From owner-rapd@net.bio.net Thu Aug 01 23:00:00 1996 Path: biosci!bloom-beacon.mit.edu!spool.mu.edu!uwm.edu!news-res.gsl.net!news.gsl.net!nntp.coast.net!news.sprintlink.net!news-stk-200.sprintlink.net!news.sprintlink.net!new-news.sprintlink.net!newsfeed.internetmci.com!in2.uu.net!gateway.sequent.com!newsfeed.orst.edu!news.uidaho.edu!usenet From: Josh Udall Newsgroups: bionet.molbio.rapd Subject: Re: Contamination. Date: Fri, 02 Aug 1996 01:11:09 -0600 Organization: University of Idaho Lines: 23 Distribution: world Message-ID: <3201AA0D.24DF@uidaho.edu> References: <4togj1$7ds@ox.mc.edu> Reply-To: judall@uidaho.edu NNTP-Posting-Host: abd1.aberdeen.uidaho.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0b5aGold (Win95; I) Robert Hamilton wrote: > > We have been trying amplify some fern DNA for an ecological study. We > optimized our reactions..everything fine, and then when we began, started > getting strong evidence of contamination. We replaced everything..but the > Taq, and associated reagents (magnesium chloride and buffer). Perkin > Elmer won't consider the possibility their product is contaminated. > > However, has anyone else had a problem with Stoffel fragment Taq? > > Our lot number for the Taq is F0560, Mgl2 is F0409 and buffer is F0468. > > If someone has used these lots and they have worked, I would appreciate > hearing from you, and, of course, I would also appreciate hearing from > you if you have had trouble with these lots. > > Thank you. (you might have to respond to rhamilto@ox.mc.edu) Specifically, what was your evidence for contamination? I have found the greatest part of this sort of variation mostly due to extraction procedure. Did your reactions just stop working? Josh From owner-rapd@net.bio.net Thu Aug 01 23:00:00 1996 Path: biosci!agate!newsgate.duke.edu!solaris.cc.vt.edu!homer.alpha.net!uwm.edu!vixen.cso.uiuc.edu!newsfeed.internetmci.com!battery.awod.com!usenet From: arthurv@awod.com Newsgroups: bionet.molbio.rapd Subject: Re: Contamination. Date: 2 Aug 1996 23:34:06 GMT Organization: ipomoea Lines: 21 Message-ID: <4tu39e$5c3@battery.awod.com> References: <4togj1$7ds@ox.mc.edu> Reply-To: arthurv@awod.com NNTP-Posting-Host: chs0193.awod.com X-Newsreader: IBM NewsReader/2 v1.2 >getting strong evidence of contamination. We replaced everything..but the >Taq, and associated reagents (magnesium chloride and buffer). Perkin >Elmer won't consider the possibility their product is contaminated. Robert, How did your group determine that you had contamination? What is the nature of this contamination, i.e., how do the bands look in your lanes? I assume that you used control lanes (e.g., absence of template DNA). Are you using PE's pcr base kit? -- if so, isn't there a sample DNA template that you can play around with? Just curious, Arthur e-mail: arthurv@awod.com the sweetpotato homepage: http://www2.linknet.net/s_potato ====================================================== From owner-rapd@net.bio.net Sun Aug 04 23:00:00 1996 Path: biosci!daresbury!not-for-mail From: Roland Koelliker Newsgroups: bionet.molbio.rapd Subject: RAPD Trouble!!! Date: 5 Aug 1996 08:36:59 +0100 Lines: 25 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <4u48ar$jv@mserv1.dl.ac.uk> X-Sender: rkoellik@patho1 content-length: 865 Original-To: rapd@dl.ac.uk Dear netters I'm using RAPD to detect genetic variability in festuca pratensis (meadow fescue). All worked fine so far, after initial problems I found a set of 20 primers that produce nice polymorphic bands. All of a sudden these primers stop to work. I tried everything (new polymerase, new buffer, mgcl, dntp, and so on) but still I do not get the same nice results as before. Since it once worked and I already have some nice pictures I don't want to play around with the reaction conditions again. Does anybody have an idea what's going on here? I'm quite desperate. Thank your help Roland Roland Koelliker Swiss Fed Inst Technol Plant Sciences ETH-Zentrum, LFW-C47 CH-8092 Zurich Tel ++41-1-632 4890 Fax ++41-1-632 1153 e-mail: koelliker@ipw.agrl.ethz.ch From owner-rapd@net.bio.net Mon Aug 05 23:00:00 1996 Path: biosci!rutgers!uwm.edu!chi-news.cic.net!News1.mcs.net!van-bc!newsfeed.direct.ca!news From: mckaya@sfu.ca (Sheldon McKay) Newsgroups: bionet.molbio.rapd Subject: Re: RAPD Trouble!!! Date: 6 Aug 1996 19:01:35 GMT Organization: Internet Direct Inc. Lines: 17 Message-ID: <4u84qf$c54@aphex.direct.ca> References: <4u48ar$jv@mserv1.dl.ac.uk> Reply-To: mckaya@sfu.ca NNTP-Posting-Host: van-as-05b05.direct.ca X-Newsreader: WinVN 0.92.6+ In article <4u48ar$jv@mserv1.dl.ac.uk>, Roland Koelliker says: > >Dear netters >I'm using RAPD to detect genetic variability in festuca pratensis (meadow >fescue). All worked fine so far, after initial problems I found a set of 20 >primers that produce nice polymorphic bands. All of a sudden these primers >stop to work. I tried everything (new polymerase, new buffer, mgcl, dntp, >and so on) but still I do not get the same nice results as before. Since it >once worked and I already have some nice pictures I don't want to play >around with the reaction conditions again. Does anybody have an idea what's >going on here? I'm quite desperate. > I've seen primers' performance work well the first time, then deteriorate after time and sometimes crap out entirely. What has helped for me is take many small aliquots from the concentrated primer stock solution and minimize freeze/thaw cycles, also thawing on ice instead of at RT. From owner-rapd@net.bio.net Tue Aug 06 23:00:00 1996 Path: biosci!agate!newsgate.duke.edu!solaris.cc.vt.edu!homer.alpha.net!uwm.edu!cs.utexas.edu!news.sprintlink.net!news-stk-200.sprintlink.net!newsreader.sprintlink.net!news.sprintlink.net!news-dc-10.sprintlink.net!news.edu.tw!nctuccca.edu.tw!spring.edu.tw!serv.hinet.net!netnews.hinet.net!news From: 忘憂草 Newsgroups: bionet.molbio.rapd Subject: Re: Contamination. Date: Wed, 07 Aug 1996 19:35:54 -0700 Organization: DCI HiNet Lines: 45 Message-ID: <3209528A.6B88@ms1.hinet.net> References: <4togj1$7ds@ox.mc.edu> <3201AA0D.24DF@uidaho.edu> NNTP-Posting-Host: 168.95.231.24 Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 2.01 (Win16; I) Josh Udall wrote: > > Robert Hamilton wrote: > > > > We have been trying amplify some fern DNA for an ecological study. We > > optimized our reactions..everything fine, and then when we began, started > > getting strong evidence of contamination. We replaced everything..but the > > Taq, and associated reagents (magnesium chloride and buffer). Perkin > > Elmer won't consider the possibility their product is contaminated. > > > > However, has anyone else had a problem with Stoffel fragment Taq? > > > > Our lot number for the Taq is F0560, Mgl2 is F0409 and buffer is F0468. > > > > If someone has used these lots and they have worked, I would appreciate > > hearing from you, and, of course, I would also appreciate hearing from > > you if you have had trouble with these lots. > > > > Thank you. (you might have to respond to rhamilto@ox.mc.edu) > > Specifically, what was your evidence for contamination? I have found > the greatest part of this sort of variation mostly due to extraction > procedure. Did your reactions just stop working? > > Josh Yes, extrachtion porcedure is one of the problems. Sometimes Primers will be part of the problems Josh Huang Taiwan R.O.C. -- 忘憂草 ☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆ 邏輯是科學的骨幹 知識是科學的血肉 ☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆ From owner-rapd@net.bio.net Tue Aug 06 23:00:00 1996 Path: biosci!bcm.tmc.edu!cs.utexas.edu!howland.erols.net!vixen.cso.uiuc.edu!newsfeed.internetmci.com!cpk-news-hub1.bbnplanet.com!thorn.cc.usm.edu!news From: Shiao Wang Newsgroups: bionet.molbio.rapd Subject: Re: RAPD Trouble!!! Date: Wed, 07 Aug 1996 14:05:32 -0500 Organization: University of Southern Mississippi Lines: 14 Message-ID: <3208E8FC.3196@whale.st.usm.edu> References: <4u48ar$jv@mserv1.dl.ac.uk> NNTP-Posting-Host: 131.95.113.27 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Win95; I) To: Roland Koelliker > All worked fine so far, after initial problems I found a set of 20 > primers that produce nice polymorphic bands. All of a sudden these primers > stop to work. I tried everything (new polymerase, new buffer, mgcl, dntp, > and so on) but still I do not get the same nice results as before. I had similar problems once. Turned out to be the primers which apparently degraded over just months in the freezer. New primers solved the problem for me. -- ******* Shiao Y. Wang Univ. of Southern Mississippi sywang@whale.st.usm.edu From owner-rapd@net.bio.net Wed Aug 07 23:00:00 1996 Path: biosci!CATI.CSUFRESNO.EDU!aelashr From: aelashr@CATI.CSUFRESNO.EDU (Abd elnasser Elashry) Newsgroups: bionet.molbio.rapd Subject: Ascochyta Date: 8 Aug 1996 05:48:36 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <199608081249.FAA16253@CATI.CSUFresno.EDU> NNTP-Posting-Host: net.bio.net Dear netters: I am a master student and I used RAPD-PCR for differentiating three species of Ascochyta In addition to one isolate of Phoma spp.. Could I use the pattern of Phoma spp. during the construction of dendrogram to assess the relatedness between the isolate or that is wrong. Please , write your Email which I can reply using it in the body of the Email because of something wrong in my Email . Thanks for your cooperation. Nasser From owner-rapd@net.bio.net Mon Aug 12 23:00:00 1996 Path: biosci!PIRA.CENA.USP.BR!DAVHMOON From: DAVHMOON@PIRA.CENA.USP.BR Newsgroups: bionet.molbio.rapd Subject: Polysaccharides Date: 13 Aug 1996 07:30:47 -0700 Organization: CENA/USP Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <1DD3BA42878@pira.cena.usp.br> NNTP-Posting-Host: net.bio.net Dear Netters, We are having great problems with polysaccharides during our plant DNA extractions. Does anyone know of a relatively simple way of getting rid of them. We have tried one method based on CTAB precipitation and another using low levels of SDS. Any help that anyone can give would be greatly received. Yours faithfully David H. Moon From owner-rapd@net.bio.net Tue Aug 13 23:00:00 1996 Path: biosci!rutgers!csn!nntp-xfer-1.csn.net!carbon!night.primate.wisc.edu!sdd.hp.com!swrinde!cs.utexas.edu!chi-news.cic.net!news.newnorth.net!news From: Craig Echt Newsgroups: bionet.molbio.rapd Subject: Re: Polysaccharides Date: Tue, 13 Aug 1996 11:18:49 -0500 Organization: USDA Forest Service Lines: 33 Message-ID: <3210AAE9.2528@newnorth.net> References: <1DD3BA42878@pira.cena.usp.br> Reply-To: cecht@newnorth.net NNTP-Posting-Host: rhin-cs-4.newnorth.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; PPC) To: DAVHMOON@PIRA.CENA.USP.BR DAVHMOON@PIRA.CENA.USP.BR wrote: > > Dear Netters, > We are having great problems with polysaccharides during our plant > DNA extractions. Does anyone know of a relatively simple way of > getting rid of them. We have tried one method based on CTAB > precipitation and another using low levels of SDS. > Any help that anyone can give would be greatly received. > > Yours faithfully > David H. Moon I have used both the following methods and both work well, although I prefer the NaCl method. Michaels et al. 1994 BioTechniques 17:274 Removal of polysaccharides from plant DNA by ethanol precipitation. Fang et al. 1992 BioTechniques 13:52 A quick and inexpensive methods for removing polysaccharides form plant genomic DNA. Some labs use ethanol ppt in the presence of 2M CaCl to remove pectin, but I do not know the citation. -- ===================================== Craig S. Echt Research Geneticist, USDA Forest Service Forestry Sciences Lab e-mail: cecht@newnorth.net 5985 County Rd. K Ph: (715) 362-1114 Rhinelander, WI 54501 fax: (715) 362-1166 USA From owner-rapd@net.bio.net Thu Aug 15 23:00:00 1996 Path: biosci!rutgers!uwm.edu!vixen.cso.uiuc.edu!newsfeed.internetmci.com!in3.uu.net!EU.net!Norway.EU.net!online.no!nntp-oslo.UNINETT.no!nntp-trd.UNINETT.no!daresbury!not-for-mail From: Roland Koelliker Newsgroups: bionet.molbio.rapd Subject: Possible reasons for RAPD failure Date: 16 Aug 1996 16:04:48 +0100 Lines: 266 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <4v22mg$366@mserv1.dl.ac.uk> X-Sender: rkoellik@patho1 content-length: 12977 Original-To: rapd@dl.ac.uk Dear Netters I did get so many nice suggestions how to solve my RAPD problem, that I think it's helpful for many of us to see them all together. Unfortunately my reactions still don't work, so if you have any further suggestions please help. Possible reasons for RAPD failure: After my experience it may be caused by: 1. degradation,precipitation or imperfect mixing of your stock DNA (most probably), test it on elpho and in various concentrations on PCR (0.1ng - 250ng per 25ul reaction mixture) 2. substantial changes in your water quality 3. precipitation of your Mg stock after repeated freezing/thawing 4. (very probable) degradation of dNTPs (splitting off the third "P" stops chain extension, aliquots should be used 5. your PCR machine good luck! You say you have tried new taq, dNTP's, buffer etc, but have you tried testing the pH of your water. We had a similar problem in our lab a few months agoe and the cause was our distilled water being too low pH. Other possibilities are your PCR machine..they need a regular service or the temperatures may be inacurate, have you changed the source of your gilson tips, microcentrifuge tubes, PCR tubes. The possibilities are almost infinite. Good luck, RAPD is a great technique untill something goes wrong, and then its a pig. Occasionally primers "go bad", although this is not very common. Also check your thermocycler - did someone change your cycling program? Does the machine work well for other people? - maybe it needs fixing. My RAPDS stopped working once when I made new dilutions of my concentrated DNA stocks. It turns out the tubes I was using to dilute the DNA were inhibiting the reaction (don't know if the DNA was binding to the tube, some manufacturing oil was in the tubes or what). Once I diluted the DNA in a pcr tube, it all started working again. Good luck! Have you ever tried a brnad new primer? The problem of appear and disappear bands mayb a primer problem. Good Look Do you know the phase of the moon, while you were doing the amplifications that worked out? Was it raining or did the sun shine? Just kidding! I'm working with Sugarcane Mapping using RAPD for more than 2 years and some times that happens without any reason. What I do is start all over again. I had more problems when I used a home made Taq Polymerase, but even with BRL material it didint work some perfectly. What I do is always have some standard DNA in my reaction. I always use mix that doesnt need small amounts of reagents so the errors are low. And cross my fingers..... I've had similar problems with RAPDs and tried changing everything I could think of as you've been doing. RAPDs are finiky, so I began looking at things I could not change, but may have changed on its own...such as the water that you use for your reactions. I've correlated my problems with the time of year and the spring water runoff. Although I use nanopure autoclaved water, there may have been changes occurring that were greater than the changes I made to the water (ie. pH, ion concentration). Even though distillation etc. would supposedly take care of those things, if the changes were greater than the consistent level of distilling and autoclaving that I do to the water, then 'leftover' effects may contribute to an ultimate change in water quality. This may not be the answer that you would like to hear, but it has repeatedly happened to me, and in different provinces. My solution was to wait several weeks, and try again with new water. It worked. Alternatively, it may not be the water. Have you tested your electrophoresis apparatus? ...used a positive control? ...tested consistency in temperatures of the thermal cycler? ...are you in a lab which might have power interuptions or inconsistencies in current? A friend of mine discovered his problem was the inconsistency in current being delivered to the thermal cycler. Try a positive control with other primers ie. ribosomal or mitichondrial primers. They always work for me. ....that's a lot to test. Or maybe you've already tested it all. Are you using the same template? If not, extractions vary greatly from one plant to another, from one extraction to another. If you are using the same template, have you tried a fresh extraction? Sometimes the template degrades. I have also had the water cause problems such as those you describe. The last thing I can think of is the thermal cycler...have you checked the temperatures on the thermal cycler. We have had the same problem with RAPDS. The solution is simple. Boil the primer(s) for 10 minutes and snap freeze in liquid nitrogen. Trust me, this works every time. Multiple freeze/thaws and long-term storage, even at -20, results in the formation of primer secondary structure. The symptoms usually appear as random RAPDS, some work some don't. Boiling results in linearizing the primer, and then the reactions work. Good Luck. I read your RAPD trouble in rapd@net.bio.net. I have much experience in RAPD application in many plant species. If you have ever tried to change enzymes, dNTPs and primers, I think that your problem is not in enzymatics but in mechanics. Please check the followings whether or not; 1) The oil in the tube was highly pure? 2) The tube was nicely fit in the PCR machine? If you have purchased microcentrifuge tubes from different company recently, you would better check them. Tubes not fit in holes (slots) of the machine can not be received the real temperature from the machine. 3) If you put oil in slots of the machine, the oil was not too much? When oil in slots is so much, it will make air bubble at 94 degree of denaturation time. Again, it will make poor temperature control in the tube. You can clean it with cotton ball, and add just a little bit of oil to adjust proper contact a tube with a slot. 4) The DNA content was accurate (means too small or a lot)? 5) Pipetting was accurate? You would better make master mix rather than pipetting one by one. 6) If you have no problem in above mentions, please try to use 1.25X buffer rather than 1X buffer. I do not know why, though but, I usually get better and reliable results with 1.25X buffer. If you have further question, please feel free contact with me. Good luck. We had a similar experience. After sucessfully using RAPD PCR (to study Epichloe endophytes and Bromus erectus grass) for more than two years we began having trouble with our reactions last October. After much wasted time and effort (and money) we found that our enzyme manufacturer had re-formatted the enzyme they were selling in response to a "crack-down" by the holder of the Taq polymerase patent. We subsequently tried two other enzymes (from two other small companies) and had the same thing happen (for the same reason). Unfortunatly, the enzyme sold by the patent holder doesn't work (in our hands) for RAPD PCR. We have yet to solve our problem! I would ask around to see whether anyone in Europe has found a dependable enzyme (that they have bought recently and which is liscensed for PCR) that works for RAPDs. Let me know if you find one!!! Try increasing the concentration of your primers, test conc. of primers, test them on a different species, buy new set of primers from the same supplier. A couple of guesses for you; 1. Water quality can affect things a lot, and if you've started a new batch, or you prepare your own (distilling is not good enough for reliable PCR results) your source may itself be variable (e.g. some farmer may have just sprayed something on the fields ...). 2. Lab conditions in summer are often hostile to reliable PCR (dust, heat etc), especially if you lab is not air conditioned, or someone turns it off at night while your PCR is running. You'll probably find the conditions in your PCR tubes is wildy different from the winter conditions, ramping times etc... Find a cooled room, or incubator to put you PCR machine in. We now use water that has been purified by reverse osmosis and then ultrafiltered and autoclaved. We have also purchased some very expensive water (for water) that is highly purified, which has worked the best. Quite often if it becomes humid the PCR machines have trouble. Things can go very wrong for no apparent reason. Might be your problem. I am working on RAPDs in trees such as Prosopis and Neem. I too have observed this kind of a problem a couple of time in my work. In both the instances, the causes for the lack of RAPDs all of a sudden were seemingly different. 1. The first time we suddenly ran out of RAPDs was when apparently our DNAs were old (more than 4 months old). At that time we either rephenolised the samples once again or we simply reprecipitated them in presence of sodium acetate. These samples gave normal RAPD profiles. 2. The second instance this happened was very recently, after we had made fresh batches of DNA. All of the new DNAs simply failed to give RAPDs with any primer. In most cases at the end of PCR we were getting smears and not bands, so apparently the primers were being extended. We were at this stage quantifying DNA by the EtBr on gel method. We diluted the DNAs to 25 ng/ul according to concentration as determined by OD at 260 nm and after that 25 to 200 ng of the DNA has consistently given good RAPDs. Have you tried new dilutions of your template DNA? I experienced this kind of trouble that were completely eliminated with fresh dilutions... Have you checked the water? It has given us trouble occasionally. I've seen primers' performance work well the first time, then deteriorate after time and sometimes crap out entirely. What has helped for me is take many small aliquots from the concentrated primer stock solution and minimize freeze/thaw cycles, also thawing on ice instead of at RT. I had similar problems once. Turned out to be the primers which apparently degraded over just months in the freezer. New primers solved the problem for me. I had the same problem whilst working on RAPD's in maize a couple of years ago. I found the only way to repeat my results were to remake the primers and re-prepare the DNA. I did not think that this would be the problem because I was careful with my DNA and primers. However, once I had remade the primers and reprep'd my DNA I was able to repeat the result. It's not your reaction conditions unless you have changed polymerase or thermocycler How do you store your primers and how old are they? They should be stored at -20C minimum, being thawed out as little as possible. It's a good idea to make two sets of primers to minimize the thawin and refreezing. I have my primers at 10 uM solutions stored at -80C and then dilute them to 2 uM working stocks which are kept refrigerated. Also, you might want to check your water quality, something simple which can really fowl things up. We use RO water thats purified to remove 99% of impurities, which is then autoclaved. I then store my water in 1ml portions at -20C. The best solution is to start everything from scratch, except the primers. Make sure you keep all reagents frozen at atleast -20C. I hope this helps some, I can't say that I've never been in your position working with RAPDs. Try recalibrating your pipetters. What size reaction mix are you using? Very small volumes make pipetting error a serious problem, according to responses on the 'net. This has happened to everyone, and rarely does one find a definitive answer such as bad taq, primers, etc. Primers apparently can lose potency over time due to annealing, which has reportedly been solved by immersion of the tube of primer in a hot water bath followed by snap-chilling. You might inquire about this from others in the newsgroup. I would check the pipetters (equipment and persons!) first, though. One of the most important factors in the success of RAPDs is the concentration and purity of the DNA. Have you change any of these, has the original sample evaporate a little bit in the refrigerator? You appear to be having the same problem that I have found. Basically the primers degrade with time and probably freeze-thawing. I have read somewhere that if you boil the primers for five minutes in a boiling water bath then quick freeze in liquid nitrogen this somehow restores the primers. We have tried this and so far have had one hundred percent success with primers that for some reason stopped working. Alternatively you can buy fresh primers which is more expensive. Roland Koelliker Swiss Fed Inst Technol Plant Sciences ETH-Zentrum, LFW-C47 CH-8092 Zurich Tel ++41-1-632 4890 Fax ++41-1-632 1153 e-mail: koelliker@ipw.agrl.ethz.ch From owner-rapd@net.bio.net Fri Aug 16 23:00:00 1996 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.rapd Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 17 Aug 1996 02:00:05 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199608170900.CAA21934@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. Moderation means, however, that someone has to take the time to review each message before it goes out. We have set up software here that simply allows the moderator to forward to an address at net.bio.net messages that (s)he wishes to have distributed. This takes no more time than that needed to read the message and pass it on, say about 1 min. per message. Most newsgroups currently have a discussion leader who is responsible for their newsgroup. The discussions leaders and their e-mail addresses are listed in the BIOSCI Information Sheet which is available on the Web at http://www.bio.net/. If a newsgroup is being hit with too many junk postings, please contact the discussion leader for that group and see if there is interest in moderating the group. Please do not assume that by simply posting a complaint to the newsgroup itself, anyone on the BIOSCI staff will act on your complaint. With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. C) In the body of your message put one or more of the following commands with an "end" command on the last line, e.g., subscribe methods unsubscribe methods end Do NOT put your e-mail address or other text on these lines. The server only allows you to cancel your subscription if the address on your mail header matches the address on our mailing list. Please ask for help at biosci-help@net.bio.net if your address has changed, e.g., if you know you are on the list but the server tells you that you are not a member. Users in Europe, Africa, and Central Asia who use the BIOSCI node at -------------------------------------------------------------------- computer daresbury.ac.uk (also known as dl.ac.uk): ------------------------------------------------- To subscribe and unsubscribe to/from the BIOSCI lists, you need to specify the full USENET newsgroup name with "bionet-news." prepended. The USENET newsgroup names are listed in the BIOSCI Information sheet on the Web at http://www.bio.net/. For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the body of the message as text on the Subject: line is ignored. To unsubscribe from all the lists at the UK node, use unsub bionet-news Please note that if the address in the list is different than the one in your mail message header, you will not be able to unsubscribe by this method. If you have problems, please mail biosci@daresbury.ac.uk. 4) The BIOSCI user address and research interest directory. ----------------------------------------------------------- Please take this opportunity to add your name, address, and research interest information to the BIOSCI User Address Database if you have not already done so. You can fill out the address form directly through our Web page at URL http://www.bio.net/adrform.html. The address database is reindexed nightly for WWW access (the URL is http://www.bio.net/). If you are not directly on the Internet but can reach it by e-mail, please use our waismail server to access the user directory. waismail use is described above. You can also request a user address form by e-mail from biosci-help@net.bio.net. Please check your database entry from time-to-time to see if your address information is still up-to-date. Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-rapd@net.bio.net Sat Aug 17 23:00:00 1996 Path: biosci!rutgers!rockyd!cmcl2!panix!newsfeed.internetmci.com!news.ac.net!news.cais.net!newshub.sdsu.edu!newsfeeder.sdsu.edu!news.sgi.com!csulb.edu!news.uoregon.edu!newsfeed.orst.edu!news.orst.edu!ucs.orst.edu!fjellstr From: Robert Fjellstrom Newsgroups: bionet.molbio.rapd Subject: Re: Polysaccharides Date: Sat, 17 Aug 1996 22:09:48 -0700 Organization: Oregon State University Lines: 28 Message-ID: References: <1DD3BA42878@pira.cena.usp.br> NNTP-Posting-Host: ucs.orst.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <1DD3BA42878@pira.cena.usp.br> I have had good luck in removing polysaccharides after ethanol precipitation (in 35% EtOh, as in the biotechniques article) but recently have gotten "even better" results from doing a PEG ppt. Briefly, add an equal amount of 13% PEG (8000), 1.6M NaCl to your DNA (in ca. 0.5 ml TE, the volume doesn't really matter) and spin down for 10-15 min. in a microfuge. Wash the ppt. in 70% EtOh (or if your a purist, resuspend the pellet in TE and do an ammonium acetate ppt.) and dissolve your pellet in TE for PCR. This tends to remove a lot of inhibitory compounds that prevented RADP reactions in a number of legumes we have worked with and it also removes polysacchrides (which were not a PCR problem but were a problem for Southern digestions). Good Luck - Bob F. On 13 Aug 1996 DAVHMOON@PIRA.CENA.USP.BR wrote: > Dear Netters, > We are having great problems with polysaccharides during our plant > DNA extractions. Does anyone know of a relatively simple way of > getting rid of them. We have tried one method based on CTAB > precipitation and another using low levels of SDS. > Any help that anyone can give would be greatly received. > > Yours faithfully > David H. Moon > > > From owner-rapd@net.bio.net Sun Aug 18 23:00:00 1996 Path: biosci!ml.csiro.au!Peter.Grewe From: Peter.Grewe@ml.csiro.au (Peter Grewe) Newsgroups: bionet.molbio.rapd Subject: yet another get rich quick....NOT! Date: 18 Aug 1996 22:36:32 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 13 Sender: daemon@net.bio.net Distribution: world Message-ID: <199608190535.PAA03890@strait.ml.csiro.au> NNTP-Posting-Host: net.bio.net unsubscribe ______________________________________________________ Peter Grewe International - Phone: +61-02-32-5374 FAX: +61-02-32-5000 Local - Phone: 002-32-5374 FAX: 002-32-5000 Internet : Grewe@ML.Csiro.AU http://www.ml.csiro.au/~grewe/grewehp.html Mail: CSIRO Fisheries GPO Box 1538 / Castray Esplanade Hobart, Tasmania Australia 7001 From owner-rapd@net.bio.net Mon Aug 19 23:00:00 1996 Path: biosci!rutgers!uwm.edu!msunews!agate!newsgate.duke.edu!news-server.ncren.net!taco.cc.ncsu.edu!faeppc2.cvm.ncsu.edu!user From: david_ley@ncsu.edu (David Ley) Newsgroups: bionet.molbio.rapd Subject: DNA bands in Neg. Control lane Date: Tue, 20 Aug 1996 15:40:38 -0500 Organization: NCSU-CVM Lines: 23 Message-ID: NNTP-Posting-Host: faeppc2.cvm.ncsu.edu We use the RAPD technique to perform epidemiological studies on poultry mycoplasmas. We have used the test for approximately one year w/o major problems. Recently, though, we have been amplifying DNA in our negative control. Our negative is aliquoted from a master mix of nuclease-free water (promega), 10X PCR buffer (Promega), MgCl2 (Promega), Taq (Promega), and primers (Life Technologies, stored at -20 in one-use aliquots). We do add nuclease-free water to QS our negative to 100ul. We use dedicated pipettors (Gilson) and plugged tips (USA/scientific). We also extract DNA and use amplified DNA in two separate areas of the lab. The bands that appear do not correspond to bands in our samples; a few also carry over from gel to gel, but this can vary. This is particularly annoyning since the results on our test samples are good. Has anyone experienced this? Any ideas? David H. Ley, DVM, PhD //// College of Veterinary Medicine < * ) North Carolina State University \ \___/// david_ley@ncsu.edu 4700 Hillsborough St. ( --- ) Raleigh, NC 27606 USA \/ \/ From owner-rapd@net.bio.net Thu Aug 22 23:00:00 1996 Path: biosci!agate!spool.mu.edu!usenet.eel.ufl.edu!tank.news.pipex.net!pipex!usenet2.news.uk.psi.net!uknet!usenet1.news.uk.psi.net!uknet!EU.net!nntp04.primenet.com!nntp.primenet.com!newspump.sol.net!news.inc.net!it!delp From: delp@it.uwp.edu (Jacqueline Delp) Newsgroups: bionet.molbio.rapd Subject: Bands present in Neg Control Date: 23 Aug 1996 16:25:50 GMT Organization: University of Wisconsin - Parkside Lines: 15 Message-ID: <4vkm2e$8r1@news.inc.net> NNTP-Posting-Host: 131.210.1.100 X-Newsreader: TIN [version 1.2 PL2] In reference to an article posted on 20 Aug 96 from Dr. David Ley: I too have been having problems with my negative control. I have attempted every possible procedure to eliminate this problem. The bands I am getting in my negative control are not present in any of my samples. I have no idea where they are coming from. I use Taq from Promega also and I am just curious, what lot number are you using? My lot number is #57319120. The last two numbers (20) refer to alliquot but the first 6 numbers identify the batch, therefore are the important numbers. I am going to try to UV my master mix prior to template addition in hopes of eliminating the neg control bands. Baffled!!! Jackie From owner-rapd@net.bio.net Thu Aug 22 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!netnews.nwnet.net!news.u.washington.edu!homer14.u.washington.edu!ottok From: "Kevin G. Otto" Newsgroups: bionet.molbio.rapd Subject: Re: DNA bands in Neg. Control lane Date: Fri, 23 Aug 1996 16:04:48 -0700 Organization: University of Washington Lines: 41 Message-ID: References: NNTP-Posting-Host: homer14.u.washington.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII NNTP-Posting-User: ottok To: David Ley In-Reply-To: In response to your apparent problem with neg. controls amplifying, I, too, have an occasional amplification in my negs.. I know the problem comes from the BSA that I use in the reaction buffer but I typically don't worry about it. If the bands are different than those of your experimentals you won't score them and furthermore they won't segregate so there shouldn't be a problem. If you're really bothered by this amplification problem, there is the possibility that something(like bacteria) has grown in your reaction buffer. Either way...good luck. Kevin Otto On Tue, 20 Aug 1996, David Ley wrote: > We use the RAPD technique to perform epidemiological studies on poultry > mycoplasmas. We have used the test for approximately one year w/o major > problems. Recently, though, we have been amplifying DNA in our negative > control. > > Our negative is aliquoted from a master mix of nuclease-free water > (promega), 10X PCR buffer (Promega), MgCl2 (Promega), Taq (Promega), and > primers (Life Technologies, stored at -20 in one-use aliquots). We do add > nuclease-free water to QS our negative to 100ul. We use dedicated > pipettors (Gilson) and plugged tips (USA/scientific). We also extract DNA > and use amplified DNA in two separate areas of the lab. > > The bands that appear do not correspond to bands in our samples; a few also > carry over from gel to gel, but this can vary. This is particularly > annoyning since the results on our test samples are good. > > Has anyone experienced this? Any ideas? > > David H. Ley, DVM, PhD //// > College of Veterinary Medicine < * ) > North Carolina State University \ \___/// david_ley@ncsu.edu > 4700 Hillsborough St. ( --- ) > Raleigh, NC 27606 USA \/ \/ > > From owner-rapd@net.bio.net Thu Aug 22 23:00:00 1996 Path: biosci!LIFSCI.SDSU.EDU!MCCLELLAND From: MCCLELLAND@LIFSCI.SDSU.EDU Newsgroups: bionet.molbio.rapd Subject: bands in negative controls Date: 23 Aug 1996 12:50:28 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 14 Sender: daemon@net.bio.net Distribution: world Message-ID: <960823125046.20202836@LIFSCI.SDSU.EDU> NNTP-Posting-Host: net.bio.net I am not as upset as some when arbitrarily primed PCR yields some bands in the negative control unless these bands are due to contamination from a previous fingerprint. Arbitrary primers are capable of amplifying from extremely poor matches so sporadic amplification may occur, but extremely inefficiently. In the presence of millions of molecules of the intended target these sporadic products have no chance. The time to get worried is when the negative control gives reproducible patterns. Then it may compete with the intended products and cause a problem. Otherwise, I would not worry about it very much. TO be on the safe side we have always fingerprinted at two nucleic acid concentrations to ensure that the products we were getting were reproducible. Michael McClelland From owner-rapd@net.bio.net Fri Aug 23 23:00:00 1996 Path: biosci!agate!howland.erols.net!cam-news-hub1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!newsfeed.internetmci.com!in3.uu.net!gateway.sequent.com!newsfeed.orst.edu!news.orst.edu!ucs.orst.edu!fjellstr From: Robert Fjellstrom Newsgroups: bionet.molbio.rapd Subject: Re: Bands present in Neg Control Date: Fri, 23 Aug 1996 22:39:00 -0700 Organization: Oregon State University Lines: 21 Message-ID: References: <4vkm2e$8r1@news.inc.net> NNTP-Posting-Host: ucs.orst.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII In-Reply-To: <4vkm2e$8r1@news.inc.net> I think this topic has come up before in this or the molbio methods bulletin board and the most educated guess was that the polymerase itself carries enough contaminating DNA from Thermus aquaticus to get amplified. This DNA would be present in such little amounts that as soon as any other template is available it out competes the Taq DNA and becomes the only source for amplification (hence these bands do not correspond to any bands seen in the organism under study). There are generally few bands in these "control" lanes which is also typical of RAPD amplification of bacterial genomes. If this hypothesis is true, all the purification techniques you may try would not eliminate the bands in the control lanes. I actually believe this is common and most people just ignore them and take it for granted that the bands with the host DNA added are real and are not due to contaminants. All the bands I have scored in the legumes I work with are heritable and that would not be predicted with random contamination events. Regards, Robert Fjellstrom Research Geneticist USDA-ARS From owner-rapd@net.bio.net Sat Aug 24 23:00:00 1996 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!cpk-news-hub1.bbnplanet.com!cpk-news-feed1.bbnplanet.com!ox.mc.edu!usenet From: Robert Hamilton Newsgroups: bionet.molbio.rapd Subject: Re: Bands present in Neg Control Date: 25 Aug 1996 00:25:23 GMT Organization: Mississippi College Lines: 15 Message-ID: <4vo6hj$oao@ox.mc.edu> References: <4vkm2e$8r1@news.inc.net> NNTP-Posting-Host: mobo.mc.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.22 (Windows; I; 16bit) Robert Fjellstrom wrote: >I think this topic has come up before in this or the molbio methods >bulletin board and the most educated guess was that the polymerase itself >carries enough contaminating DNA from Thermus aquaticus to get >amplified. This DNA would be present in such little amounts that as soon >as any other template is available it out competes the Taq DNA and >becomes the only source for amplification (hence these bands do not >correspond to any bands seen in the organism under study). We get a specific set of bands in our controls and our samples when we have used our current lot of Perkin-Elmer Taq. We use plant DNA as a template, and maybe the affinity of the primers for the template is low, and any given bacterial contamination is dominating the reaction, regardless of concentration. From owner-rapd@net.bio.net Sat Aug 24 23:00:00 1996 Path: biosci!SERVIDOR.DGSCA.UNAM.MX!carvalho From: carvalho@SERVIDOR.DGSCA.UNAM.MX ("Emyra e Alex") Newsgroups: bionet.molbio.rapd Subject: primer extension Date: 25 Aug 1996 10:10:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 16 Sender: daemon@net.bio.net Distribution: world Message-ID: <9608251812.AA19117@servidor.dgsca.unam.mx> NNTP-Posting-Host: net.bio.net Dear netters, I am looking for a good paper describing the primer extension tecnique. Please if someone knows something send to me. Thank you very much, Alexandro Cassio T. Carvalho Department of Infectious Diseases INNSZ-Mexico City Calle Vasco de Quiroga 15. Tlalpan. FAX 525 513 0010 CP 14000 From owner-rapd@net.bio.net Mon Aug 26 23:00:00 1996 Path: biosci!OREO.CHEM.UOTTAWA.CA!djohnson From: djohnson@OREO.CHEM.UOTTAWA.CA ("Doug Johnson") Newsgroups: bionet.molbio.rapd Subject: RAPD markers Date: 27 Aug 1996 11:24:35 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <1E556A6AF5@oreo.uottawa.ca> NNTP-Posting-Host: net.bio.net Could anyone suggest an alternative to HaeIII digested phiX174 as a marker for RAPD analysis? I would prefer to make my own, and in my hands a plasmid gives more DNA per cell than a virus. This is really an effort to save money. All suggestions are welcome. Doug Johnson djohnson@oreo.uottawa.ca From owner-rapd@net.bio.net Tue Aug 27 23:00:00 1996 Path: biosci!EM.AGR.CA!vincentd From: vincentd@EM.AGR.CA (Daniel Vincent) Newsgroups: bionet.molbio.rapd Subject: RAPD markers -REPONSE Date: 28 Aug 1996 16:03:11 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Look for 123 bp DNA Ladder from Gibco BRL From owner-rapd@net.bio.net Tue Aug 27 23:00:00 1996 Path: biosci!MBRSWI.AGR.CA!SCLOUTIER From: SCLOUTIER@MBRSWI.AGR.CA Newsgroups: bionet.molbio.rapd Subject: Post-doc position in gene cloning - wheat Date: 28 Aug 1996 10:34:45 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: <01I8T7G37GTE001Y7S@VMGW.AGR.CA> NNTP-Posting-Host: net.bio.net POST-DOCTORAL POSITION IN MOLECULAR GENETICS The Cereal Research Centre of Agriculture and Agri-food Canada in Winnipeg, CANADA, is presently seeking a post-doctoral visiting fellow with experience in molecular genetics including PCR and cloning to conduct a three year project on cloning disease resistance genes in wheat. Experience working with plants and knowledge of pathology and plant breeding would be an asset. CRC is devoted to developing wheat cultivars. Its biotech and chemistry section is involved in mapping, marker development, marker assisted selection, gene cloning and transformation. Post-doctoral visiting fellowship: 35 000$CAN/year If interested, please send your resume to: S. Cloutier CRC - AAFC 195 Dafoe Road Winnipeg, Manitoba CANADA R3T 2M9 or via the internet: scloutier@mbrswi.agr.ca or scloutier@em.agr.ca or by fax: 204-983-4604 From owner-rapd@net.bio.net Thu Aug 29 23:00:00 1996 Path: biosci!agate!spool.mu.edu!news.sgi.com!swrinde!cs.utexas.edu!howland.erols.net!newsxfer2.itd.umich.edu!netnews.worldnet.att.net!news.u.washington.edu!homer14.u.washington.edu!ottok From: "Kevin G. Otto" Newsgroups: bionet.molbio.rapd Subject: Re: RAPD Beads Date: Thu, 29 Aug 1996 22:30:45 -0700 Organization: University of Washington Lines: 36 Message-ID: References: <505687$7bk@doc.jmu.edu> NNTP-Posting-Host: homer14.u.washington.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII NNTP-Posting-User: ottok In-Reply-To: <505687$7bk@doc.jmu.edu> Curtis, I was one of the individuals that Pharmacia had perform comparative tests of their new product "RAPD Beads". For our research purposes, molecular mapping of large pedigrees, the beads turned out to be rather limited. Typically, we make a master mix containing everything except the template DNA. With this new product there would actually be twice as many pipetting steps to perform the same mapping experiments. However, for other projects(those using a small number of templates and primers..etc) these might work well. Our testing showed that they at least work. If cost is truly an issue though, I bet that purchasing the individual components would be considerably cheaper. Good Luck! Kevin Otto University of Washington On 29 Aug 1996, Curtis M. Pasfield wrote: > Upon returning to school this year my mentor brought it to my attention > that a company ( I do not remember the name), is producing beads for use > with RAPD-PCR. These beads contain all of the necessary components > including possibly primers. These seem like a wonderful tool. The > project that I have been working on has been plagued with problems with > the various components of the reactions. I was wondering if anyone has > used these beads and what kinds of results have been obtained. Should I > spend the money from my very limited research budget or should I just try > to iron out the bugs from my protocols? Thanks in advance. > > Curtis Pasfield > James Madison University > pasfiecm@jmu.edu > > From owner-rapd@net.bio.net Thu Aug 29 23:00:00 1996 Path: biosci!rutgers!uwm.edu!cs.utexas.edu!howland.erols.net!newsfeed.internetmci.com!in3.uu.net!hearst.acc.Virginia.EDU!news.jmu.edu!pasfiecm From: pasfiecm@falcon.jmu.edu (Curtis M. Pasfield) Newsgroups: bionet.molbio.rapd Subject: RAPD Beads Date: 29 Aug 1996 22:40:07 GMT Organization: James Madison University, Harrisonburg, VA Lines: 13 Sender: @JMU.EDU Message-ID: <505687$7bk@doc.jmu.edu> NNTP-Posting-Host: falcon.jmu.edu X-Newsreader: TIN [version 1.2 PL2 JMU4] Upon returning to school this year my mentor brought it to my attention that a company ( I do not remember the name), is producing beads for use with RAPD-PCR. These beads contain all of the necessary components including possibly primers. These seem like a wonderful tool. The project that I have been working on has been plagued with problems with the various components of the reactions. I was wondering if anyone has used these beads and what kinds of results have been obtained. Should I spend the money from my very limited research budget or should I just try to iron out the bugs from my protocols? Thanks in advance. Curtis Pasfield James Madison University pasfiecm@jmu.edu