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 Molted Body Contour Feathers
(1) Cut the tip of the feather sample into a labeled 1.5ml microcentrifuge tube with sterile fine tipped scissors. For molted feathers, use one feather per DNA extraction, as not to cross contaminate samples. If the feather tip is very large (e.g. from a wing or tail feather), cut the tip into several pieces so that it can be fully immersed in the liquid for the overnight incubation step.Do not handle the tip of the feather, as not to remove the DNA containing cells.
(2) Add 200µl of Buffer ATL to each microcentrifuge tube.
(3) Add 20µl Proteinase K and vortex for 5-20 seconds. Incubate the samples at 50˚C for 24-48 hours. Extended incubations can lead to more effective lysis of the DNA containing cells and a higher end product DNA concentration.
(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 10,000 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 10,000 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 any kind of feather produces low quality DNA and 5-10µL of DNA is required per 15µL PCR reaction.