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 genomic DNA from whole nucleated or non-nucleated animal blood, pp. 30. QIAGEN (Valencia, California, U.S.A.).
Protocol for Avian Blood
(1) Add 10µl of whole blood into a labeled 1.5mL microcentrifuge tube.
(2) Add 200µl of Avian Phosphate Buffered Solution (PBS) to each sample (see below for avian PBS recipe).
(3) Add 20µl Proteinase K to each sample and mix by vortexing for 5-15 seconds.
(4) Add 200µl of Buffer AL and mix by vortexing for 5-15 seconds. Incubate the samples at 50˚C for 10 minutes.
(5) Add 200µl of cold 100% ethanol to each sample and mix by vortexing for 5-15 seconds.
(6) Transfer the mixture from Step 5 into the labeled spin columns and centrifuge at 8000 x gravity for 1 minute. Discard the tubes 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 8000 x gravity for 1 minute and discard the spin columns. Freeze the extracted DNA samples at -20˚C for future use. Avian blood produces the best quality DNA and only 1-3µL of DNA are required per 15µL PCR reaction.
Recipe for Avian Phosphate Buffered Solution (PBS)
Avian PBS is composed of:
1.08g Sodium phosphate dibasic (Na2HPO4)
4.0g Sodium chloride (NaCl)
0.1g Potassium phosphate monobasic (KH2PO4)
0.1g Potassium chloride (KCl)
Double distilled water to bring the total volume to 500ml.