Dear Colleagues, Thanks to the many, many people who replied to my query about using acrylamide and EtBR as an alternative to modern capillary arrays. I apologize for not replying to each of you individually; the number of responses and requests for copies of the answers necessitate a mass- mailing. It seems that most everyone agrees that analysis in a capillary array using fluorescent-labelled primers is always preferable. However, a number of you suggested alternatives. The most popular were silver- staining of acrylamide gels, and the Elchrom Scientific 'origins' system. My current plan is to try out demo-models of each of these before making a final purchase. Below, I have posted the responses I received, with identifying information removed out of respect for our colleagues' privacy, If you would like to write directly to any of the respondents, please let me know and I will pass along their contact information. Best regards, and thanks to all, Chris. Christopher Irwin Smith Assistant Professor Department of Biology Willamette University Salem, OR 97301 ph: 503-370-6181 fax: 503-375-5425 www.willamette.edu/~csmith/ChrisSmith.htm email: csmith@willamette.edu csmith@uidaho.edu chris_smith@post.harvard.edu ---- Elchrom (Switzerland) used to make a system with horizontal agarose gels that may help. But I think that nothing beats labelled primers and sizing on sequencer. Dear Chris, I have been using for this purpose precast gels from Elchrom scientific, which also ship a electrophoresis apparatus. This is in a reasonalbe prize range and staining is easy with Sybergold (or if you want with EtBr). I prefer the Syber as it is more sensitive. You can check out details on http://www.elchrom.com/ Cheers ---- Hi Chris, I don't know anything about the methods you've asked about. But I just wanted to say that genotyping in core facilities using capillary machines isn't expensive. You probably know this already, but here in PA we do multiplex PCRs and send them to Penn state for about $200 for a 96 well plate. Total cost per locus per individual can be as low as abou $0.50. This is a great and worthy price, given the complication of any alternatives. Best, ---- hey there, no reason this shouldn't work. you previous experience with acrylamide was tainted by getting the toxic powder into solution, at the right concentration, etc. There are premade solutions available now, so the "toxicity" and difficulty is significantly reduced. check out biorad, I think they were the ones making this stuff. best of luck C ---- Dear Cris, Just thought I would let you know I have used agarose gels to visualize microsat variation and staining them with EtBr. We used 3 or 5 % agarose gels (which you need to play with to get it right, and must have a degassing step to get rid of many trapped bubbles). We were quite happy with the results. However, I must qualify that we picked and choose microsats where the difference in sizes between the alleles were larger than 15 bp, and we were scoring progeny from a F2 cross (only two alelles present). I don't think it works as well if you have more than 3 alleles segregating, since you may only see a blur. Best wishes, ---- Dear Chris, There is a system from Elchrom Scientific. The gels are stained with Sybr Gold, it works good but: only 2bp differences can be detected unambigously, you can do max. 3 loci at once if you have approx. 100bp difference. I have used it some years ago, if you do e.g. paternity analysis using only a few microsatellite loci (3-4) it is ok otherwisse you have to run many many gels and the gels are plus minus expensive. Anyway it is very easy and undergrads could easily handle it, you only need a good geldoc sytem for detection of alleles.The best: you don?t have to produce gels, you can just buy them and you don?t need flourescent labelling of primers. Just visit their home page (http://www.elchrom.com/public/index.php) there is information available and you can ask me for more information. I must say I have a ABI sequencer now and do not use the Elchrom apparatus any more... hope to have helped you. cheers, ---- Dear Christopher Such staining methods are difficult because if running denaturing gels the 2 DNA strands run differently and there are 2 bands for each allele. If you do non denaturing you get heteroduplex bands as well making it difficult to interpret. I tried the later many years ago with silver staining, colleagues tried silver staining denaturing gels and blotting and probing with biotin labelled probes and gave up for research purposes. Good luck ---- Hello Chris, I did quite a bit of microsatellite work while a postdoc at Duke generating a linkage map of a medically important fungus. For work I'm now doing on a forest pathogenic fungus, I'm using the fluor-tagged M13 system with capillary sequencers (3730) at Yale. The acrylamide setup is really not that bad, and for microsatellites you can use the Bio-Rad Sequi-Gen GT system designed for marker analysis. The rig is wide and short, and their gel system is very easy to handle. Rarely did we have failures of the gel. After running the gels, we did not stain with Eth-Br but rather used a very easy silver-staining technique. Once silver-stained, we'd dry the gels onto the plate (we used something called bind-silane to make the gel stick to one plate, and silicon, e.g. Rain-X, to separate it from the other plate), photograph them over a white light box, then score the pictures. We switched to a system where we used radiography copy film that we exposed directly on the dried gel, then developed in the darkroom. One problem with Eth-Br is having a large enough UV box to get a picture of the entire run. The combs for the rig have 51 wells, so we were able to run an entire 96-well plate's worth of reactions by loading the first 48, running it for 1/2 hr or so, then loading the next 48. Since the progeny could only segregate for a maximum of two alleles (haploid), there was no issue with overlap between the two loads. I have this whole process written up in a protocol, and if you're interested, I can send it to you. It's so easy that I had several undergraduates at Duke helping out, and they all became quite proficient at it. The trickiest part is loading the gel, but that just takes getting used to. You'll need the Sequi-Gen GT setup, the power supply (a pretty high-powered one), and if you don't have the photography equipment, you can score the runs directlyon the plate, then wash off the gel (using NaOH and detergent), and do the next run. We kept several plates circulating through the process. ---- Hi Christopher Re: message on Evoldir I have had in the past undergrad students carry out microsat projects visualise PCR products using Metaphor (very high resolution) agarose and staining with EtBr or Sybr green. With the latter stain it's totally non-toxic and great for the beginning molecular ecologist. However, Metaphor is quite expensive, and we found that it detects differences of at least 4bp between alleles most consistently. I tend to use the Metaphor agarose system now as a precursor to a confirmatory run on our sequencer/genotyper, as at least it ensures that the student has some useful variable product. Hope this helps and good luck ---- Chris, I do not have any experience with the system you described in your post, but do have a little experience with the Elchrom systems. Thought I would mention it in case you have not heard of them. The initial investment is about twice the system you mention, and the system works on pre-cast proprietary gels that are a little costly, but it is an alternative. I use the system for SSCP, and intend to use it to screen a microsat library. You stain the gel using SYBERGreen, which is (supposedly) less mutagenic than EtBr. The big plus, why I point it out to you, is that it is incredibly easy to use, perfect for an undergraduate setting. Here is the web site for the authorized domestic vendor that I have dealt with: https://www.geneseesci.com/highresgels.php And the Elchrom website: http://www.elchrom.com/public/index.php We purchased an ORGINS system for SSCP analysis for exactly the reasons you mention in your email concerning acrylamide and undergraduates. Feel free to give a call or drop a line if you want. ---- You could concider staining with GelRed with is an, if not non- toxic, then at least less toxic alternative to EthBr. In it's newest version it stain PA gels. Cheers ---- Dear Dr. Smith, I have similar interest in a similar set up (for, unsurprisingly, similar reasons). I would like to enquire as to what responses you got, if any, from your evoldir post? Best wishes, csmith@willamette.edu