2.5 ml 1M Tris, pH 8
10.0 ml 0.5M EDTA
5.0 ml 10% SDS
31.5 ml ddI H20
The final volume is 50 ml. To the amount needed for the number of tails at hand, add 35 µl of a 10.0 mg/ml solution of Proteinase K (if you have trouble with excess protein, more Proteinase K may be added at this stage). RNAse may also be added, but generally we have not experienced problems with RNA in these preps. You might also add the RNAse later, during the restriction digestion.
Add 0.5 ml to each tube, then incubate overnight at 55C in a water bath. The tail should be nicely dissolved so that only hairs and occasional pieces of cartilage and bone are present. The solution may also look a bit dirty due to the dissolved pigments in the tail hair and skin.
Spin the tubes for 10 minutes in an Eppindorf centrifuge (about 12000 x G), then decant the supernate into new 1.5 ml tubes. During this transfer, it is very important to keep the pellet of hair and debris out of the transfer tube. Sometimes, addtional protein and other materials will be present at the top of the pellet, and you must take care when decanting to not transfer this material. Remember that you will end up with plenty of DNA for the Southerns, so it is advised to leave a little with the pellet for the sake of keeping the transferred solution clean. Add 0.5 ml of room-temp isopropanol to each tube (the salts in the tail-buffer are sufficient, but you may wish to add a 1/10 dilution of 5M NaCl or 3M NaOAc to help in the precipitation of the DNA), and invert the tubes a few times to produce cottony precipitates. It is a good idea to not let the precipitates get too tightly packed, because it will be difficult to return them to aqueous solution if this is the case. Now, transfer the precipitate with a flamed glass Pasteur pipette to a tube filled with 70% EtOH. Next, remove as much EtOH from the precipitate as possible by rubbing the DNA along a dry side of the tube or the rim. Now, the precipitate is transferred to an Eppindorf with 0.2 ml of TE buffer, and the precipitate should release from the pipette tip and go into solution with gentle agitation. If you have trouble getting the DNA back into solution, you can place the tubes in the 55C bath for about 30 minutes, and agitate them a bit until they go into solution.
Since this prep is not clean and there will be extra protein in the DNA, you may see some carryover of the pigment, which will make the DNA precipitate and subsequent solution look a little dirty. This is generally ok, but in the future, you may wish to spin the tubes a bit longer, or use more Proteinase K during the tail digestion. From an average tail piece of about 1.0 cm, we usually get about 200 µg or so, thus for restriction digestion, we usually use 10 µl.
Add 10 µl from each tube to wells in a round-bottom 96 well microtiter plate. Next, prepare a digestion buffer so that the total volume to be digested is around 30 µl. We usually make the digestion buffer up in bulk and then add it to the wells with a multipipettor. Both EcoRV and PvuII may be used, but we generally like to use EcoRV for our analyses. We add 50U of EcoRV per well to the bulk digestion buffer, along with the vendor's recommended digestion buffer concentrate and water. It may seem a bit expensive, but we really do recommend at least 5U/µg DNA. Skimping on restriction enzyme is a good way to get uncut DNA and a big waste of time. After the digestion buffer has been added to each well, add the plate cover and seal the sides with parafilm, and place it in a tissue-culture incubator for digestion overnight. Following digestion, add sample buffer (we use 10 µl of 20% glycerol with bromophenol blue in TE) and add to wells of a 0.9% agarose gel, and continue with standard southern techniques.
We label 50 ng of our probe (the EcoRI fragment) with Amersham's Megaprime random-nonamer kit and clean it up on little Sephadex G50 spin columns, after which we add it to the blots in sealed bags, and let the blots hybridize overnight at 65C. We also like to help things out a bit by making up our hyb buffer with 10% dextran sulfate. Our hyb buffer uses Denhardt's, and we do prehybs and hybs with Salmon sperm DNA to block.
Craig Walsh, Ph.D.