Dear all A few days ago I posted a request concerning efficient DNA extractions in plates. Below you find my original e-mail: > I like to do DNA extractions from single Daphnia using the chelex > method for economical reasons. As I have to proceed a high quantity of > samples, I would like to do the extractions directly in > 96-well-plates. Does anyone have any experiance if this works and do > you have found a good systems for grinding the animals directly in the > plate? Please find below a selection of the answers I received. Many thanks for all your help and the useful information! Best regards, Barbara Barbara Walser University of Fribourg E-mail: barbara.walser@unifr.ch From: Ben Longdon (b.longdon@ed.ac.uk) We do Drosophila chelex extractions using heavy duty 96 well plates with deep wells, putting ball bearings in the wells, then shaking the flies on a homemade device which consists of a sheet sander, which we've adapted to strap the plates to. Can then transfer the DNA to a standard 96 well plate for ease of use. From: Brant Faircloth (brant@ucla.edu) you may wish to look into the use of steel beads (1-2 per well; BioSpec Products, Inc. SKU 11079132c; you may prefer stainless beads, however) along with a paint-shaker or reciprocating saw to do your tissue disruptions (i can send PDFs if you don't have access): 1) reciprocating saw methodology - http://www3.interscience.wiley.com/journal/118523651/abstract 2) paint shaker - see http://www.springerlink.com/content/d70250873105gq05/ once the tissues are disrupted, you can chelex normally. The stainless beads are less likely to introduce nasties to your samples, but they are more expensive and non-disposable (treat with 10% bleach and a few ultra-pure water washes prior to re-use). if you wish to separate the solution from the chelex (it is hard to remove supernatant from chelex extracted samples in plates and chelex can cause problems with some tips/needles in liquid handlers), you can filter it out using Millipore plates (MAHVN4510) or similar suspended over inexpensive skirted plates + centrifugation at 900 G for 5 m. Use filtrate from the plates normally in downstream applications (PCR, etc.). From: Lena Bayer-Wilfert [lb445@cam.ac.uk] for single Drosophila flies, we grind the flies in a deep 96 well plate with the help of ball bearings and a home-made contraption using a sander and some bits of wood. Not sure whether that would grind single Daphnia well enough. I guess that you'll get some more useful feedback from the Daphnia community, but if not let me know and I can try to get you a better description of what we do. Tom Little [tlittle1@staffmail.ed.ac.uk] We use the CTAB method, which is very reliable. Chelex used to cause some trouble (I think especially if the samples were to be stored, probably Chelex is fine if you use the DNA right away). John Colbourne at Indiana would be a good source of update information. From: Africa Gomez [A.Gomez@hull.ac.uk] May I suggest that you consider an alternative method to Chelex which we have shown to work on Daphnia and allows for easy high-throughput (and can be carried out in 96-well plates)? Paper freely available at: http://www.aslo.org/lomethods/free/2008/0218.pdf From: Michael Monaghan [monaghan@igb-berlin.de] I have used 96-well plates extensively for DNA extraction, mostly with insects but also with Daphnia. The animals can be placed in lysis buffer + protK in a 96-deep-well plate overnight in a water bath. there is no need to grind them. i use the deep well plate available from VWR (cat. nr. 732-0585) and cover it with plastic film or a PCR plate cover to minimize evaporation. From: Joaquin Munoz [quini@ebd.csic.es] I have no experience making DNA extraction with Chelex in 96-well plates, but I suggest to read our recent paper describing a new cheaper method which use HotSHOT protocol in resting eggs and pieces of adults from Daphnia, Artemia, Rotifers.... From: natassa [natassa_g_2000@yahoo.com] I used Chelex a long time ago for the same economic reasons, and I just wanted to say that I am not convinced it is worth it. I was screening a lot of transformants to find mutants, and I realized a long time later that I had far more than i estimated (due to poor DNA quality). It all depends on your goal... I used heat shock for lysing cels, but well, it is far easier in the fungus I was working on rather than in Daphnia I suppose. From: Alice Dennis [alicebdennis@gmail.com] I use Chelex for extractions in the snail's I'm working on, but I use 8-well strip tubes because they have a cap that snaps tighter than anything I've found for a 96-well plate. This makes vortexing and storing the tubes easier. The strip tubes can also be labeled down the side, which helps me keep track of the individual extraction numbers. I haven't tried this with animals as small as daphnia, but if you used small volumes, I don't see why it won't work. The only step I've added to a standard protocol is that I leave my samples out at room temperature overnight before I boil and PCR them. From: Jeremy De Waard (jeremy.dewaard@gmail.com) We used chelex in my previous lab for a variety of tissue types and in 96-well format. It worked quite well, including for Daphnia, although the DNA degraded rapidly relative to other methods. We found that grinding did not provide a significant increase in yield (as measured by spec or subsequent PCR success). The protocol we used is given in the attached book chapter from last year. From: John Robinson [jdrobins@uga.edu] I'm a graduate student at the University of Georgia working on Daphnia magna, and I've had some success isolating DNA using a chelex protocol. My isolations have been done so far in small batches, but I plan to expand it to larger batches. Currently I've been using 100 microliters of a 7% chelex solution for my isolations. I've had some success sequencing mtDNA and genotyping microsatellites from these isolations. I haven't been grinding the Daphnia at all in the individual tubes. Here's the protocol I've used so far. 100 uL 7% chelex into individual wells of a strip tube (hopefully full 96-well plates in the future) + single Daphnia I vortex this mix for 30 seconds then boil the mix in a thermal cycler at 99-100 degrees Celsius for 10 minutes vortex again for 30 seconds another 10 minute boil at 99-100 degrees then centrifuge the plate @ max rpm for 3 minutes WALSER Barbara