Hello all. I would like to thank all the members of the list who took the time to respond. I had quite a few protocols to test, and I think I have found one that works really well for my fish. A slightly modified protocol based on what Sarah Helyar (thank you so much, and yes we did meet in Seattle) sent me worked really well. CTAB, which was also another common suggestion, didn't work so well. Upon adding isopropanol, I get a viscous liquid that comes out of solution, and no DNA at the end of the elution step. Depending on who you talk to they say this viscous substance is either SDS or polysaccharides. In any case, with a little RNAse, I was able to obtain about 15-20µg of DNA from 15-20mg of muscle with A260/A280 of around 1.8-2. Running a gel showed high molecular weight with little degradation. So, I was really pleased, as before I was not able to get any DNA at all. Thanks again everyone, below I have posted all responses, which will hopefully be a resource to others. All the best. Anders. -- If I hear "slimy" or "oily" I have to think of this procedure: Bahl, A., Pfenninger, M. A rapid method of DNA isolation using laundry detergent (1996) Nucleic Acids Research, 24 (8), pp. 1587-1588. Cited 30 times. http://www.scopus.com/inward/record.url?eid=3D2-s2.0-0029923281&partnerID=40&md5=60a69b5b3d97efc5de234209d7c40d04 Might be worth a try. The 2nd author is still active and might be responsive upon direct contact. -- Have used a mixture of techniques with fish, from a high salt to using some other mystic witchcraft broth! I should add, all these techniques seemed to work on blood, muscle tissue, and fin samples...if I was to choose one for muscle, perhaps the high salt one... -- Maybe you could try the CTAB 2X protocol. It is a very efficient one and works very good on a wide range of organisms although initially used for plants. Landsnails that are covered with mucus are working fine with the CTAB protocol so maybe the oily substances of the fishes will be dealt with too. A google search with CTAB 2x will provide several protocols and you could use the one that better suits your needs. I wish you good luck. -- I did extract DNA from frozen muscle with phenol cloroform protocol with excellent results, I also used QIAGEN DNeasy tissue kit and no problem. -- we normally use the hexadecyltrimethylammonium bromide (CTAB) protocol for genomic DNA extraction from fish muscle preserved in ethanol. See attached an example of the protocol. It works also on very degraded samples, I used it also for total DNA extraction from fish gut content, following Deagle BE, Jarman SN, Pemberton D, Gales NJ (2005) Genetic screening for prey in the gut contents from a giant squid (Architeuthis sp.). Journal of Heredity 96: 417-423 -- Have you tried Chelex? I just started working on fish about a year ago, and the lab I am working in uses Chelex for fish muscle and scales--and it seems to work great. It's cheap and requires very little tissue and takes much less time than the Quiagen protocol. -- am not sure that its the oil that's causing the problem if the liver was extracting ok, but anyhow, I generally use a very basic high salt protocol, so no phenol/chloroform steps, the yield from this is usually pretty high, it tends not to be the cleanest DNA, but for general applications like msats and SNPs, theres been no problem. We currently working on herring - so they're pretty oily too. -- I used to work with muscle samples of Yellowfin Tuna. We used a regular precipitation with isopropanol and ethanol (a normal High salt protocol). I don't have the protocol just in hand but I think that you can find it everywhere. Consists of incubation with PK and Buffer, two precipation with isopropanol, and one with ethanol... and finally elution with water or TE... -- You might consider rehydrating the samples by rinsing, then soaking them in water for ~15 minutes prior to using the kit. Sometimes this helps, and it is easy enough to try. -- I would try one of the older chloroform or chloroform/phenol methods. Those reagents should get rid of extra lipids early on in the extraction process. Also, you might want to try increasing the detergent concentration a bit in the initial homogenates. -- We do tons of fish here at the Smithsonian - muscle, eyes, fins, anything really. We basically have a DNA barcoding factory and having been going nuts with Caribbean fish. Not sure what's going on with your extractions, but thought I'd share a few things that we've learned. You may already be doing all this. -It is important to dry off as much of the ethanol from the sample as possible - dry it on a chem-wipe and maybe even let it air dry a bit. Or it sounds like you're already rinsing it - we sometimes do that with the extraction solution - sort of dunk it. But air drying works well. -Digest overnight at least - the SDS in the digestion solution should take care of the oil - you can add more proteinase k which might help with the yield, or add some more half-way through the digestion. It is hard to have too much protk, you can safely add 3-4X the usual amount. -We generally use a phenol-chloroform technique (we have an Autogen robot that does this), but have certainly done plenty of Qiagen extracts. And we generally preserve tissue in a salt buffer rather than ethanol. It keeps the tissues soft and definitely preserves very well. And now that there are restrictions on shipping ethanol (at least in the States where the gov't does as many stupid things as possible), the salt buffer passes under the radar. -But we extract DNA successfully from preserved in alcohol all the time (most phyla for example). -We also never quantify DNA! -So your fish samples don't amplify at all? What species are you working on? And, if you'd be interested, we have some primers for CO1 that seem to work on every fish we've come across. But, perhaps you are not doing PCR anyway. -- Have you tried 'smashing' the muscle? what we do is cut it in the smallest pieces we can, smashed it a little, and use also very little of the tissue to do the extraction ... we always get nbetter results with very little amount of tissue than with more. -- I worked on fish (marlin) too and at times had similar problems. If the muscle tissue is particularly oily then there may not be much you can do. I had some muscle tissue samples that contained a lot of orange-ish colored oil and I was never able to get DNA from those samples. But you could try squeezing a piece of the tissue between Kimwipes to try to absorb some of the oil. However, for the other muscle tissue samples, I would extract them overnight in a CTAB solution with a good amount of Prot. K in a water bath at 37C. I would basically wait until the tissue dissolved, vortexing every hour or so while I was in the lab. Sometimes this would take longer than 1 night. Then I would follow through with the PCI extractions. A long extraction like that might be worth a shot. Good luck and let me know if there's any way I can help! -- We also use the Qiagen DNeasy extraction kits on fish tissue, usually fin instead of muscle, though. One thing we have found with ethanol- preserved tissues is that if the ethanol is not allowed to completely dry before starting the lysis step, the digestion does not work well and hence the yield from the extraction is low. I think it has something to do with the lysis buffer also having ethanol in it, which throws off the ratio if the tissue also contains some ethanol. We usually let such tissue samples evaporate in a hood before starting the digestion. You could also try adding more proteinase K to the digestion buffer; we've found that this helps some stubborn samples break down more fully. -- Hi Anders, I used to work with fish and we extracted hundreds of DNA samples without any problem. The protocol we used is from: Taggart, J.B., Hynes, R.A., Prodohl, P.A. and Fergusson, A. (1992). A simplified protocol for routine total DNA isolation from salmonid fishes. Journal of Fish Biology 40: 963-965. I also used this protocol with modifications where necessary to extract DNA from other organisms's tissues. It works really fine. -- Though I am not expert i animal tissue DNA isolation, I could suggest that, prior crushing make the sample alcohol free, probably by keeping in water and After homogenization in Liquid N2, try to vortex the sample with oil extracting substances like Chloroform or any organic solvent for 5 to 10 minutes. these two steps help you to get rid of alcohol and oil. Then by completely removing the organic solvent (keeping at room temp ot 40-50 C in oven) you subject the sample to DNA extraction buffer. -- Did you try salting it out? Maybe tweaking a deparafinization protocol? http://dx.doi.org/10.1016/j.prp.2008.04.005 http://dx.doi.org/10.1016/j.ab.2009.08.016 --