In reply to the query: ----------------------------- Does anyone use buffered 70% - 90% ethanol for the preservation of specimens for DNA-based work and, if so, with what recipe, rationale and results? ----------------------------- I received several replies (not listed) from readers who thought that buffering ethanol would be a sensible and logical procedure but who had no actual experience to contribute. There were two replies from people who do buffer their ethanol, as follows: 1. From: "Joe Staton" We used to add about 0.1 ml of 1/10x TE (pH 8.0) to help with this. I think Tim Collins taught me this. As I understand it, many inverts release acids when preserved which leads to DNA destruction. Others I know change out the EtOH after 3 days to help with the same problem. and 2. From: "Blair Hedges" I usually buffer my 70-80% ethanol for specimen/DNA preservation (of reptiles and amphibians) with TE (Tris EDTA) pH 8.0. I buy 100X concentrate from Sigma and bring the concentration up to 1X TE, although for large volumes (liters) it may be a weaker TE concentration. My reason for doing so is not so much for pH but simply as a proven DNA buffer. Also, I use 151 proof rum (75% ethanol) instead of laboratory grade ethanol because the latter sometimes includes a touch of methanol which could harm DNA. I work in the West Indies so the rum is easy to get. --------------------------- My conclusion is that buffering the ethanol is a sensible precaution that may be particularly valuable for calcite-shelled animals and I intend in future to dilute ethanol with 1 x TE rather than water. To Blair Hedges' reasons for using rum I will mention my experience of methanol-denatured ethanol. This is the standard shipboard fixative for the brachiopods I receive from the IRD/ORSTOM Pacific sampling campaigns, and brachiopods preserved in this have yielded amplifiable DNA after a decade or so at -20. The only difficulty so far has been with some crinoids that contain quinone pigments. These (I assume) converted the 10% methanol to formaldehyde, the vapour of which caught me unawares when repacking the specimens in an enclosed, hot environment.. Thank you all. BLC Dr B. L. Cohen Molecular Genetics University of Glasgow, Pontecorvo Building, 56 Dumbarton Rd Glasgow G11 6NU Scotland, UK. Phone: (+44) (0)141 330 5103 (direct line) 330 6219 (secretary) 339 8855 (switchboard) Fax: 330 4878 http://www.gla.ac.uk/ibls/molgen/staff/cohen-bl.html "B.L.Cohen"