > as we know it would not be possible in Peru at all! The reason less > energy and, hence lower temperatures, are needed at high altitudes is > the vapour pressure of the liquid does not need to be as high before > it matches atmospheric pressure. As the temp. of a liquid rises, so > does its vapour pressure. Once it reaches atmospheric pressure, voila, > boiling. Putting a lid on the vesselwill raise the pressure of the gas > over the liquid, raising its boiling point. > > -Jim > > -- > --------------------------------------------------------------------- > Jim Mansfield internet: mansfield@ibd.nrc.ca > Institute for Biodiagnostics, NRCC phone: (204) 984-5191 > http://www.ibd.nrc.ca/~mansfield/ fax: (204) 984-5472 > > Well, we have to keep in mind that PCR is an in vitro assay. Eventough cells use most of the same components we add to the tube, there are cell mechanims (not boiling, naturally) to unwind and melt DNA strands. We have to also remember that organisms and their cells adapt to enviromental pressure changes and that live sistems regulate their own presure to keep the whole sistem "on wheels". Diana Sara Leal Klevezas Centro de Investigacion Biomedica del Noreste Monterrey, N.L. Mexico. From owner-diagnostics@net.bio.net Sun Mar 26 23:00:00 1995 Path: biosci!bcm!cs.utexas.edu!uunet!newsfeed.pitt.edu!quadra800.pathology.pitt.edu!user From: rapr@med.pitt.edu (Robert Preston) Newsgroups: bionet.diagnostics Subject: Re: low boiling point Date: Mon, 27 Mar 1995 12:41:12 -0500 Organization: U.Pittsburgh Sch. Med. Lines: 27 Message-ID: NNTP-Posting-Host: quadra800.pathology.pitt.edu forbes@cip.org.ec wrote: > >We would like to initiate some PCR based methods near Quito, > >Ecuador at an altitude of 3050 m above sea level. Water boils > >here at 89 C which will not allow us to bring the vials up above > >this temperature. Simple capping of the tubes (this is essential anyway, to prevent water loss by evaporation even in the absence of boiling) will be sufficient to prevent boiling at temperatures up to 95-100C or higher. If your reagents are pure, and your equipment is clean (and they have to be, if you intend to do successful PCR) the reactions would probably superheat above the boiling point even if the tubes had no caps at all. Capping will allow pressurization at high temp that will prevent both superheating and boiling. BUT, you have to keep those caps CLOSED despite the pressurization, so plan to add whatever weight you need to the tops of the tubes to prevent them from popping open. (which they tend to do even in a boiling water bath at sea level, due to the pressure of the heated air ABOVE the liquid phase!) Bring a small clothing iron to the lab, or pour a custom lead (Pb) cap-weight, or use screw-cap tubes (baad idea, unless you're only doing a few reactions at a time) or best of all, use a cycler that has a heated-cap/pressure plate assembly, in which case you would also eliminate the messy oil overlay. -- Robert Preston rapr@med.pitt.edu University of Pittsburgh Pittsburgh PA 15261 From owner-diagnostics@net.bio.net Sun Mar 26 23:00:00 1995 Path: biosci!agate!library.ucla.edu!europa.chnt.gtegsc.com!howland.reston.ans.net!cs.utexas.edu!uunet!newsfeed.pitt.edu!quadra800.pathology.pitt.edu!user From: rapr@med.pitt.edu (Robert Preston) Newsgroups: bionet.diagnostics Subject: PCR Inhibition Water Chloramines Date: Mon, 27 Mar 1995 12:40:10 -0500 Organization: U.Pittsburgh Sch. Med. Lines: 39 Message-ID: NNTP-Posting-Host: quadra800.pathology.pitt.edu > > giving reproducible results - it was driving us mad. Eventually, we trac= > > ed it to > > the *water*. The water used was filtered and autoclaved but when run = > > side by > > side with reactions using water from a different source it was clear that= > > it was > > indeed the culprit. Why? We don't know. Depending on the source of the water, it is possible for it to contain high levels of chlorinated nitrogenous compounds that neither show up on conductivity meters nor are removed by distillation. In fact, distillation actually CONCENTRATES monochloramine in the distillate by a steam distillation mechanism. Over several days time, the monochloramine in the "purified" water tank reacts with itself, catalyzed by acidity due to CO2, to make dichloramine in varying amounts. FRESH charcoal filtration as a final step (after distillation or deionization) removes this cantaminant (and any other chloraminated material) but if your municipal purification is chloramine-based rather than chlorine-based, your charcoal filter will be saturated in a short time and the problem will return. If you have a chloramine-based municipal treatment system, you need to know that fact and avoid using the water for reagents unless you watch the charcoal filter situation religiously. Chloramine-related problems tend to be highly sporadic and irreproducible because of the dynamics of the many variables involved. The simplest way to test for the stuff (at least in the levels that would be causing problems) is to use the starch+iodine+KI test for chlorine that any high-school chemistry student should know. It also has a UV absorption peak around 245, as I recall, which is weak but useful for detecting the material if 10 cm pathlength cuvets are available. You should also be aware that autoclave steam supplies are not beyond suspicion as sources of contamination, depending on how well-managed your steam department is. -- Robert Preston rapr@med.pitt.edu University of Pittsburgh Pittsburgh PA 15261 From owner-diagnostics@net.bio.net Mon Mar 27 23:00:00 1995 Path: biosci!cbr.for.csiro.au!charlie.bell From: charlie.bell@cbr.for.csiro.au (Charlie Bell) Newsgroups: bionet.diagnostics Subject: Re: PCR at high altitude Date: 27 Mar 1995 18:10:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 35 Sender: daemon@net.bio.net Distribution: world Message-ID: <199503280207.AA08823@acacia.cbr.for.csiro.au> NNTP-Posting-Host: net.bio.net >Dear Netters: > >A quick observation (somebody correct me if this is not accurate): > >Taq is an enzyme, albeit one with a very high temperature optimum. >Therefore, at a temp slightly below boiling in Quito (89 C, was it?), >shouldn't Taq still work, even if slightly less than optimally, such that >routine amplifications could still be accomplished? Does anyone know how >rapidly Taq's efficiency falls off either side of 94 C? I assume the dropoff >is steeper above 94 C? > |===========================================| > David A. Watson, Ph.D. (dwatson@plains.nodak.edu) > Asst. Prof. of Veterinary and Microbiological Sciences > North Dakota State University > --------------------------------------------------------------------------- > "Some are wise, some otherwise." > |===========================================| > > The 94C temperature is to denature the double stranded DNA. The Taq polymerase reaction is routinely run at about 72C. The question to be answered is; is necessary to reach 94C to single strand the DNA? I think the addition of salt or sugar to raise the boiling point will interfere with the polymerase reaction. The use of DMSO sounds like the best idea to me, if anything is necessary. Another possibility is just to increase the denature time at a lower temperature (say 2 mins at 90C instead of 1 min at 94C). ****************************************************** Charlie Bell Phone 06-2818324 (International) +616-2818324 CSIRO Fax 06-2818312 (International) +616-2818312 Division of Forestry E-mail charlie.bell@cbr.for.csiro.au PO Box 4008 QVT Canberra ACT 2600 Australia ****************************************************** From owner-diagnostics@net.bio.net Tue Mar 28 23:00:00 1995 Path: biosci!III.NET!irc From: irc@III.NET Newsgroups: bionet.diagnostics Subject: MULTIPLE SCLEROSIS CONFERENCE Date: 28 Mar 1995 16:23:53 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 106 Sender: daemon@net.bio.net Distribution: world Message-ID: <199503290023.TAA23956@iii.net> NNTP-Posting-Host: net.bio.net Forwarded message: From irc Sun Mar 19 19:12:18 1995 From: irc Message-Id: <199503200012.TAA13785@iii.net> Subject: MULTIPLE SCLEROSIS CONFERENCE To: irc@iii.net Date: Sun, 19 Mar 1995 19:12:16 -0500 (EST) X-Mailer: ELM [version 2.4 PL23] Content-Type: text Content-Length: 2963 ********************************* ADVANCES IN THE DIAGNOSIS AND TREATMENT OF MULTIPLE SCLEROSIS ********************************* ************** *JUNE 19-20, 1995* The Charles Hotel Cambridge, Massachusetts ********************** sponsored by NATIONAL MULTIPLE SCLEROSIS SOCIETY International Research Communications _________________________________________ The exponential growth in our knowledge of multiple sclerosis has led to exciting new diagnostic and treatment methodologies. This program brings together the most prominent scientists from both clinical and basic research set- tings to discuss current progress in the diagnosis and treatment of multiple sclerosis. Topics covered at this conference will provide a comprehensive survey of specific laboratory and clinical issues within the context of four broad sessions; novel diagnostic procedures, experimental animal models, leukocyte trafficking and specific treatments. This conference will be of interest to re- searchers and physicians involved in the study of multiple sclerosis, autoimmune diseases and other neuroimmunological disorders. The program is designed to encourage interaction between clinicians and basic researchers. The laboratory re- search and clinical findings presented at this conference will help to form the basis for new diagnostic and treatment strategies into the next century. _________________________________________ ***************************************** KEYNOTE ADDRESS Dr. Abul K. Abbas, Harvard Medical School DIAGNOSIS AND COURSE OF MS Dr. John F. Kurtzke, VAMC, Washington DC (chair) Dr. William A. Sibley, University of Arizona Dr. Gary Birnbaum, University of Minnesota Dr. Donald W. Paty, University of British Columbia EXPERIMENTAL MODELS OF MS Dr. Stephen D. Miller, Cleveland Clinic Foundation (chair) Dr. William J. Karpus, Northwestern University Dr. Vijay Kuchroo, Harvard Medical School Dr. Arthur A. Vandenbark, VAMC, Portland, Oregon Dr. Caroline Whitacre, Ohio State University LEUKOCYTE TRAFFICKING IN MS AND EAE Dr. Richard M. Ransohoff, Cleveland Clinic Foundation (chair) Dr. Bruce D. Trapp, Cleveland Clinic Foundation Dr. William F. Hickey, Dartmouth Medical School Dr. Mariano Elices, Cytel Corporation Dr. Donald E. Staunton, ICOS Corporation SPECIFIC TREATMENTS FOR MS Dr. Anthony T. Reder, University of Chicago (chair) Dr. Robert Knobler, Thomas Jefferson University Dr. Jeffrey A. Cohen, Cleveland Clinic Foundation Dr. John H. Noseworthy, Mayo Clinic Dr. Howard L. Weiner, Harvard Medical School ********************************************* For more information about the conference and how to register, please call International Research Communications at (617) 489-0443 or (800) 495-1073 or send E-mail to IRC@iii.net From owner-diagnostics@net.bio.net Tue Mar 28 23:00:00 1995 Newsgroups: bionet.virology,bionet.parasitology,bionet.diagnostics Path: biosci!agate!howland.reston.ans.net!cs.utexas.edu!news.sprintlink.net!uunet!in1.uu.net!news2.new-york.net!news From: grenard@herpmed.com Subject: Snakebite WebPage Nntp-Posting-Host: herpmed.com Content-Type: TEXT/PLAIN; charset=US-ASCII Sender: news@news2.new-york.net (Network News) Mime-Version: 1.0 Organization: Misconfigured client newsreader Date: Wed, 29 Mar 1995 05:02:45 GMT X-Newsreader: NEWTNews & Chameleon -- TCP/IP for MS Windows from NetManage Message-ID: Lines: 6 Xref: biosci bionet.virology:2048 bionet.parasitology:623 bionet.diagnostics:48 Field workers who may be at risk of venomous snakebite are invited to check the new werbpage: North American Snakebite Emergency: First Aid/Contact Info: Point your web browsers at: http://www.xmission.com/~gastown/herpmed From owner-diagnostics@net.bio.net Tue Mar 28 23:00:00 1995 Path: biosci!sasa.gov.uk!browning From: browning@sasa.gov.uk (Isla Browning) Newsgroups: bionet.diagnostics Subject: (none) Date: 29 Mar 1995 07:50:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net unsubscribe virology From owner-diagnostics@net.bio.net Tue Mar 28 23:00:00 1995 Path: biosci!CIP.ORG.EC!forbes From: forbes@CIP.ORG.EC Newsgroups: bionet.diagnostics Subject: low boiling point and highland coffee Date: 29 Mar 1995 06:19:46 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 33 Sender: daemon@net.bio.net Distribution: world Message-ID: <9503280934.AA00565@cip.org.ec> NNTP-Posting-Host: net.bio.net > I can't believe the amount of discussion that has been generated by > this low boiling point question. Surely, with all of our collective > knowledge we have all the right answers. My only question now is what > happened to Greg Forbes? Are you taking all of this in Greg? Did you get > your PCR machine yet? Do you have any results yet? And... does coffee taste > as good if the water used in preparation only gets to 89 C ? Please, > please, please, let us know how you're doing and how PCR works there in > Quito. I'm on the edge of my seat. #8-) Admittedly, coffee is best here when it comes from an expresso machine where the water is heated under pressure - perhaps a lesson for PCR. Well, thanking all the people on the list who have contributed is about # 8 on my "to-do" list for today, but I'll jump ahead to it now. We have also been amazed at the number of interesting suggestions that have been posted and are very appreciative! The PCR machine is somewhere between the US and Quito right now and in a day or so we will start the process of getting it out of customs (should a similar fate never befall any of you!). I hope we can try out PCR at 3058 masl within 2 wks. Again, many thanks for the suggestions! Greg Greg Forbes Tel. Of: +593-2-690362 Plant Pathologist Tel. Hm: +593-2-330971 International Potato Center Fax. +593-2-692604 P.O. 17-21-129 CEQ, Quito, Ecuador Internet: forbes@cip.org.ec From owner-diagnostics@net.bio.net Tue Mar 28 23:00:00 1995 Path: biosci!ZEUS.DATASRV.CO.IL!ila2027 From: ila2027@ZEUS.DATASRV.CO.IL (Falk Fish) Newsgroups: bionet.diagnostics Subject: Re: low boiling point and highland coffee Date: 29 Mar 1995 13:48:58 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <9503280934.AA00565@cip.org.ec> NNTP-Posting-Host: net.bio.net On 29 Mar 1995 forbes@cip.org.ec wrote: > Admittedly, coffee is best here when it comes from an expresso machine > where the water is heated under pressure - perhaps a lesson for PCR. > > Well, thanking all the people on the list who have contributed is > about # 8 on my "to-do" list for today, but I'll jump ahead to > it now.We have also been amazed at the number of interesting > suggestions that have been posted and are very appreciative! The > PCR machine is somewhere between the US and Quito right now and > in a day or so we will start the process of getting it out of > customs (should a similar fate never befall any of you!).I hope > we can try out PCR at 3058 masl within 2 wks. > > Again, many thanks for the suggestions! > > Greg > > > Greg Forbes Tel.Of: +593-2-690362 > Plant Pathologist Tel.Hm: +593-2-330971 > International Potato Center Fax.+593-2-692604 > P.O. 17-21-129 CEQ, Quito, Ecuador Internet:forbes@cip.org.ec > > So, how is the coffee in Ecuador?