Historic Samples

The following DNA extraction protocol should be referenced as: Bush, K.L. 2007. DNA extraction technique for plucked historic feathers using the QIAamp® DNA Micro Kit. Last modified August 15, 2007. http://www.aviangenetics/DNA_extraction_index.htm


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 Historic Feathers


DAY 1

(1) Cut the tips of 5 plucked feather samples into small pieces (~1mm) into a labeled 1.5ml microcentrifuge tube with sterile fine tipped scissors. Use 100% ethanol and DNA Away® to clean scissors in between samples.


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


(3) Add 20µL of Proteinase K to each sample and vortex for 30 seconds.


(4) Incubate at 50˚C for 48-72 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 30 seconds.  Incubate the samples at 70˚C for 10 minutes.


(6) Add 200µl of cold 100% ethanol and vortex for 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 10,000 x gravity for 1 minute.  Discard the tube containing the flow through.


(10) Add 500µl of Buffer AW2 to each sample and centrifuge at 10,000 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 110µl 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. If you have more than 5 feathers from the same individual, save the spin column to extract the second set of DNA (I have used spin columns to successfully extract 4 rounds of DNA from individual samples).

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