Plucked Contour Feathers

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 Plucked Body Contour Feathers


DAY 1

(1) Cut the tip of the feather sample into a labeled 1.5ml microcentrifuge tube with sterile fine tipped scissors. For plucked feathers, use 2-3 feather tips per DNA extraction. 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.


DAY 2

(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 any kind of feather produces low quality DNA and 5-10µL of DNA is required per 15µL PCR reaction. 


QIAamp® DNA Micro Kit - Isolation of Genomic DNA from Tissue Samples

(Qiagen (2003) QIAamp® DNA Micro Handbook. Protocol for isolation of genomic DNA from tissues, pp. 35-37. QIAGEN (Valencia, California, U.S.A.).


Protocol for Plucked Body Contour Feathers, Molted Body Contour Feathers, & Plucked Chick Down


The following DNA extraction protocol should be referenced as: Bush, K.L. 2005. DNA extraction technique for plucked body contour feathers, molted body contour feathers, and plucked chick down using the QIAamp® DNA Micro Kit. Last modified August 22, 2005. http://www.aviangenetics/DNA_extraction_index.htm


DAY 1

(1) Cut the tip of the feather sample into small pieces (~1mm) into a labeled 1.5ml microcentrifuge tube with sterile fine tipped scissors. For plucked feathers, use 2-3 feathers per DNA extraction and for molted feathers, use one feather per DNA extraction. For chick down, put 5-6 whole down feathers into a 1.5ml microcentrifuge tube.


(2) Add 200µl of Buffer ATL to each sample.


(3) Add 20µL of Proteinase K to each sample and vortex for 5-20 seconds.


(4) Incubate at 50˚C for 18-48 hours. Extended incubations can lead to more effective lysis of the DNA containing cells and a higher end product DNA concentration.


DAY 2

(5) Add 2µl of carrier RNA dissolved in Buffer AE to every 200µl of Buffer AL to be used in the extraction process.  Add 200µl of the Buffer AL/carrier RNA mix to each sample and vortex for 5-20 seconds.  Incubate the samples at 70˚C for 10 minutes.


(6) Add 200µl of cold 100% ethanol and vortex for 5-20 seconds.


(7) Incubate the samples at room temperature for 5 minutes.


(8) Transfer the solution from the microcentrifuge tube to a labeled spin column and centrifuge at 10,000 x gravity for 1 minute. Discard the tube containing the flow through.


(9) Add 500µl of Buffer AW1 to each sample and centrifuge at 8000 x gravity for 1 minute.  Discard the tube containing the flow through.


(10) Add 500µl of Buffer AW2 to each sample and centrifuge at 8000 x gravity for 1 minute. Discard the tube containing the flow through.


(11) Put each spin column in a new collection tube and centrifuge at 14,000 x gravity for 3 minutes.


(12) Put the spin column into a labeled microcentrifuge tube and add 100µl of Buffer AE.


(13) Incubate the samples at room temperature for 5 minutes.


(14) Centrifuge at 7,000 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. .

Contact Me - Krissy Bush ~ Last Modified January 10, 2011