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 Hatched Eggshell Membranes
(1) For hatched eggshell membranes, remove a piece of vascularized membrane (~1cm x 4cm) from the bottom of the egg with a pair of sterile tweezers. The bottom of the egg contains a larger amount of vascularized membrane than the egg top and generally has enough tissue for ~3-6 DNA extractions (based on an egg the size of a chicken egg). Place the membrane in a labeled 1.5ml microcentrifuge tube and crush with tweezers until the fragments are ~2mm2 in size (small enough for effective lysis, but large enough so that they can not be drawn up into the pipette tip in Step 6).
(2) Add 400µl of Buffer ATL to each microcentrifuge tube.
(3) Add 40µl Proteinase K and vortex for 5-20 seconds. Incubate the hatched eggshell membrane samples at 50˚C for 12-24 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 11,000 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 11,000 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 hatched eggshell membranes is of high quality and only 3µL of DNA per 15µL PCR reaction is required.