From owner-rapd@net.bio.net Tue Mar 01 22:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!EU.net!howland.reston.ans.net!usenet.ins.cwru.edu!magnus.acs.ohio-state.edu!mgrebus From: mgrebus@magnus.acs.ohio-state.edu (Marcella E Grebus) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.rapd Subject: no product, Promega kit? Date: 2 Mar 1994 16:17:16 GMT Organization: The Ohio State University Lines: 24 Message-ID: <2l2e6c$dnc@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: beauty.magnus.acs.ohio-state.edu Xref: biosci bionet.molbio.methds-reagnts:11983 bionet.molbio.rapd:457 hey folks, i have a question for anyone who uses Taq polymerase from Promega. our laboratory has been using Promega's Taq kits which include the Taq polymerase, MgCl2, and 10x buffer. we've gotten good amplification for the past year using these. but in the past few weeks, our pcr runs have been yielding no products, and we don't know why. we haven't changed anything in our protocols. our loss of amplification coincides with the use of a new Promega kit... and we were wondering if anyone has had problems with recent batches of Taq, 10x buffer, or MgCl2 from them. might they have made recent changes in their suppliers or ingredients? all our other conditions, dna primers, etc. are exactly the same as thye were when we got products. any suggestions/thoughts welcome! i'll be following this group, but e-mail is also welcome. thanks, --marcy ps. does anyone know of a "trouble-shooting" guide or some similar document for pcr? is there an ftp site or faq file available? From owner-rapd@net.bio.net Tue Mar 01 22:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!EU.net!howland.reston.ans.net!torn!falcon.ccs.uwo.ca!marvin.mbl.uwo.ca!ggloor From: Greg Gloor Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.rapd Subject: Re: no product, Promega kit? Date: 2 Mar 1994 17:10:32 GMT Organization: University of Western Ontario Lines: 8 Distribution: world Message-ID: <2l2ha8$knq@falcon.ccs.uwo.ca> References: <2l2e6c$dnc@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: julian.uwo.ca X-UserAgent: Version 1.1.3 X-XXMessage-ID: X-XXDate: Wed, 2 Mar 94 12:12:26 GMT Xref: biosci bionet.molbio.methds-reagnts:11988 bionet.molbio.rapd:458 Subject: no product, Promega kit? Marcella, Have you tried a new set of dNTP's? We've found that when our PCR's start to poop out it is time to break out a fresh batch of dNTP's. This has always been the reason in our case in about 5 years of PCR. Regards.........Greg From owner-rapd@net.bio.net Wed Mar 02 22:00:00 1994 Path: biosci!ABRSLE.AGR.CA!DEMEKE From: DEMEKE@ABRSLE.AGR.CA Newsgroups: bionet.molbio.rapd Subject: STOFFEL VS CONVENTIONAL TAQ Date: 3 Mar 1994 17:27:17 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01H9J9YCK09U000H8K@GW.AGR.CA> NNTP-Posting-Host: net.bio.net Hi: We have used stoffel and Taq on wheat. The advantage of the Stoffel fragment was in the case of faint bands that are hard to reproduce. They either totally disappear or show well with the stoffel. We were able to get a reproducible polymorphic band with the Stoffel fragment. One of the problems with the Stoffel fragment is the more intense background produced. Otherwise it has some advantages over Taq. Tigst From owner-rapd@net.bio.net Wed Mar 02 22:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: Stoffel Date: 3 Mar 1994 21:24:12 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9403032130.AA12910@phibred.phibred.com> NNTP-Posting-Host: net.bio.net There was a meesa From owner-rapd@net.bio.net Wed Mar 02 22:00:00 1994 Path: biosci!MAROON.TC.UMN.EDU!stolz002 From: stolz002@MAROON.TC.UMN.EDU (Allison M Stolz-2) Newsgroups: bionet.molbio.rapd Subject: stoffel fragment? Date: 3 Mar 1994 21:45:16 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 7 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Hello, Can someone back up a little? What exactly is Stoffel Fragment? Who sells it? How expensive is it? How widely used is it in comparison to regular taq? Thanks, Allison Stolz Forestry, University of Minnesota From owner-rapd@net.bio.net Wed Mar 02 22:00:00 1994 Path: biosci!bloom-beacon.mit.edu!gatech!europa.eng.gtefsd.com!emory!news.cc.emory.edu!news.cc.emory.edu!not-for-mail From: vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) Newsgroups: bionet.molbio.rapd Subject: Stoffel vs Conventional Taq Date: 3 Mar 1994 07:14:42 -0500 Organization: Emory University Lines: 5 Message-ID: <2l4kbj$maq@emoryu1.cc.emory.edu> NNTP-Posting-Host: emoryu1.cc.emory.edu X-Newsreader: TIN [version 1.2 PL2] Has any one compared Stoffel fragment with the usual taq for RAPDs and found it to improve reproducibility. Thanks in advance From owner-rapd@net.bio.net Wed Mar 02 22:00:00 1994 Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.rapd Path: biosci!daresbury!trane.uninett.no!sunic!EU.net!howland.reston.ans.net!sol.ctr.columbia.edu!usenet.ucs.indiana.edu!bronze.ucs.indiana.edu!jgraham From: jgraham@bronze.ucs.indiana.edu (the End) Subject: Re: no product, Promega kit? Message-ID: Sender: news@usenet.ucs.indiana.edu (USENET News System) Nntp-Posting-Host: bronze.ucs.indiana.edu Organization: Indiana University Affiliation: Kit Busters R Us References: <2l2e6c$dnc@charm.magnus.acs.ohio-state.edu> Date: Thu, 3 Mar 1994 15:57:35 GMT Lines: 29 Xref: biosci bionet.molbio.methds-reagnts:12032 bionet.molbio.rapd:460 Sure, Make your 10X salts solution and make it at 5X, and don't freeze it, but filter sterilze and store at room temperature. 200 mM KCl 50 mM Tris-Cl (8.3) 7.5 mM MgCl 0.05% gelatin We also add 0.05% NP-40 deteregent and 5% Acetamide (10/100 ul of 50%) to all final reactions. I add MgCl to 2.5 mM when using plasmid targets. If that fails, reconstitute a new batch of dNTPs If that fails, purchase some new Taq polymerase (fully licensed for PCR of course ;) Now throw away the silly "PCR Kit". It is essential that you be able to trouble-shoot the components of the reaction yourself (at this stage), as later on that skill will be necessary in your own experiments. Best wishes, Jim J. Graham From owner-rapd@net.bio.net Wed Mar 02 22:00:00 1994 Path: biosci!ACD.TUSK.EDU!PRAKASH From: PRAKASH@ACD.TUSK.EDU Newsgroups: bionet.molbio.rapd Subject: RE:Stoffel Vs Conventional Taq Date: 3 Mar 1994 20:02:51 -0000 Organization: Tuskegee University Lines: 27 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01H9JDICADKW000ZKK@Acd.Tusk.Edu> NNTP-Posting-Host: net.bio.net Recently Varma of Emory asked a question whether any one has tested Whether Stoffel Fragment was better in resulting reproducible RAPD patterns than regular Taq. We have consistently used Stoffel fragment in our RAPD researh with sweetpotato and peanut, and found it to result in a greater number of bands than regular Taq. Stoffel can tolerate higher temperature (96C is the temp for denaturation we use) and needs higher Mg (4-5 mM). We have conducted several tests on its reproducibility using multiple extracts of DNA from single genotypes, and have had no problem in reproducing even the faint bands. We have compared to thermal cyclers and found similar patterns. This still doesn't say that Stoffel produces more reproducible bands than Taq, but it is certainly more informative. C. S. Prakash ********************************************************************* C. S. PRAKASH PHONE (205) 727 8023 SCHOOL OF AGRICULTURE FAX (205) 727 8067 TUSKEGEE UNIVERSITY PRAKASH@ACD.TUSK.EDU ********************************************************************* From owner-rapd@net.bio.net Thu Mar 03 22:00:00 1994 Path: biosci!ACD.TUSK.EDU!PRAKASH From: PRAKASH@ACD.TUSK.EDU Newsgroups: bionet.molbio.rapd Subject: Answers to Frequently Asked questions on Stoffel Fragment Date: 4 Mar 1994 19:41:06 -0000 Organization: Tuskegee University Lines: 47 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <01H9KQKS5YFC000UVT@Acd.Tusk.Edu> NNTP-Posting-Host: net.bio.net I saw the following note today: Hello, Can someone back up a little? What exactly is Stoffel Fragment? Who sells it? How expensive is it? How widely used is it in comparison to regular taq? Thanks, Allison Stolz Forestry, University of Minnesota Dear Allison: Stoffel fragment is a truncated (lacks the 289 aminoacids at the N-terminal region) AmpliTaq polymerase sold by Applied Biosystems (#N80880038) (Phone 1-800-327-3002). Cetus has not published much on this but they cite a reference in their product literature: Lawyer et al. 1989 J. Biol Chem. 264: 6427 -6437. Stoffel fragment has higher thermostability than native or amplitaq, and thus more stable at higher denaturation temps (96-98C). I think this is the primary reason it gives more bands in RAPDs. I am not aware of detailed studies on Stoffel (neither does Cetus), but Barnes 1992 Gene 112: 29-35 paper talks in details about a similar truncated Taq. Two others companies sell truncated Taqs: Delta Taq by USBiochemicals, KlenTaq by AB Peptides (St. Louis, Missouri). Please do remember that Stoffel has lower processivity and thus you need to use more of it (we use 0.2 units per microliter) Also, You need to include Mg at 4-5 mM. I rememberthat Dr. Chris Cullis of Case Western U had a note in the Plant Molecular Biology REporter during Summer 1993 comparing these Taqs and also found that it was critical to use certain type of buffer (Ican't lay my hands on the article now). Hope this helps. -Prakash ********************************************************************* C. S. PRAKASH PHONE (205) 727 8023 SCHOOL OF AGRICULTURE FAX (205) 727 8067 TUSKEGEE UNIVERSITY PRAKASH@ACD.TUSK.EDU ********************************************************************* From owner-rapd@net.bio.net Thu Mar 03 22:00:00 1994 Path: biosci!LIFSCI.SDSU.EDU!SOBRAL From: SOBRAL@LIFSCI.SDSU.EDU Newsgroups: bionet.molbio.rapd Subject: STOFFEL FRAGMENT Date: 4 Mar 1994 22:15:38 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 29 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <940304141715.20207fd7@LIFSCI.SDSU.EDU> NNTP-Posting-Host: net.bio.net Stoffell seems to have recently generated a lot of interest....We published a paper on its use for arbitrarily primed PCR (known to some as RAPDs or DAF or whatever-you-like): TAG 86:105-112 (1993). We have used it and compared results with AmpliTaq and the other truncated polymerases and found that it gives more amplified fragments/primer, that these fragments are of smaller size (expected because of lower processivity), that they are more robust (i.e., they are always reproducibly there), and that more product is made. For fingerprinting, Stoffel is great. The KlenTaq gives much the same result, with even more fragments/primer, but batch-to-batch variation (on the enzyme preps) was observed, too, as well as more than one band for the enzyme on polyacrylamide gels. Please feel free to contact us if you have questions regarding the use of Stoffel. (By the way, we also used Stoffel to generate a genetic map of sugarcane, reported in GENETICS 134:1249-1260 (1993)).\ Good luck! Sincerely, Bruno WS Sobral, Ph.D. Staff Scientist California Institute of Biological Research (CIBR) 11099 N. Torrey Pines Road, Suite 300 La Jolla CA USA 92037 (619)535-5483 (office) (619)535-5491 (lab) E-mail: sobral@lifsci.sdsu.edu or brunosfly@aol.com (619)535-5472, 535-5490 or 535-5481 (faxes) From owner-rapd@net.bio.net Fri Mar 04 22:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!pipex!howland.reston.ans.net!news.intercon.com!panix!ddsw1!news.cic.net!magnus.acs.ohio-state.edu!mgrebus From: mgrebus@magnus.acs.ohio-state.edu (Marcella E Grebus) Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.rapd Subject: SUMMARY: no pcr products, Promega kits? Date: 5 Mar 1994 16:41:26 GMT Organization: The Ohio State University Lines: 161 Message-ID: <2lacnm$49@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: bottom.magnus.acs.ohio-state.edu Xref: biosci bionet.molbio.methds-reagnts:12121 bionet.molbio.rapd:467 hey folks, *thanks* to all those who responded to my plea for suggestions regarding our problem with pcr [no amplification products]. we have not yet gotten the problem figured out, as to whether it's the Promega kits or something else. but i'm working on it, following a number of the suggestions you all sent me. the following, as requested, are the responses i received via e-mail. if anyone has any additional suggestions, i'd be elated to hear from you. thanks again! --marcy --------------------------------------------------------------- Marcy, I am responding to your posting on the methods-and-reagents billboard about Promega Taq Polymerase. We have been using it to do simple PCR amplifications for about a year so we have gone through the change in kits as well. We have done two amplifications with the new reagents and saw no difference in yeild. Again, the PCR we do is real simple amplification off of Qiagen prepped DNA. I have saved some of the old buffers in the case of something screwy going on, perhaps you still have some kicking around your lab. At least you can see if it is a buffer problem. Good luck - Brian Zeiler brianz@lifesci.microbio.ucla.edu --------------------------------------------------------- From: Marcella, A brief reply. We use promega kits a LOT, and know that one kit can differ considerably from another. For that reason we keep kits separate and record lot numbers as routinely as any other component of the reaction. We've had some extremely crappy 10X kits from promega that we've simply discarded. We've also done the experiments to compare buffers: different lot numbers can and do give different results - another unwanted variable. Usually the 10X is the cause of variation, rather than magnesium. What is the lot number of your new kit - we may have a similar lot with equally bad results, or alternatively we could eliminate that from your list of possibilities. A tip - try 1 mg/ml non-acetylated BSA (from NEB) - it works extremely well for improving the level of amplification and repeatibility. Phillip Wilcox Forest Biotech NCSU. ---------------------------------------------------------- Have you contacted tech/sales rep at Promega regarding your faulty Taq? Also, could you mention the lot number? Sounds like a faulty batch of Taq. Was it send on dry ice? regards, ******************** Andre Hamel email: hamel@cc.umanitoba.ca Manitoba Government Veterinary Services lab tel.: (204) 945-7630 545 University Crescent, FAX: (204) 945-8062 Winnipeg, Manitoba, CANADA R3T 5S6 ******************** -------------------------------------------------------------- From: "FrohlichM" Marcy, I had similar unexplained total failure with a kit from Perkin-Elmer a couple years ago, that turned out to be caused by the MgCl2- The actual concentration of MgCl2 IN SOLUTION was about 1/4 what it should have been. After I found the problem I asked their rep, who said the MgCl2 precipitates on freezing and may not redissolve when you thaw the tube, even if the tube stays thawed for a long time. Mixing my own MgCl2, which I never freeze, solved the problem completely. Of all the reagents this is the simplest, so I wasted a lot of time trying other manipulations before I checked the MgCl2. I expect the same problem could also happen with solutions from other manufacturers. Good Luck, Michael Frohlich --------------------------------------------------------------- From: Louis van de Zande Hi, Well first try some of the old batch (or has that been used totally), Then try some Taq from the nextdoor lab and finally contact Promega and confront them with the problem. Yes, I know of "The simple fools guide to PCR" which has some good advice aln also a list of useful primers. As the titel suggests, it is a lucid protocol, but I find it very useful. If you are interested, give me the address and I will send you a photocopy. Good luck L.van de Zande Dept. of Genetics University of Groningen P.O. Box 14 9750 AA Haren The Netherlands Tel. +31 50 632126 Fax. +31 50 632348 ------------------------------------------------------------ From: jgraham@bronze.ucs.indiana.edu (the End) Sure, Make your 10X salts solution and make it at 5X, and don't freeze it, but filter sterilze and store at room temperature. 200 mM KCl 50 mM Tris-Cl (8.3) 7.5 mM MgCl 0.05% gelatin We also add 0.05% NP-40 deteregent and 5% Acetamide (10/100 ul of 50%) to all final reactions. I add MgCl to 2.5 mM when using plasmid targets. If that fails, reconstitute a new batch of dNTPs If that fails, purchase some new Taq polymerase (fully licensed for PCR of course ;) Now throw away the silly "PCR Kit". It is essential that you be able to trouble-shoot the components of the reaction yourself (at this stage), as later on that skill will be necessary in your own experiments. Best wishes, Jim J. Graham ---------------------------------------------------------- From: Stephen T Simpson Marcy, Forgive me if I send this note the wrong way, but I believe you asked about possible causes for PCR reactions dying out. Well, I have always found this sensitive reaction to change by some unknown force. I try to do everything the same (even the PCR chant and dance) each time but still get varying results. It must be my poor method, because people are publishing papers that include quantitative PCR (are you as skeptical as I?). But back to your question, I know you said your reaction buffers were the same, but I understand that Invitrogen (I think) has created a PCR optimizer kit. It came out after I was done with PCR, but a girl in my lab used it and said it worked well. It basicly just comes with a set of different buffers with varying Mg, salts, etc. Though I hope you find the problem (like the old dNTPs, that was a good thought), I just thought I'd share that with you in case you have to compleatly reconstruct the reactions. Good luck... and post or mail me a summary of responses, please. Stephen -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=- Stephen T. Simpson Scott-Ritchey Research Center (205) 844-5951 College of Veterinary Medicine simpsst@mail.auburn.edu Auburn University, AL 36892 From owner-rapd@net.bio.net Sun Mar 06 22:00:00 1994 Path: biosci!daresbury!not-for-mail From: nfix@virgo.jcu.cz (Ivo Wiesner) Newsgroups: bionet.molbio.rapd Subject: summary-too high RAPD background Date: 7 Mar 1994 10:16:31 -0000 Lines: 211 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2leutv$e08@mserv1.dl.ac.uk> X-Char-Esc: 29 X-Charset: ASCII Original-To: rapd@dl.ac.uk @CT 1 @LM 6 @RM 65 @PL 63 @TB -----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T-----T @MT 3 @MB 3 @PO 10 @PN 1 @OP @LH 6 -------SUMMARY - TOO HIGH BACKGROUND IN RAPD-------------------- A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: THANKS MUCH TO ALL OF YOU WHO RESPONDED TO MY PROBLEM WITH TOO HIGH BACKGROUND in RAPD: A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: : Hello Netters! : Could somebody out there share with me his/her : experience in RAPD reaction? : I work on RAPDs on fescue and pea and have constant : problems with too high background in lines on : gel which covers more or less the bands themselves. : I use200uM dNTPs, 3mMMgCl2, 1U Taq polymerase/25ul, : 1uM primer (10 bases), template within 100ng-10pg/25ul, : so usual reaction : temperature profile 45 cycles with 30deg.annealing : orusual 37deg.annealing and originates with denaturation : at 95 deg. for 5 min., ending with 10 min.at 72 deg. : I tested various concentrations of template, primer (o.2uM-4uM), : only 35 cycles with 37 deg.annealing. : I constantly receive high background which prevent : evaluation of products... : Has somebody out there any idea what should be changed : or what is going wrong???? : Any suggestion highly appreciated!!!!!!!!!!!!!! : Greetings : Ivo Wiesner : nfix@virgo.jcu.cz --------------------------------------------------- BEGIN REPLY --------------------------------------------------- Hi! About your background problem. I've done some work with strawberry and I've noticed that more than 50ng template cause problem and I used only 1U of polymerase for 50 ul reaction. Cycle was first denat. 1min then only 20s 94C,1min 40C and 2min 72C. Comparable results were obtained. Other conditions were about the same as yours. Wish you luck! Kari A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: Re: background You didn't mention what buffer you are using. We use 80 mM Tris-HCL pH9 with 20 mM ammonium sulfate. If we use the same amplification procedure but change the buffer to 10 mM Tris-HCl pH 8.3 with 50 mM KCl, all we get is a smear. Could be that your conditions are in between, and therefore you see some bands with a smear in the background. Your DNA concentration sounds high - we routinely use 10 ng/25 ul reaction. For Arabidopsis, we use: 80 mM Tris-HCl pH9 20 mM ammonium sulfate 3.5 mM magnesium chloride 100 uM each dNTP pH7 0.4 uM primer 1 unit AmpliTaq polymerase 10 ng genomic DNA 25 ul final volume We are using a complicated program in the P-E 9600 that mimics the old BIOS oven. Suffice it to say it is a fairly permissive program that slowly ramps down to 32 C, up to 70, and then to 88. We do 45 cycles and it takes about 12 hours. We haven't attempted to shorten it. Best of luck, Sandra Russell DuPont Life Sciences russelsh@esvax.dnet.dupont.com A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: Hi, There has been a discussion on this in this group. It turned out that in MOST cases contamination does the background. When you use vials, pipette tips etc that have been autoclaved in the same autoclave where also stuff from DNA extraction is sterilized, some DNA might settle down in your "clean" tips and ruin all your reactions. Furthermore it can be useful to re-evaluate the DNA extraction procedure that you use. If all this does not solve the problem, than it is just trial and error to find out the optimal setting for the cycler. Good luck Louis van de Zande A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: The following two articles could be useful in finding out the problem that you are facing; 1) Weeden et al. 1992. Inheritance and reliability of RAPD markers. Application of RAPD Tech. to Plant Breeding. pp. 12-17. Joint Plant Breeding Symposia Series of Crop Sci. Soc. of Amer., Amer. Soc. Hort. Sci. and Amer. Genet. Asoc. 2) Williams et al. 1993. Genetic analysis using random amplified polymorphic DNA markers. Methods in Enzymology, 218:705-740. I have observed that the reason for high background is usually higher primer and Taq conc. and lower magnesium conc. These differences are mentioned in the first article. I use 0.5 unit of Taq, 2-3 mM Mg and 120 uM primers. The other possible reasons could be high template DNA conc., over-exposure or over-developing of the film and excessive EtBr staining. You may want to test all the components not only individually but also in combinations. This takes some time but helps in the long run for reliability and repeatability of RAPDs, thats what I found in grapes. Good luck! Muhammad A. Lodhi Cornell University A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: Hi fellow rapd I just read your letter to bio-net. It sounds like you are very colode to the right compostion. I myself have been using rapd for the past three years with excellent success. My only queries are what method do you isolate your dna with. we use a modified dellaprta which has a phenol:chloroform extraction after koac and centrifugation. We follow this at the end of the protocol with a precipitation in 2.5 M NaCl. this gives good quality of dna for rapd in B. napus (canola) The only other differences we use are 1.9 mM MgCl and dna template concentrations of 5 -25 ng with 10 being optimal. I hope this helps you in your work. If you have any other querries plaease feel free to contact me best regards Glen hawkins A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: If you are getting smeared RAPD products (high background), it means that your annealing is not specific enough, so that your primers are annealing to some non-specific sites. I would suggest that you raise your annealing temperature in increments of 1 degree. If that does not work, try reducing your MgCl2 concentration to 2mM, since that will also reduce the specificity. Finally, I would try reducing your DNA concentration, only because this seems to be a good solution to any PCR problems. Owain Edwards edwards@nature.berkeley.edu > Hi, there: I think it is too much MgCl2, and/or too much DNA. I use only 1.5- 2 mM MgCl2, and 15-25 ng DNA/15 ul, and it works fine. Good luck. Jingzhong Lin U of Toronto Too high amount of genomic DNA frequently results in elevated "semary" background. Titrate amt. of genomic DNA between 2-50 nanograms/25 ul reaction. You may also try a different barand of DNA polymerase. We are currently using Stoffel fragmernt of Taq from Perkin-Elmer (needs higher Mg++ then Taq and a different buffer). Antoni Rafalski A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: Hello never despair. Things will work out eventuallHere's what I suggest. 1)Dtermine the best template and primer concentrations 2) Perform a magnesium chloride titration I think that the smearing may be due to too high MgCl2 conc. Try to vary the concentrations of Mg 0,1.0, 1.25, 1.5, 1.75. 2, 2.5, 5, 10 mM. Then zero in on the best banding with 0.2 mM. Reference: Oste, Christian (1989)Optimization of Magnesium Concentration in the PCR reaction. Amplifications: a forum for PCR users. Perkin Elmer Cetus. Feb, issue 1. pp 10-11. Here is the address Perkin-Elmer Corporation, 761 Main Avenue, Norwalk, CT 06859-0251, USA I hope that his helps. Too low Mg conc auses band drop outs and too high causes smearing. That's all that I can think of for now. Good luck.Z A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A:A: ThanX once again !!!!!!!!! World Netters are really exciting community !!!!!!!!!!!!!!!!!!!!! Ivo WIESNER nfix@virgo.jcu.cz From owner-rapd@net.bio.net Sun Mar 06 22:00:00 1994 Path: biosci!daresbury!doc.ic.ac.uk!agate!overload.lbl.gov!bks From: bks@s27w007.pswfs.gov (Bradley K. Sherman) Newsgroups: bionet.agroforestry,bionet.plants,bionet.molbio.rapd Subject: Institute of Forest Genetics on the World-Wide Web Date: 7 Mar 1994 01:04:34 GMT Organization: Dendrome, A Genome Database for Forest Trees Lines: 35 Distribution: world Message-ID: <2lduj2$r09@overload.lbl.gov> NNTP-Posting-Host: s27w007.pswfs.gov Xref: biosci bionet.agroforestry:570 bionet.plants:2740 bionet.molbio.rapd:468 The Dendrome project of the Institute of Forest Genetics has an experimental http server on the World-Wide Web (WWW). The Universal Resource Locator (URL) for this service is: http://s27w007.pswfs.gov/ A Gopher server is also available at s27w007.pswfs.gov, port 70. The Dendrome project hopes to become the primary electronic resource for the study of the molecular genetics of forest trees. Criticism and suggestions for services are welcome. The project is directed by David B. Neale; the software engineer is Bradley K. Sherman. The Institute of Forest Genetics is located in Placerville and Albany, California, and is part of the Pacific Southwest Research Station of the USDA Forest Service. Funding for the project is from the Office of the Plant Genome, USDA Agricultural Research Service. Dendrome is also a contributor to the Plant Genome Database of the National Agricultural Library. [There is a variety of free software available for browsing the World-Wide Web, the best know of which is Mosaic from the National Center for Supercomputing Applications --an Internet connection is necessary. WWW, which began at CERN in Geneva, Switzerland, allows for the presentation of data in a variety of media, including text, graphics, sound and animated graphics.] --bks -- Bradley K. Sherman P.O. Box 245 Computer Scientist Berkeley, CA, 94701 Dendrome Project 510-559-6437 FAX: 510-559-6440 Institute of Forest Genetics Internet: bks@s27w007.pswfs.gov From owner-rapd@net.bio.net Mon Mar 07 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!gatech!newsxfer.itd.umich.edu!news.cic.net!magnus.acs.ohio-state.edu!mgrebus From: mgrebus@magnus.acs.ohio-state.edu (Marcella E Grebus) Newsgroups: bionet.molbio.rapd,bionet.molbio.methds-reagnts Subject: "air" type thermal cyclers Date: 8 Mar 1994 17:13:55 GMT Organization: The Ohio State University Lines: 20 Message-ID: <2liboj$e5t@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: beauty.magnus.acs.ohio-state.edu Xref: biosci bionet.molbio.rapd:471 bionet.molbio.methds-reagnts:12218 hey folks, does anyone here work with one of those "air" type thermal-cyclers? the kind that cycles air instead of using a heat block. our lab is interested in looking into them, because we need to get another thermal-cycler, and i've heard that the ones that rely on air circulation reduce ramping times significantly but may have some disadvantages. i'd appreciate any advice, experience, comments, ordering/manufacturer information [eg, 1-800 number]. if there's interest, i'd be happy to post a summary of e-mails received. also, if you feel strongly that a particular type of thermal-cycler is the "best", let's discuss pro's and con's. i'm really open to any brand/model. thanks in advance, --marcy From owner-rapd@net.bio.net Mon Mar 07 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!sol.ctr.columbia.edu!news.kei.com!yeshua.marcam.com!zip.eecs.umich.edu!umn.edu!msus1.msus.edu!vax1.mankato.msus.edu!vengeance Newsgroups: bionet.molbio.rapd Subject: Omaha Project Message-ID: <1994Mar8.085037.2375@vax1.mankato.msus.edu> From: vengeance@vax1.mankato.msus.edu Date: 8 Mar 94 08:50:37 -0500 Organization: Mankato State University Lines: 9 I am trying to build a list of names and E-Mail addresses of people in the Omaha Nebraska area for a school related project. If you live in Omaha or go to school there or know someone that does and will be around for three months or more, please reply via E-Mail to Vengeance@vax1.mankato.msus.edu. Thank you very much! Ryan Krueger From owner-rapd@net.bio.net Tue Mar 08 22:00:00 1994 Path: biosci!MED.PITT.EDU!bsh From: bsh@MED.PITT.EDU (Basavaraju Shankarappa) Newsgroups: bionet.molbio.rapd Subject: Re: Centrifuge for microtiter plates Date: 9 Mar 1994 14:41:43 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 23 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9403091441.AA13850@mercury.med.pitt.edu> References: <199403090041.AA23629@diemen.utas.edu.au> NNTP-Posting-Host: net.bio.net Hi: Almost all the table top centrifuges come with an option to buy the rotor that can accept microplates. If you have any table top centrifuge in the lab, I suspect you will definitely find a microplate rotor in the manufacturer's catalog. If you don't have the table top, than you can buy one with all. Raj Shankarappa bsh@med.pitt.edu > Could any one suggest a supplier for a benchtop centrifuge that can handle > microtiter plates. > > Thanks in advance, > > Rene E. Vaillancourt > CRC for Temperate Hardwood Forestry > Hobart, Tasmania 7005, AUSTRALIA > fax: 61 02 202703 > r.vaillancourt@bioch.utas.edu.au > > > From owner-rapd@net.bio.net Tue Mar 08 22:00:00 1994 Path: biosci!bioch.utas.edu.au!R.Vaillancourt From: R.Vaillancourt@bioch.utas.edu.au Newsgroups: bionet.molbio.rapd Subject: Centrifuge for microtiter plates Date: 9 Mar 1994 00:41:08 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199403090041.AA23629@diemen.utas.edu.au> NNTP-Posting-Host: net.bio.net Could any one suggest a supplier for a benchtop centrifuge that can handle microtiter plates. Thanks in advance, Rene E. Vaillancourt CRC for Temperate Hardwood Forestry Hobart, Tasmania 7005, AUSTRALIA fax: 61 02 202703 r.vaillancourt@bioch.utas.edu.au From owner-rapd@net.bio.net Wed Mar 09 22:00:00 1994 Path: biosci!daresbury!keele!uknet!pipex!howland.reston.ans.net!news.moneng.mei.com!uwm.edu!post.its.mcw.edu!not-for-mail From: abuhajir@post.its.mcw.edu (Majed Abu-Hajir) Newsgroups: bionet.molbio.rapd,bionet.molbio.methds-reagnts Subject: Re: "air" type thermal cyclers Followup-To: bionet.molbio.rapd,bionet.molbio.methds-reagnts Date: 10 Mar 1994 12:23:29 -0600 Organization: Medical College of Wisconsin; Milwaukee Wisconsin Lines: 45 Message-ID: <2lnoj1$mh1@post.its.mcw.edu> References: <2liboj$e5t@charm.magnus.acs.ohio-state.edu> NNTP-Posting-Host: post.its.mcw.edu X-Newsreader: TIN [version 1.2 PL1] Xref: biosci bionet.molbio.rapd:474 bionet.molbio.methds-reagnts:12298 Marcella E Grebus (mgrebus@magnus.acs.ohio-state.edu) wrote: : hey folks, : does anyone here work with one of those "air" type thermal-cyclers? : the kind that cycles air instead of using a heat block. : our lab is interested in looking into them, because we need to : get another thermal-cycler, and i've heard that the ones that : rely on air circulation reduce ramping times significantly but : may have some disadvantages. : i'd appreciate any advice, experience, comments, ordering/manufacturer : information [eg, 1-800 number]. if there's interest, i'd be happy : to post a summary of e-mails received. : also, if you feel strongly that a particular type of thermal-cycler : is the "best", let's discuss pro's and con's. i'm really open to : any brand/model. : thanks in advance, : --marcy I am looking into using one myself. I have not started yet. The way I understand it: the Air Thermal cyclers have the advantage of faster cycling, as you mentioned and the capability to run several types of reactions (ie: in tubes, or plates or slides for In Situ amplificatin). A disadvatage is that some people feel you should avoid radioactive materials because of dificulty in cleaning the air thermal cycler and that may become a source of contamination. One resource for such cyclers is Integrated Separation Systems, Tel 1-800-433-6433 or 508-655-1500 Fax 508-655-8501. These guys have acquired a product that used to be marketed by BIOS in the past (BIOSycler) and call it ISS Pro-Oven I ($3290) and they also are advertising a new enhanced product they call ISS Pro-Oven III ($4290), both are programmable and the more enhanced model seems to be impressive (at least in their advertising). My only experience with ISS was calling them for technical support for the old BIOSycler I found laying around in our lab, they have been very prompt and helpful in their replies to me although we never bought anything from them. This all what I know about this issue. I will appreciate a summary posted to the Newsgroup. Thanks in advance. From owner-rapd@net.bio.net Fri Mar 11 22:00:00 1994 Path: biosci!MOE.UCR.EDU!brunell From: brunell@MOE.UCR.EDU (Mark Brunell) Newsgroups: bionet.molbio.rapd Subject: high background for rapds Date: 12 Mar 1994 04:21:20 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9403120419.AA01864@moe.ucr.edu> NNTP-Posting-Host: net.bio.net If anyone is still interested in this, I have been plagued with high background in my rapd reactions. What finally worked was raising the annealing temp to 52 deg C. This is very high for 10-mers, and you will find that only a few primers will give any amplification, but the ones that do will be very clear and sharp. I have tried all of the tactics mentioned in the summary and have had little luck with them. Mark S. Brunell University of California, Riverside From owner-rapd@net.bio.net Tue Mar 15 22:00:00 1994 Path: biosci!PHIBRED.COM!murrayian From: murrayian@PHIBRED.COM (IAN MURRAY) Newsgroups: bionet.molbio.rapd Subject: Re: Summary concerning smearing of RAPD bands Date: 16 Mar 1994 18:53:56 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9403161900.AA09362@phibred.phibred.com> NNTP-Posting-Host: net.bio.net Hello Netters, I lost the smmary of the RAPD smearing. I have had trouble retrieving it through waismail (I only get the FAQ ). Could someone send a copy of it to me? My address is: Murrayian@phibred.com Thankyou very much. From owner-rapd@net.bio.net Wed Mar 16 22:00:00 1994 Path: biosci!GPU.SRV.UALBERTA.CA!dchong From: dchong@GPU.SRV.UALBERTA.CA (Daniel Chong) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 17 Mar 1994 23:06:38 -0000 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Hi Netters; I have a discussion with a friend on the topic of Bulk Segregant Analysis (BSA) and target genes. He thinks that any unique differences (markers) detected between two bulks with sufficient number of individuals are surely linked to the target gene. You do not need to screen the segregating population for linkage analysis if you do not care the map distances between marker loci and the target gene. I do not agree with him. Am I wrong or is he wrong? Daniel Chong U of A From owner-rapd@net.bio.net Thu Mar 17 22:00:00 1994 Path: biosci!MUCC.MAHIDOL.AC.TH!scbst From: scbst@MUCC.MAHIDOL.AC.TH (Burachai Sonthayanon - SCBC) Newsgroups: bionet.molbio.rapd Subject: Can we use RAPD for human fingerprinting ? Date: 17 Mar 1994 22:49:11 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: bionet Message-ID: NNTP-Posting-Host: net.bio.net Hello Netters, =09We would like to know if someone has already worked on=20 fingerprinting of human DNA with RAPD technique ? Can someone please=20 kindly inform us of some references that we may look up ?=20 =20 =09A colleague of mine tried 200 random primers to try to amplify=20 human DNA samples and found only around 20+ primers to be successful. I=20 think this is an abnormally low figure and his condition seems not to be=20 optimum. However, I have no experience with animal DNA samples. Can someone= =20 kindly help by suggesting conditions ? =09Thanks. ***************************************** From=20=09B. Sonthayanon =09Department of Biochemistry, =09Faculty of Science, Mahidol U., =09Rama 6 Rd., Bangkok 10400 =09Thailand =09FAX 66 2 248-0375 E-mail : scbst@mucc.mahidol.ac.th ***************************************** From owner-rapd@net.bio.net Fri Mar 18 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!darwin.sura.net!nntp.st.usm.edu!whale.st.usm.edu!sywang From: sywang@whale.st.usm.edu (Shiao Y. Wang) Newsgroups: bionet.molbio.rapd,bionet.molbio.methds-reagnts Subject: Re: "air" type thermal cyclers Date: 19 Mar 1994 04:49:08 GMT Organization: University of Southern Mississippi Lines: 38 Message-ID: <2me084$j20@server.st.usm.edu> References: <2liboj$e5t@charm.magnus.acs.ohio-state.edu> <2lnoj1$mh1@post.its.mcw.edu> NNTP-Posting-Host: whale.st.usm.edu X-Newsreader: TIN [version 1.2 PL1] Xref: biosci bionet.molbio.rapd:479 bionet.molbio.methds-reagnts:12588 Majed Abu-Hajir (abuhajir@post.its.mcw.edu) wrote: : Marcella E Grebus (mgrebus@magnus.acs.ohio-state.edu) wrote: : : hey folks, : : does anyone here work with one of those "air" type thermal-cyclers? : : the kind that cycles air instead of using a heat block. : : our lab is interested in looking into them, because we need to : : get another thermal-cycler, and i've heard that the ones that : : rely on air circulation reduce ramping times significantly but : : may have some disadvantages. : : i'd appreciate any advice, experience, comments, ordering/manufacturer : : information [eg, 1-800 number]. if there's interest, i'd be happy : : to post a summary of e-mails received. : : also, if you feel strongly that a particular type of thermal-cycler : : is the "best", let's discuss pro's and con's. i'm really open to : : any brand/model. : : thanks in advance, : : --marcy : I am looking into using one myself. I have not started yet. The way I : understand it: the Air Thermal cyclers have the advantage of faster cycling, : as you mentioned and the capability to run several types of reactions (ie: in : tubes, or plates or slides for In Situ amplificatin). A disadvatage is that : some people feel you should avoid radioactive materials because of dificulty : in cleaning the air thermal cycler and that may become a source of : contamination. The cycling time is actually slower because air transfers heat less efficiently than mineral oil found in more traditional thermal cyclers. However, a major advantage is the much greater number of samples that you can handle at one time (literally hundreds). Another advantage is the more uniform heating and cooling. If you have large numbers of samples or have multiple formats, air thermal cycler is the way to go. From owner-rapd@net.bio.net Sat Mar 19 22:00:00 1994 Path: biosci!daresbury!trane.uninett.no!sunic!pipex!zaphod.crihan.fr!jussieu.fr!univ-lyon1.fr!swidir.switch.ch!scsing.switch.ch!urz.unibas.ch!steinger From: steinger@urz.unibas.ch Newsgroups: bionet.molbio.rapd Subject: Re: "air" type thermal cyclers Message-ID: <1994Mar20.145714.43628@urz.unibas.ch> Date: 20 Mar 94 14:57:14 MET References: <2liboj$e5t@charm.magnus.acs.ohio-state.edu> <2lnoj1$mh1@post.its.mcw.edu> <2me084$j20@server.st.usm.edu> Organization: University of Basel, Switzerland Lines: 20 In article <2me084$j20@server.st.usm.edu>, sywang@whale.st.usm.edu (Shiao Y. Wang) writes: > The cycling time is actually slower because air transfers heat less > efficiently than mineral oil found in more traditional thermal cyclers. > However, a major advantage is the much greater number of samples that you > can handle at one time (literally hundreds). Another advantage is the > more uniform heating and cooling. If you have large numbers of samples or > have multiple formats, air thermal cycler is the way to go. I wouldn't say that temperature uniformity is generally higher in air thermal cyclers. I tried the ProOven (Biosycler) from Integrated Separartion Systems some time ago and measured temp. gradients of several degrees C within the oven cavity. RAPD amplification was reproducible between four wells in a corner of the microtiter plate but not between opposite corners of the same plate! So check first if you observe any positional effects in your amplifications before buying an air thermal cycler (or any thermal cycler). Thomas Steinger, Botanical Institute, Basel From owner-rapd@net.bio.net Sun Mar 20 22:00:00 1994 Path: biosci!CABELL.VCU.EDU!bio3tcm From: bio3tcm@CABELL.VCU.EDU (Theodore C. Muller III) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 20 Mar 1994 17:04:26 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <9403210105.AA18592@cabell.vcu.edu> NNTP-Posting-Host: net.bio.net How do I sign to communicate with others utilizing RAPD? From owner-rapd@net.bio.net Sun Mar 20 22:00:00 1994 Path: biosci!MIRANDA.UMDS.AC.UK!fsymons From: fsymons@MIRANDA.UMDS.AC.UK (Fernley Symons) Newsgroups: bionet.molbio.rapd Subject: (none) Date: 21 Mar 1994 12:19:12 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <2593.9403212004@miranda.umds.ac.uk> NNTP-Posting-Host: net.bio.net unsubscribe From owner-rapd@net.bio.net Mon Mar 21 22:00:00 1994 Path: biosci!bcm!cs.utexas.edu!usc!yeshua.marcam.com!charnel!xmission!u.cc.utah.edu!ls-10.biology.utah.edu!user From: Rasmussen@Bioscience.utah.edu (Randy P. Rasmussen) Newsgroups: bionet.molbio.rapd Subject: Re: "air" type thermal cyclers Followup-To: bionet.molbio.rapd,bionet.molbio.methds-reagnts Date: 22 Mar 1994 18:50:52 GMT Organization: University of Utah Lines: 24 Message-ID: References: <2liboj$e5t@charm.magnus.acs.ohio-state.edu> <2lnoj1$mh1@post.its.mcw.edu> <2me084$j20@server.st.usm.edu> NNTP-Posting-Host: ls-10.biology.utah.edu In article <2me084$j20@server.st.usm.edu>, sywang@whale.st.usm.edu (Shiao Y. Wang) wrote: > The cycling time is actually slower because air transfers heat less > efficiently than mineral oil found in more traditional thermal cyclers. > However, a major advantage is the much greater number of samples that you > can handle at one time (literally hundreds). Another advantage is the > more uniform heating and cooling. If you have large numbers of samples or > have multiple formats, air thermal cycler is the way to go. This information only applys to the old toaster oven type cycler not the rapid air cycler that the original question referred to. I have two Idaho Technology air cyclers on my bench and they are not slower - they are ten times faster than most heat block instruments. These machines take 15 to 30 minutes to do an average 30 cycle reaction. They can do this because the samples are held in glass or plastic capillary tubes which have very high surface area to volume ratios. The Idaho Tech machines hold a maximum of 48 samples. -- Randy P. Rasmussen Department of Biology Division of E. coli Psychobiology University of Utah From owner-rapd@net.bio.net Tue Mar 22 22:00:00 1994 Path: biosci!TAUNIVM.TAU.AC.IL!VHSTRI%VOLCANI.bitnet From: VHSTRI%VOLCANI.bitnet@TAUNIVM.TAU.AC.IL Newsgroups: bionet.molbio.rapd Subject: Subscribe Date: 23 Mar 1994 03:03:57 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <199403231103.DAA20619@net.bio.net> NNTP-Posting-Host: net.bio.net subscribe From owner-rapd@net.bio.net Fri Mar 25 22:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!daresbury!not-for-mail From: Julian Paul Robinson Newsgroups: bionet.molbio.rapd Subject: AMOVA- where?, how? Date: 26 Mar 1994 10:40:38 -0000 Lines: 12 Sender: daemon@mserv1.dl.ac.uk Distribution: bionet Message-ID: <2n13f6$k67@mserv1.dl.ac.uk> Original-To: rapd@dl.ac.uk Dear all, I've heard that a program called AMOVA is available. Does any know how to get hold of it ? I am able to use ftp to get it if it is available. Many thanks, Julian e-mail:jpr@st-and.ac.uk From owner-rapd@net.bio.net Sun Mar 27 23:00:00 1994 Path: biosci!MERCURY.UARK.EDU!DRHOADS From: DRHOADS@MERCURY.UARK.EDU ("Doug Rhoads") Newsgroups: bionet.molbio.rapd Subject: Re: RNA RAPDs Date: 28 Mar 1994 07:12:59 -0800 Organization: University of Arkansas Lines: 25 Sender: daemon@net.bio.net Distribution: bionet Message-ID: <296802F4F82@uamercury.uark.edu> NNTP-Posting-Host: net.bio.net >To: rapd@net.bio.net >From: vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) >Subject: RNA RAPDs >Date sent: 28 Mar 1994 08:27:32 -0500 > >I would like to hear from any one with experience in RAPDs for RNA. Does >the usual excess of ribosomal RNA cause problems when one is looking for >differences in mRNA. Is it better to separate the mRNA first? > >I am planning to set this up with human cells and would be greatful for any >advice. > We have been performing differential display on yeast RNAs. WE have at present had no problem running the reactions on first-strand synthesized from total RNA. We synthesize first strand using MMLV-RT on about 100 ug total RNA. We generally get a couple micrograms of first strand which we then either throw 5-10ng into the RAPD/differential display reaction. We have used this approach to pull out a few gene sequences that are differentially regulated and would recommend it to anyone. Doug Rhoads || Dept. of Biological Sciences drhoads@mercury.uark.edu || 601 Science Engineering drhoads@uafsysb.uark.edu || University of Arkansas 501-575-3251 || Fayetteville, AR 72701 From owner-rapd@net.bio.net Sun Mar 27 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!europa.eng.gtefsd.com!emory!news.cc.emory.edu!news.cc.emory.edu!not-for-mail From: vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) Newsgroups: bionet.molbio.rapd Subject: RNA RAPDs Date: 28 Mar 1994 08:27:32 -0500 Organization: Emory University Lines: 7 Message-ID: <2n6m04$bg3@emoryu1.cc.emory.edu> NNTP-Posting-Host: emoryu1.cc.emory.edu X-Newsreader: TIN [version 1.2 PL2] I would like to hear from any one with experience in RAPDs for RNA. Does the usual excess of ribosomal RNA cause problems when one is looking for differences in mRNA. Is it better to separate the mRNA first? I am planning to set this up with human cells and would be greatful for any advice. From owner-rapd@net.bio.net Tue Mar 29 23:00:00 1994 Path: biosci!agate!dog.ee.lbl.gov!overload.lbl.gov!deh From: deh@s27w007.pswfs.gov (Dave Harry) Newsgroups: bionet.molbio.rapd Subject: Re: AMOVA- where?, how? Date: 30 Mar 1994 16:00:26 GMT Organization: Dendrome, A Genome Database for Forest Trees Lines: 36 Distribution: bionet Message-ID: <2nc7mq$765@overload.lbl.gov> References: <2n13f6$k67@mserv1.dl.ac.uk> NNTP-Posting-Host: s27w007.pswfs.gov >> Dear all, >> I've heard that a program called AMOVA is >> available. >> Does any know how to get hold of it ? I am >> able to use ftp to get it if it is available. >> > >What exactly is this AMOVA program supposed to do? > AMOVA is analogous to ANOVA in certain ways in that it partitions variation within and among groups. Check out the following paper for further information. I've also heard the program is available, but I've not tried to locate it. Excoffier, L., P.E. Smouse, and J.M. Quattro 1992 Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitrochondrial DNA restriction data Genetics 131:479-491 The procedure was applied to RAPD data in the following paper. Huff, D.R.; Peakall, R.; Smouse, P.E. 1993 RAPD Variation Within and Among Natural Populations of Outcrossing Buffalograss [Buchloe dactyloides (Nutt) Engelm] Theor Appl Genet 86(8): 927-934 Dave Harry From owner-rapd@net.bio.net Tue Mar 29 23:00:00 1994 Path: biosci!GPU.SRV.UALBERTA.CA!ryang From: ryang@GPU.SRV.UALBERTA.CA (Rong Yang) Newsgroups: bionet.molbio.rapd Subject: Re: AMOVA- where?, how? Date: 30 Mar 1994 08:46:02 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 50 Sender: daemon@net.bio.net Distribution: bionet Message-ID: References: NNTP-Posting-Host: net.bio.net On 30 Mar 1994, Anthony Hilton wrote: > In article <2n13f6$k67@mserv1.dl.ac.uk>, Julian Paul Robinson > wrote: > > > Dear all, > > I've heard that a program called AMOVA is > > available. > > Does any know how to get hold of it ? I am > > able to use ftp to get it if it is available. > > > > Many thanks, > > > > Julian > > > > e-mail:jpr@st-and.ac.uk > > What exactly is this AMOVA program supposed to do? > > Wig. > > Hi, Tony: My reply seems to be more than what you want to know, but I thought it may be a good idea to share my experience with others. The AMOVA functions like the usual ANOVA, partitioning total variation into, say, among- and within-population components, but it was designed specifically for analysing molecular data such as RFLP haplotypes. All the analysis is based on the pre-defined inter-individual Euclidean distance matrices or alike. Because there is no error term in AMOVA, significance level for variance component estimates were calculated using a non-parametric permutational procedure (see Excoffier et al. Genetics 131:479-491 for details). As for its application to analysing RAPD data, some inference can be made about population structure. However, one has to be cautious to interpret his/her results because of dominance problem with RAPDs. In other words, with RAPDs, all we have are a set of phenotypes and we know nothing about the genetics unless conventional segregation analysis is conducted. Therefore, prior to invoking any genetic interpretation of estimated variance components from AMOVA, one has to establish the genotype-phenotype relationship. One obvious exception is that if you work with haploid magegametophytes in conifer species or others, you should be able to make the genetic interpetation without problem. Hope this will help. Rong-Cai Yang From owner-rapd@net.bio.net Tue Mar 29 23:00:00 1994 Path: biosci!agate!howland.reston.ans.net!pipex!uknet!bhamcs!bham!bcs88.bham.ac.uk!user From: A.C.Hilton@bham.ac.uk (Anthony Hilton) Newsgroups: bionet.molbio.rapd Subject: Re: AMOVA- where?, how? Followup-To: bionet.molbio.rapd Date: 30 Mar 1994 10:45:15 GMT Organization: University of Birmingham Lines: 18 Distribution: bionet Message-ID: References: <2n13f6$k67@mserv1.dl.ac.uk> NNTP-Posting-Host: bcs88.bham.ac.uk In article <2n13f6$k67@mserv1.dl.ac.uk>, Julian Paul Robinson wrote: > Dear all, > I've heard that a program called AMOVA is > available. > Does any know how to get hold of it ? I am > able to use ftp to get it if it is available. > > Many thanks, > > Julian > > e-mail:jpr@st-and.ac.uk What exactly is this AMOVA program supposed to do? Wig. From owner-rapd@net.bio.net Wed Mar 30 23:00:00 1994 Path: biosci!bcm!cs.utexas.edu!howland.reston.ans.net!torn!utnut!utcsri!newsflash.concordia.ca!canopus.cc.umanitoba.ca!hamel From: hamel@cc.umanitoba.ca (Andre Hamel) Newsgroups: bionet.molbio.rapd Subject: AMOVA Date: 30 Mar 1994 23:41:57 GMT Organization: The University of Manitoba Lines: 19 Message-ID: <2nd2o5$l1b@canopus.cc.umanitoba.ca> NNTP-Posting-Host: toliman.cc.umanitoba.ca Archie search yielded; Host fly.bio.indiana.edu Location: /biology/ibmpc FILE -rw-r--r-- 3043 Sep 8 1992 winamova.readme FILE -rw-r--r-- 7923 Sep 7 1992 winamova.txt FILE -rw-r--r-- 98120 Sep 10 1992 winamova.zip For MS-Windows no doubt. ....... suffice? regards, ******************** Andre Hamel email: hamel@cc.umanitoba.ca Manitoba Government Veterinary Services lab tel.: (204) 945-7630 545 University Crescent, FAX: (204) 945-8062 Winnipeg, Manitoba, CANADA R3T 5S6 ******************** From owner-rapd@net.bio.net Wed Mar 30 23:00:00 1994 Path: biosci!UNAMVM1.DGSCA.UNAM.MX!carvalho From: carvalho@UNAMVM1.DGSCA.UNAM.MX (Carvalho Torres Alexandro cassio-UACPYP) Newsgroups: bionet.molbio.rapd Subject: Re: RNA RAPDs{ Date: 31 Mar 1994 11:33:40 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 27 Sender: daemon@net.bio.net Distribution: bionet Message-ID: References: <2nds88$58v@nkosi.well.com> NNTP-Posting-Host: net.bio.net On 31 Mar 1994, Matthew J. Brauer wrote: > vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) writes: > > > >I would like to hear from any one with experience in RAPDs for RNA. Does > >the usual excess of ribosomal RNA cause problems when one is looking for > >differences in mRNA. Is it better to separate the mRNA first? > > >I am planning to set this up with human cells and would be greatful for any > >advice. > There's a similar, if not exactly the same, procedure which is loosely > analogous to RAPDs, called "differential display". The original > paper is by Liang and Pardee, in SCIENCE 1993 sometime. I have a > preprint, so I'm not sure of the exact citation. > > The complete reference is: Liang, P.& pardee, A.B. Differential display of eukariotc messenger RNA by means of the polymerase chain reaction. Science 257:967-71, 1992. Best regards. Alexandro carvalho Instituto Nacional de l aNutricion Salvador Zubiran. FAX 655 96 75 From owner-rapd@net.bio.net Wed Mar 30 23:00:00 1994 Path: biosci!agate!ihnp4.ucsd.edu!swrinde!sgiblab!barrnet.net!well!mjbrauer From: mjbrauer@well.sf.ca.us (Matthew J. Brauer) Newsgroups: bionet.molbio.rapd Subject: Re: RNA RAPDs Date: 31 Mar 1994 06:57:12 GMT Organization: The Whole Earth 'Lectronic Link, Sausalito, CA Lines: 13 Message-ID: <2nds88$58v@nkosi.well.com> References: <2n6m04$bg3@emoryu1.cc.emory.edu> NNTP-Posting-Host: well.sf.ca.us X-Newsreader: NN version 6.5.0 #1 (NOV) vvarma@emoryu1.cc.emory.edu (Vijay A. Varma) writes: >I would like to hear from any one with experience in RAPDs for RNA. Does >the usual excess of ribosomal RNA cause problems when one is looking for >differences in mRNA. Is it better to separate the mRNA first? >I am planning to set this up with human cells and would be greatful for any >advice. There's a similar, if not exactly the same, procedure which is loosely analogous to RAPDs, called "differential display". The original paper is by Liang and Pardee, in SCIENCE 1993 sometime. I have a preprint, so I'm not sure of the exact citation.