Plucked Chick Down

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 Chick Down


DAY 1

(1) For chick down, put 5-6 whole down feathers into a 1.5ml microcentrifuge tube with sterile forceps.


(2) Add 200-400µl of Buffer ATL to each microcentrifuge tube (you want enough ATL to cover all of the down and have some liquid visible above the down).


(3) Add 20-40µl 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. C


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