ANSWERS USEFUL PAPERS LISTED AT THE END (1) Here is a paper about turkey DNA, some of the samples used were toe clippings. you should email Dr. Camilla Speller for more questions, she did the research. I am sure she can help you with this. her email is: (2) We extracted DNA from foot pad tissue by using a traditional Proteinase K digest, followed by a PureGene protocol, but any other protocol for ProtK and tissue sampels should work well. Before the procedure starts you should let the tissue soak for 1-2 days in the ProtK buffer (ProtK added). Then do a few cycles of freezing +thawing the tissue with the buffer in liquid nitrogen to break cell walls (say, 10 times or so). Then probably a few more days with ProtK and the tissues will dissolve (add ProtK newly after every night or so.). We will soon submit a manuscript in which that method is used, please let us know if you want to try it out. Robert H. S. KRAUS PhD student Resource Ecology Group Wageningen University, The Netherlands Email robert.kraus@wur.nl (3) Have a look at the papers by Giri Athrey; he did the same kind of work very recently. His email: giri.athrey@tamu.edu Eric Pante Department of Biology University of Louisiana at Lafayette P.O. Box 42451, Lafayette, LA 70504 Lab: (337) 482-6494 Web: www.ull.edu/~egp0628 (4) I haven't worked on birds, but I have extracted DNA from equivalent age mouse specimens, using the Qiagen Blood and Tissue DNA kits. Iused pretty much the standard protocol, leaving the tissue to lyse overnight in the proteinase K (rather than the minimum 3 hrs) and, to get more concentrated DNA at the end, eluted the sample with half the amount of elution buffer. Obviously, contamination is potentially a huge problem to be aware of. Good luck, Eleanor eleanor.jones@ebc.uu.se (5) I haven't used the kit you are asking about, but I have had success with the Chargeswitch Forensic DNA Putification Kit from Invitrogen and with the DNeasy Tissue kit from Qiagen (see attached paper). Incubations with prot K were overnight. Note that I rehydrated the samples first, this makes it a lot easier to cut them up into very small pieces. Sanne Boessenkool Postdoctoral Researcher NCB - National Centre for Biosystematics Natural History Museum University of Oslo P.O. Box 1172 Blindern NO-0318 Oslo, Norway Visiting address: Sars' gate 1, Tøyen Phone: +47 22851777 www.nhm.uio.no/forskning-samlinger/forskning/forskningsgrupper/ncb/ (6) What kind of markers do you intend to use? We have used similar samples for SNPs and the Puregene DNA isolation kit worked fine, although I advise you to test on the fragment length and amount of DNA always and assume that half of your samples may have been degraded too much. Rudy Jonker M.Sc. Ph.D. Candidate Resource Ecology Group Wageningen University e: rudy.jonker@wur.nl i: www.reg.wur.nl/UK/Staff/Jonker t: +31(0)317-485304 Skype: rudyjonker (7) I have ventured on a similar thing a couple years ago (see attached paper). We used the Quiagen Kit (I think you should be able to find my query in the archives of evoldir, from somewhen between summer 2007 and summer 2008). DNA extraction was no problem, contamination was one. Archive DNA usually comes in small quantities, and is easily contaminated. To ensure that your results are really from your samples, you might want to make use of a lab that is specialised on ancient and archive DNA processing. We ended up having the samples run in Allan Bakers Lab (Toronto), but the facility here in Sheffield can also do such things. Julia Schroeder Research Associate Dept of Animal and Plant Sciences University of Sheffield Sheffield, S10 2TN Tel: +44 (0)114 222 0112 http://julia-schroeder.staff.shef.ac.uk/JSchroederSite/ Research_interests.html julia.schroeder@gmail.com (8) I used the Qiagen DNeasy spin columns purification kit for extracting DNA from bird foot pads 40 - 120 years of age. Sometimes it worked quite well, sometimes worse, depending on the museum where I got the samples from. The amount of DNA was always low, the concentration was adjustable by using less of elution buffer down to a given minimal amount. We used very small amplicons for sequencing (150 - 300 bp). This worked quite well in most of the samples given that they came from a museum collection where it worked well at all. The reason for why my professor wanted me to use a kit instead of a manual maxiprep was that he wanted to have very standardised and well comparable results. Severin Uebbing Dept. Evolutionary Biology Evolutionary Biology Centre (EBC) Uppsala University Norbyvägen 18D SE-752 36 Uppsala Sweden (9) I attached two papers with the protocol for DNA extraction, microsatellite and mtDNA analysis, which I used for feathers of museum specimens about the same age as yours. Should be fine for toe pads as well. Kristina Sefc Dept. of Zoology, Karl Franzens University of Graz Universitätsplatz 2, 8010 Graz, Austria Tel: +43-(0)316-3805601 email: kristina.sefc@uni-graz.at (10) I use a Qiagen DNeasy kit and the protocol in the attached paper. For me it works really well with museum specimens (some of which are 150 years old). Good luck claireraisin@hotmail.com (11) We used the Promega kits for a project on 10 ~100 year-old Ivory Gulls, and they worked fine. Major thing to watch out for is if the specimen has been dusted with arsenic power, which of course inhibits any enzymatic reaction. I remember we washed the tissues first in 100% EtOH in a tube, then rinsed them with water before extraction. Dr Steven M Carr, Professor of Biology [& Professor of Genetics, Faculty of Medicine] Department of Biology Memorial University of Newfoundland St John’s NL A1B3X9 CANADA e-mail: scarr@mun.ca http://www.mun.ca/biology/scarr/Research.html (12) I used a modified version of a Qiagen DNeasy kit for mouse skins that had been dried with butane and gasoline over 80 years ago. I think I gave my protocol to someone who worked with birds and they were also successful. I can send it to you if you are interested. Lynne Mullen, Ph.D. Preceptor for Life Sciences Department of Molecular and Cellular Biology Harvard University 1 Oxford Street Cambridge, MA 02138 lmullen@oeb.harvard.edu (13) We have not used that kit , but we had some success with DNA extractions of museum birds (raptors: bearded vulture and imperial eagles) using standard phenol-chloroform methods or Qiagen DNaseay Tissue kits. You may want to consider the blood clot in the umbiliculus of the feathers as an alternative source of DNA to footpads as described in the attached paper. godoy@ebd.csic.es (14) In the past I've used DNEasy kits from Qiagen for extracting DNA from peregrine falcon toe pads. Used for mtDNA and microsatellites. Remarkable success rate; very few did not extract, and those that failed appeared to have their legs/feet treated with some substance (arsenic?). Not sure which taxon you are working with. Peregrines are large birds, so I was able to start with a decent amount of tissue. josephwb@umich.edu (15) I have extracted DNA from cockatoo museum skins (feathers and toe pads) around the same age. I would suggest, feathers are better to work with as they yield a cleaner DNA extract (less inhibition). I followed a method on www.aviangenetics.com under the 'DNA extraction Protocols' using a Dneasy Extraction Kit (Qiagen) with great results. Nicole White (PhD candidate) BSc, Hons. (Cons Biol) Black-cockatoo Project Wildlife Genetics Lab, School of Biological Sciences & Biotechnology, Murdoch University, South Street, Perth, Western Australia, 6150 Email: n.white@murdoch.edu.au or nwhite72@iinet.net.au Phone: office +61 (08) 9360-2312 Lab +61 (08) 9360-2787 Visit our website: www.wildlifeforensics.com.au (16) I don't have much experience, but we used Qiagen DNeasy tissue extraction kits on relatively recent parakeet museum toepads (dating only back to the 1950s), combining the manufacturer's protocol with recommendations in Nisiguchi et al 2002 (below), and that worked well. Nishiguchi, M. K., P. Doukakis, M. Egan, D. Kizirian, A. Phillips, L. Prendini, H. C. Rosenbaum, E. Torres, Y. Wyner, R. DeSalle y G. Giribet. 2002. DNA isolation procedures. Pp. 249-287. En: R. DeSalle, G. Giribet y W. C. Wheeler (eds.). Methods and Tools in Biosciences and Medicine. Techniques in molecular evolution and systematics. Birkhäuser Verlag Basel, Suiza. USEFUL PAPERS: www.pnas.org/cgi/doi/10.1073/pnas.0909724107 Proc. R. Soc. B (2009) 276, 815–821doi:10.1098/rspb.2008.1246 Ibis (2010), doi: 10.1111/j.1474-919x.2009.01005.x Conserv Genet (2007) 8:879–884 DOI 10.1007/s10592-006-9240-8 The Auk 120(4):982–989, 2003 Conserv Genet (2007) 8:695–703 DOI 10.1007/s10592-006-9217-7 JOURNAL OF AVIAN BIOLOGY 36: 84 /88, 2005 Molecular Ecology (2007) 16 327–343 www.pnas.org cgi doi 10.1073 pnas.0503396102 Filippo Barbanera Researcher Department of Biology Protistology-Zoology Unit Via A. Volta, 6 I - 56126 Pisa (PI) Italy Web site: www.biologia.unipi.it Skype: barba_skype room: + 39 050 2211386 lab. : + 39 050 2211343 fax: + 39 050 2211393 Filippo Barbanera