Dear all, Thank you very much for all the input and recommendations. They were all extremely helpful. Below are all the answers I got for those of you interested in them (I included the ones already posted in evoldir for the most distracted). Once again, thank you. Sara Carvalho Evolutionary Genetics Group Instituto Gulbenkian de Ciencia Portugal saracarvalho@igc.gulbenkian.pt Sara, You might be interested in this paper: IVANOVA, NATALIAV, DEWAARD, JEREMYR, HEBERT, PAULDN (2006). An inexpensive, automation-friendly protocol for recovering high-quality DNA. Molec. Ecol. Notes 6: 998-1002. best, Kathryn Kathryn Elmer, Ph.D. Postdoctoral Researcher LS Evolutionary Biology Dept. of Biology University of Konstanz D-78457 Konstanz Germany phone: +49 (0) 75 31 88 37 10 (from abroad, no (0)) fax: +49 (0) 75 31 88 30 18 e-mail: elmerk@biology.queensu.ca website: http://www.walled.net/~kelmer/ Hi Sarah, I used the Qiagen kit for two 96-well plates in a time. It works nice and it is very easy to follow the instruction. The DNA was relatively clean. The only down-side is that the yield is relatively low - I got around 20-50 nano-gram/micro-liter in the final solution. Cheers, Yuval Hi Sara, Yes, I have used 96-wells plates from Qiagen (DNA easy 96 Tissue kit) to extract genomic DNA. It has worked fine for me. Two remarks however: 1) you need to buy a powerful and expensive centrifuge to use the kit. 2) I find the collection tubes (those in which you elute the extracted DNA at the end of the procedure) pretty bad. They come in strips of 8 tubes, with the associated caps attached to each other. This means that when you open these tubes, it is easy to contaminate the samples with one another. Hope this helps. Patrick Mardulyn Unit of Evolutionary Genetics (UEG) Institute of Molecular Biology and Medicine (IBMM) Free University of Brussels (ULB) rue Jeener et Brachet, 12 6041 Gosselies Belgium Phone: (32)(71)378-954 Fax: (32)(71)378-950 e-mail: pmarduly@ulb.ac.be http://homepages.ulb.ac.be/~pmarduly/ Hi Sara I have used the promega sv96 wizard kit - it has always given great results, even when all other extraction methods have failed! You also need a vacuum pump though. Cheers Amber Teacher Wildlife Epidemiology Institute of Zoology Regent's Park London NW1 4RY tel: 020 7449 6696 fax: 020 7483 2237 www.zsl.org email: amber.teacher@ioz.ac.uk hei, i have used a kit for the 96 well DNA extraction for plant materials 2-3 years ago. it worked very well provided that you take great care to avoid of retro-contamination among samples. This kit could be got from QIAGen, which is very likely called DNeasy Kit. it is for 6x96well preparation. but it costs a lot. good luck! jinyong We perform chelex extractions in 96 well plates. It is cheap, easy and fast and gives perfectly good DNA for microsat amplification. Our protocol is below. All the best, Andrew Pemberton Making up the chelex suspension: 6% (w/v) ?solution? (really a suspension) in ddwater (Lukas? lab).1.5gchelex beads in 23.5ml ddwater. Make up in autoclavable glass jar with lid and add (clean) magnetic stirring flea. Autoclave before first use and at intervals after that. Make up 2 or 3 bottles at a time so have spares (perhaps of x1.5 the above volumes). Use 150ul chelex for Scathophaga heads. Protocol: 1. Aliquoting out the chelex suspension: Whilst stirring on magnetic stirrer, pipette 150 ul chelex solution into each well using a wide- ended P200 tip (either then special white ?cell-saver? tips we have got or just cut the end off a regular tip with scissors or a knife). This is an inexact science as the number of beads you get will vary. Try to get roughly the same by looking at the pipette tip before dispensing. 2. Add the fly head and crush on side of well or against beads. Clean tweezers with bleach then deionised water then 70% ethanol between flies and have at least 3 sets of tweezers so the get a few minutes in the bleach. 3. Seal the plate with a clean ABI rubber sealing mat. Spin down with salad spinner. Using a PCR machine with hot lid on (105C) incubate at 55C for 1hr then (boil) 9 mins at 100C (PCR machines only go to 99.9C). Either ramp temperature down or allow to cool to room temperature (say 3 mins on bench). 5. Spin down with salad spinner to remove drops from sealing mat. 6. Replace rubber mat with aluminium sealing foil. Press down very well indeed. 8. Gently vortex and spin down. 11. Leave in fridge (4C) overnight. 12. Freeze at -20C for at least 24hours. I used to think that leaving it a few days in the freezer gave better results. No idea why it should. 13. To use. Vortex and spin down before use. You use the supernatant and must avoiding stuffing a chelex bead up your pipette tip and into your PCR! We add 1.5ul of DNA solution to 6ul total volume multiplex PCR (Quiagen multiplex PCR kit is wonderful!). Can use a multichannel pipette for this. Dr Andrew Pemberton Zoologisches Museum der Universit???Z? Winterthurerstrasse 190, CH 8057 Z? Switzerland Phone +41 (0)44 635 4772, Fax +41 (0)44 635 4780, Mobile +41 (0) 764 976 034 Hi Sara, I work on Drosophila, and have been doing fairly standard high salt extractions in 96-well format. I had 4000 samples to extract so there was no way I was doing this in single tubes. What organism are you working on? If it requires maceration before you digest the tissue, I have found that a 96-well replicating tool used in phage microbiology works wonders as a 96-well mortar and pestle. It is a bit of an expensive out-lay for this item but it was worth EVERY penny in the time it saved. The only tricky part with this method is carefully removing the supernatant after you have pelleted the cell debris. Although with practise and careful observation of marker lines on the pipette tips to gauge depth, I had no troubles. I used reagents from a kit (gentra pure gene) but you could easily make up all those components from scratch. They are nothing unusual. This method is great if you are working with low expected DNA yields. I found that all the column extraction kits (eg wizard SV and Qiagen DNeasy) required a high final elution volume (~100-200ul), which diluted my DNA to the point where it was not much use to anyone. Whereas with my high salt method I was able to re-hydrate my DNA in whatever volume I wanted (20ul). Let me know if you want the extended protocol or information on my "96-well mortar and pestle". Clare -- Clare Holleley School of Biological, Earth & Environmental Sciences (BEES) The University of New South Wales Sydney NSW 2052 Telephone: +61 (0)2 9385 2198 Fly Room: +61 (0)2 9385 2128 MEEF: +61 (0)2 9385 8236 Facsimile: +61 (0)2 9385 1558 Email: c.holleley@unsw.edu.au ola Sara, Here in the lab we have been using Nucleospin Tissue 96 kits from Macherey-Nagel. But I don t know if they are distributed in Portugal. We had good results on mollusks and polychaete. You can also try Qiagen DNeasy kits (similar to Nucleospin) that are distributed in Portugal (by Izasa when I was working in Faro, we were using them). Here in Roscoff, we no longer use them because they are more expensive than Nucleospin. For both kits you need a centrifuge that can reach 5600g with a multiplate rotor with buckets deep enough to accept the kits (Heraeus Multifuge 3SR, Sigma 4K15, or Hettich rotanta 460). You will also need a multichannel that can pipet in the bottom of 1.2ml transfer tubes, and that can go up to 1250?l (matrix Impact 2 1250?l with extended tips). They explain all that in the booklet of the kits. With such kits, you can extract 2x96 samples within an afternoon. It is quick. Boa sorte com o teu trabalho ! claire Claire Daguin-Thi???ut IE CNRS Equipe Evolution et G???tique des Populations Marines UMR 7144 CNRS UPMC Adaptation et Diversit???n Milieu Marin LIA Dispersal and Adaptation of Marine Species Station Biologique de Roscoff Place G. Teissier BP74 29682 ROSCOFF cedex T???02 98 29 23 19 Fax 02 98 29 23 36 Hi Sara, in our lab we use the 96-well Qiagen DNA extraction kits. We study vertebrates primarily and we obtain between 10-70ng/ul of DNA (quantified using a fluorometer). I hope that helps. You can download a manual (check out the protocol for "rodent tail") to determine whether the kit will require purchasing additional equipment for your lab. Chao, -Melanie -- Molecular Geneticist NMFS/ Conservation Biology Division DNA Laboratory 2725 Montlake Blvd E. Seattle WA 98112 e-mail: Melanie.paquin@noaa.gov Hi, Yes I used a lot of 96 well extraction kits. To my point of view Machery & Nagel tissues kit for 96 well worked best for this purpose, but there are also companies like Invitrogen or others (I think Qiagen and Promega also offers some kits but I not totally sure) that provide use full kits ??? depends which system you are running ??? a robot or centrifugation by precipitation or magnetic beads. Some work for both some not. Hope this helps Martin Mag. Martin Koch Department of Zoology University of Graz Universitätsplatz 2 8010 Graz Austria martin.koch@uni-graz.at tel.: +43 316 380 8756 http://www.kfunigraz.ac.at/zoowww/personal/mkoch/koch.htm Hi, we used QiaGen 96-well extraction kits and they worked fine. We were extracting both host and parasite DNA from blood, and they recovered tiny amounts of parasite DNA really well. Cheers, Kate Dear Sara, We have had great results with the Promega Wizard SV 96 Genomic DNA extraction kits - on everything from bird blood, through mammalian tissue to bee legs. You have to buy a custom-made vacuum manifold from Promega (about 500 euros from memory) and have a vacuum pump to suck the solutions (lysate, washes and elution) through the binding matrix. After that you are looking at 1.5 - 2 euros per sample - but you can easily process a 96 well plate in a morning (after an overnight proteinase K digestion). As an aside, we have had bad results with the similar Promega 96 well kit for plasmid purifications (just in case you wanted to do that as well). Good Luck, Bill Dr W C Jordan Institute of Zoology Zoological Society of London Regent's Park London NW1 4RY bill.jordan@ioz.ac.uk or w.jordan@ucl.ac.uk home page: http://www.zoo.cam.ac.uk/ioz/people/jordanb.htm Dr. Carvalho, Celex extractions can be done in 96 well plates. They're really cheap. However, the DNA doesn't last for much more than a year... see the attached protocol for details. http://www.uga.edu/srel/DNA_Lab/protocols.htm Nick Nicholas Crawford Research Technician Savannah River Ecology Laboratory Aiken, South Carolina crawford@srel.edu http://www.uga.edu/~srel/ Dear Sara, I'm extracting DNA from Scoot pine seeds and leaves. We use the Qiagen plant kit. We obtain satisfactory result but it seems that the DNA quality and quantity is better when you use an individual kit instead of a 96 well plates format. Good luck. Pascal ASPE Pascal Conservatoire Génétique des Arbres Forestiers Centre INRA 2163 Avenue de la pomme de pin BP 20619 Ardon 45166 OLIVET Cedex France *tel: 00-33-(0)2 38 41 78 32 Fax: 00-33-(0)2 38 41 48 00* * * * * ** We use the promega 96 well kit. It works well with insects, preserved through EtOH previously. I guess we get one failure per plate on average (i.e. doesn't PCR with insect conserved primers). I contemplated the Qiagen kit, but prefer a vacuum format to one requiring centrifuging. Neither are cheap, but they are efficient. An overnight digest then one mornings work. Good luck in the search. Greg Hurst have used Qiagen 96 well plant extraction kits with a Mixer Mill with great success. 192 samples in a few hours - so wonderful! * * *"Katrina.Dlugosch" dlugosch@biology.ucsc.edu* * * * * Dear Sara, we have used a 96 well set up in our lab, but it is not a kit and we still did the digests in 1.5 ml tubes. So I'm not sure wether this will be helpful to you. The advantage is that it is very cheap. The reference is Elphinstone et al. 2003 in Molecular Ecology Notes. I have attached the pdf. Also we used a simplified protocol at some points (e.g. no acid step when preparing the silica slurry). good luck, Ellen *Ellen Schluens **ellen.schluens@jcu.edu.au*** * * * * Hello, Dr. Carvalho, Our lab uses Qiagen's DNEasy-96 well procedure to extract DNA from a variety of plant species (conifers and angiosperms) and tissues (seedlings, leaves, needles). We use this method nearly exclusively, although we find it is best to have another small-scale procedure available for difficult tissues, etc. We have been generally pleased with the kit. It requires an investment in equipment (mixer mill to disrupt tissue, Matrix electric pipet, etc.), but it makes the extraction more time-efficient and consistent. Our yields are lower than with other methods, but more than sufficient for PCR-based applications (typically more than 30 ng/uL, sometimes as low as 10 ng/uL, depending on tissue type and quality). We have found that the amount of tissue used is critical - too much will clog the column membrane and result in little to no yield. We have also been able to easily adapt the procedure for tissues containing high levels of secondary compounds by adding 2-mercaptoethanol or proteinase K. Hope this helps! Cheers, Jenn Jennifer DeWoody, Biologist USDA Forest Service, NFGEL 2480 Carson Road, Placerville, CA 95667 530-295-3028 (voice), 530-622-2633 (fax) http://www.fs.fed.us/psw/programs/nfgel/ Sara Carvalho