Dear all, Thank you very much to everyone that replied to my questions about tissue preservation in DMSO. Replying to Si: we have been able to extract high molecular weight DNA from samples preserved in 20% DMSO for 8 years but some other samples gave very bad extractions and they were 6 years old. It is possible that the ratio of tissue to solution is an important factor in our case. Below are all the answers I got. Thanks again to everyone. Sofia I personally am not a fan of storing tissue in 20% DMSO as I've also had problems with extractions. Perhaps short-term storage is ok, particularly as there may be issues with samples in ethanol in the post. If I receive samples in DMSO, I rinse them with dH20 and change them into 70% or 80% ethanol. I have had no trouble with long-term storage in ethanol, even when stored at room temperature. Regards Maria In our lab, we have always stored tail tips in 100% EtOh, cutting the tissue into smaller pieces, ratio of tissue to EtOH, c. 10x Such material has been fine for 10 years plus. I have always assumed that the EtOH removes the DNA from aqueous solution, hence stopping aqueous enzymatic processes. Re: your issue, you may want to transfer the tissues into EtOH. I'm no expert on the 20% DMSO, salt issue, but the DMSO is just there to deliver the very high salt concentration into the cells of the tissue to prevent DNAases etc.. working to degrade the DNA. Just out of interest, how long have your workable samples (eg obtaining high molecular weight DNA out of them) been in the 20% DMSO solution - I've only just started working with it and would like to predict life spans of my samples. With best wishes and thanks Si Creer -- Si Creer Post Doctoral Research Fellow Molecular Ecology and Fisheries Genetics Group School of Biological Sciences University Wales, Bangor Bangor Gwynedd LL57 2UW UK e-mail: s.creer@bangor.ac.uk Tel: +1248 382302 Fax: +1248 371644 Home Page: http://biology.bangor.ac.uk/~bssa0d/ If it helps, Dawson /et al/. (1998) examined the effects of five storage solutions and three temperature regimes on marine invertebrate tissue and found that the greater the duration of storage prior to analysis, the greater the degradation of the sample, and greater degradation occurs at higher storage temperature (room temperature). The Preservation of DNA was most successful with dimethylsulfoxide and sodium chloride (DMSO-NaCl) and 70% ethanol. Dawson, M.N., Raskoff, K.A., and Jacobs, D.K. (1998) /Field preservation of marine invertebrate tissue for DNA analyses/. Molecular Marine Biology and Biotechnology. *7(2)*:145-152 Best wishes Tom We have collected cetacean and turtle tissue in salt-saturated 20% DMSO for up to 15 years. We also find that some of our older samples have degraded and we get no or little DNA out of them. We still collect some samples in DMSO, but we freeze them all now rather than storing them at room temperature. We try to keep the ratio of DMSO to sample greater than 5:1. Whenever possible we collect samples in 90% ethanol now (also stored frozen), or store samples just frozen, and changed after initial collection because it can become diluted by the water in the sample. Changing the DMSO is also a good idea, as it can become acidified. I don't know how to 'rescue' your older samples, but would recommend that you do extractions on all of them as soon as possible to obtain DNA before it becomes further degraded. Phil -- Phillip A. Morin, Ph.D. Southwest Fisheries Science Center 8604 La Jolla Shores Drive La Jolla, CA 92037, USA Phone: 858-546-7165 Fax: 858-546-7003 phillip.morin@noaa.gov http://swfsc.noaa.gov/prd-popid.aspx We at STRI use 0.25 M EDTA instead of pure water to mix up the 20% + NaCl. We add NaCl to saturation and then some more. Some people keep their samples in 4C but I keep mine in -20C or -80C if I am not using them anytime soon. I have transfered from EtOH to DMSO but never the other way around. Should be OK, if you want to do it... Let me know if you receive any other "hot tips" from EvolDir, eh? Obrigado! -Andrew Andrew J. Crawford, PhD Naos Molecular Labs Smithsonian Tropical Research Institute Republic of Panama Email: andrew@dna.ac web: http://dna.ac I am starting to use DMSO solution to preserve tissues for DNA work myself. I can therefore only recommend to use the recipe of the attached paper which seem to work nicely (NaCl saturated solution) for nematodes. I am now starting to use it for arthropods. I had no success by following the protocole given in the paper (The EDTA precipitating when I added NaCl). But it worked when I heated a bit 60�C while mixing in the NaCl. I would be very interested by any feedback you get by Evoldir members. Regards alex sgseabra@fc.ul.pt