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.
QIAamp® DNA Micro Kit - Isolation of Genomic DNA from Swabs (Omni Swab Protocol)
(Qiagen (2003) QIAamp® DNA Micro Handbook. Protocol for isolation of genomic DNA from swabs , pp. 21-24. QIAGEN (Valencia, California, U.S.A.).
Protocol Modification for Avian Mouth Swabs
(1) Q-Tip™ cotton swabs were rubbed across the inside of the bird's mouth with gentle pressure. The cotton tips were put in labeled 2ml microcentrifuge tubes, the sticks were cut off with a pair of sterile wire cutters so that the lid could be closed, and the tubes were frozen at -20˚C. These samples were then thawed for DNA extraction.
(2) Add 600µl of Buffer ATL and 40µl of Proteinase K to each sample and vortex for 10-30 seconds.
(3) Incubate the samples at 50˚C for 24 hours.
(4) Add 600µl of Buffer AL to each sample and vortex for 5-20 seconds. White precipitate will form when the Buffer AL is added to the Buffer ATL, but it will dissolve during the incubation in Step 5.
(5) Incubate the samples at 70˚C for 10 minutes.
(6) Add 300µl of cold 100% ethanol to each sample and vortex for 5-20 seconds.
(7) Transfer 700µl of the solution from the microcentrifuge tube into a labeled spin column and centrifuge at 8000 x gravity for 1 minute. Discard the tube containing the flow through. Transfer the remaining solution into the spin column, centrifuge at 8000 x gravity for 1 minute, and discard the tube containing the flow through.
(8) Add 500µl of Buffer AW1 to each sample and centrifuge at 8000 x gravity for 1 minute. Discard the tube containing the flow through.
(9) Add 500µl of Buffer AW2 to each sample and centrifuge at 8000 x gravity for 1 minute. Discard the tube containing the flow through.
(10) Put each spin column in a new collection tube and centrifuge at 14,000 x gravity for 3 minutes.
(10) Put the spin column into a labeled microcentrifuge tube and add 100µl of Buffer AE.
(12) Incubate the samples at room temperature for 5 minutes.
(13) Centrifuge at 14,000 x g for 1 minute and discard the spin columns. Freeze the extracted DNA samples at -20˚C for future use. DNA extracted from mouth swabs produces good quality DNA and 3-5µL of DNA is required per 15µL PCR reaction.