Thank you all for your answers to our question about DNA extraction of barnacles. Several methods were mentioned that could be tried. The Qiagen Dneasy tissue kit (often a very option) didn't work in our case, we had tried that already. Purgen kits (Gentra) was also suggested by several, something I want to try. Also suggested were plant CTAB methods, which we had used and didn't work in our case (with the whole animal) Using only the cirri ("feeding arms") seemed a good suggestion, avoiding problems with nasty enzymes in their intestines. So what did we do? We used only the cirri and our CTAB method (includes chloroform extractions), which gave a small amount but good DNA which appears to be much more stable than using the whole animal!! So using a part of the body, excluding intestines etc. is much better. Secondly we tried two kits (free samples!) from Microzone Ltd. The 'DYNAMITE for difficult cells' was used with a whole animal and gave loads of DNA that also appears stable. Further we used cirri with their MICROLYSIS PLUS kit, which gave a small amount, but seemingly stable DNA. The MICROLYSIS plus kit is the quickest and easiest, but gives a limited amount of DNA (good for PCRs), whereas Dynamite needs a few more steps (still very quick and easy), but gives more DNA to play with. MICROLYSIS PLUS is not hugely expensive, something like 60 pence a sample and VERY easy and quick. The DYNAMITE for difficult cells (or DYNAMITE for mouth swaps) is still in its trial phase, so we were grateful for the free sample of Microzone. This product will need some further testing, but is likely to be available after Xmas. Check their website www.microzone.co.uk (no I don't have shares). We are pleased to say that we have now passed the extraction phase, finally, and working on constructing a library. Thanks again for your suggestions. Best wishes, Kirsten Wolff List of some of the responses: do barnacles have a hepatopancreas? This organ is famed for causing problem in snails due to the number of enzymes in it (mostly anecdotal, admittedly). If so, then dissect out or use foot alone? In any case, if it is still degrading then it must be due to some kind of DNAse carry over. Cheerio, Angus We have had terrific success in extracting cirri of Balanus glandula with a 10% chelex solution (see Sotka, E.E., J.P. Wares, R.K. Grosberg and S.R. Palumbi. (2004) Strong genetic clines and geographic variation in gene flow in the rocky intertidal barnacle Balanus glandula. Molecular Ecology 13:2143-2156), as well as a Nucleospin kit. Yes, I dissect two or three pairs of cirri and place into 150 ul of 10% chelex. This represents about 1mm sq. worth of tissue. Erik Yours is a familiar tale... I have run into molluscs that do what your barnicle is doing. Have you tried crushing the tissue in liquid nitrogen and then straight into CTAB? Can you take only muscle tissue rather than the whole animal? The only other thing I can suggest is something you are undoubtedly already doing, which is to not be greedy when collecting the aqueous phase in your extractions to minimize any contaminants. Probably none of this will help -- no doubt there is an evil humour co-purifying with the DNA! Richard Which column based extractions have you used? I had similar problems when extracting DNA from mygalomorph spiders (DNA broke down after 24 hours...sometimes less) and I found that the QIAGEN kits worked nicely. If a CTAB protocol is working (albeit briefly) you could try grinding the tissue sample in liquid nitrogen (as for a standard plant extraction) - there's a paper that describes similar problems for ladybird extractions and that was their solution (Schulenburg et al in Mol. Biol. Evol. 18(4): 648-660. 2001). Otherwise there's the option of washing the sample with bleach then dd H20. In general I also found that using smaller starting quantities of tissue and ensuring that I was only using internal muscle tissue helped a great deal. All the best though... it's a nasty problem! Amber I was just sent this link by someone, and thought of you and Martha. I haven't tried it, but yet another potential source if Martha is still having lots of trouble getting usable DNA. http://www.omegabiotek.com/productsrange/genomicdnaisolation/genomicprod uctlistmain.html Reia I just saw your post on Evoldir concerning you DNA extraction problems I have not idea why the columns dont work but you could try our new DNA extraction protocol, which combined CTAB and silica purification. It was develloped for brown algae which are especialy difficult to extract but it should work on any "difficult species" I hope it will help All the best Dr. Galice Hoarau Soon to appear in Molec Ecol Notes: TECHNICAL NOTE A fast and inexpensive DNA extraction/purification protocol for brown macroalgae GALICE HOARAU, JAMES A. COYER, WYTZE T. STAM and JEANINE L. OLSEN Don't know what you are doing with A. amphitrite, ecological, systematic or what, but hope you and/or your colleagues can solved the DNA extraction business. This group of Amphibalanus (A. amphitrite, improvisus, subalbidus and eburneus) all tend to be in brackish waters and subalbidus gets closest to freshwater than any other barnacle. Could this have something to do with it? Best wishes, Bill Kirsten.Wolff@newcastle.ac.uk