The following DNA extraction protocol should be referenced as: Bush, K.L. 2005. Protocol for DNA extraction from avian blood/pin feathers using the Qiagen DNeasy® Tissue Kit. Last modified August 22, 2005. http://www.aviangenetics/DNA_extraction_index.htm
Qiagen DNeasy® Tissue Kit - Blood 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/Pin Feathers
(1) Take one frozen or dried blood feather (growing primary remige [wing feather] or retrix [tail feather]) and chop the tip of the feather into small (~2mm) thick slices with a pair of fine sterile scissors in 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 10-30 seconds.
(4) Add 200µl of Buffer AL and mix by vortexing for 10-30 seconds. Incubate the samples at 50˚C for 30 minutes to 1 hour.
(5) Vortex the samples for 10-20 seconds to ensure the sample is fully homogenized.
(6) Add 200µl of cold 100% ethanol to each sample and mix by vortexing for 10-30 seconds.
(7) 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.
(8) 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.
(9) 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.
(10) 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. 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.