The following DNA extraction protocols are the web appendix for Bush, K.L., Vinsky, M.D., Aldridge, C.L., and Paszkowski, C.A. 2005. A comparison of sample types varying in invasiveness for use in DNA sex determination in an endangered population of Greater Sage-Grouse (Centrocercus urophasianus). Conservation Genetics, 6: 867-870.
Qiagen DNeasy® Tissue Kit - Animal Tissue Protocol Modification
(Qiagen (2003) DNeasy® Tissue Handbook. Protocol for isolation of total DNA from animal tissues, pp. 18-20. QIAGEN (Valencia, California, U.S.A.).
Protocol for Fresh and Autolytic Tissue
(1) Cut up 25mg of tissue into small pieces (~1mm2) and place the tissue into a 1.5ml labeled microcentrifuge tube. For autolytic tissue, adult toes and liver work well, as do embryonic feet. Fresh heart, liver, muscle, foot, and tongue were all tried successfully with heart and liver producing DNA with the highest concentration.
(2) Add 200µl of Buffer ATL to each 1.5ml microcentrifuge tube containing the tissue.
(3) Add 20µl of Proteinase K and vortex for 5-20 seconds. Incubate the samples at 50˚C overnight (12-24 hours). Autolytic tissue gives best results if incubated for 24-48 hours.
(4) Add 200µl of Buffer AL and vortex for 5-20 seconds. Incubate the samples at 70˚C for 10 minutes.
(5) Add 200µl of cold 100% ethanol and vortex for 5-20 seconds.
(6) Carefully pipette the mixture from Step 5 into the labeled Qiagen spin column and centrifuge at 8000 x gravity for 1 minute. If any of the samples still have visible liquid in the spin column due to debris in the sample, re-centrifuge at 14,000 x gravity for 2 minutes. Discard the tube containing the flow through.
(7) Add 500µl of Buffer AW1 to each spin column and centrifuge at 8000 x gravity for 1 minute. Discard the tube containing the flow through.
(8) Add 500µl of Buffer AW2 to each spin column and centrifuge at 14,000 x gravity for 3 minutes. Discard the tube containing the flow through.
(9) Put each spin column in a labeled microcentrifuge tube and discard the tube containing the filtrate. Add 200µl of Buffer AE to the spin column and incubate at room temperature for 1 - 5 minutes. Centrifuge at 7000 x gravity for 1 minute and discard the spin columns. Freeze the extracted DNA samples at -20˚C for future use. DNA extracted from fresh and autolytic tissue samples is of high quality and only 3µL of DNA per 15µL PCR reaction is required.