Predated Eggshell Membranes

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 Predated Eggshell Membranes


DAY 1

(1) For predated eggshell membranes, remove the eggshell and membranes surrounding the point of entry with a sterile pair of tweezers.  These fragments are bent into the egg and are usually the only pieces of vascularized membrane after the predation event. Particularly in the case of mammalian predators, this is the only usable part of the egg since mammals tend to completely remove the yolk, embryo, and all of the associated membranes from the egg. Once the eggshell and membrane fragments are removed, the eggshell has to be separated from the membranes since the membranes contain the chick DNA. Place the fragments of membrane into a labeled 1.5ml microcentrifuge tube and crush with the tweezers until the fragments are ~2mm2 in size.


(2) Add 400µl of Buffer ATL to each microcentrifuge tube.


(3) Add 40µl Proteinase K and vortex for 5-20 seconds. Incubate the predated eggshell membranes at 50˚C for 24-48 hours (a longer incubation time for the predated eggshell membrane results in higher quantities of DNA in the end product).


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. Predated eggshell membranes produce lower quality DNA depending on the amount of vascularized membrane in the DNA extraction and 3-10µL 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.).


The following DNA extraction protocol should be referenced as: Bush, K.L. 2005. DNA extraction technique for predated eggshell membranes using the QIAamp® DNA Micro Kit. Last modified February 2, 2005. http://www.aviangenetics/DNA_extraction_index.htm


Protocol for Predated Eggshell Membranes


DAY 1

(1) For predated eggshell membranes, remove the eggshell and membranes surrounding the point of entry with a sterile pair of tweezers. These fragments are bent into the egg and are usually the only pieces of vascularized membrane after the predation event. Particularly in the case of mammalian predators, this is the only usable part of the egg since mammals tend to completely remove the yolk, embryo, and all of the associated membranes from the egg. Once the eggshell and membrane fragments are removed, the eggshell has to be separated from the membranes since the membranes contain the chick DNA. Place the fragments of membrane into a labeled 1.5ml microcentrifuge tube and crush with the tweezers until the fragments are ~2mm2 in size.


(2) Add 400µl of Buffer ATL to each sample.


(3) Add 40µl of Proteinase K to each sample and vortex for 15 seconds


(4) Samples were incubated at 50˚C for 18-48 hours (a longer incubation time for the predated eggshell membrane results in higher quantities of DNA in the end product).



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 15 seconds.  Incubate the samples at 70˚C for 10 minutes.


(6) Add 200µl of cold 100% ethanol and vortex for 15 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 8000 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 14,000 x gravity for 1 minute and discard the spin columns. Freeze the extracted DNA samples at -20˚C for future use. DNA extracted from predated eggshell membranes generally produces lower quality DNA and 3-10µL of DNA is required per 15µL PCR reaction. 

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