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LAB 23 - DNA Sexing of Geese

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Many species of exotic birds are sexually monomorphic: male and female birds are indistinguishable. DNA sexing utilizes the fact that male and female birds have different sex chromosomes. Bird sex determination is the reverse of that in humans; the female chromosome determines the sex of the bird. Male birds have two Z chromosomes, and are designated ZZ. Female birds have one Z chromosome and one W chromosome (ZW). The purpose of this lab was to extract DNA from blood samples of Dr. Allen’s geese, that he got by clipping a nail on the bird and letting it bleed. The next thing that we had to do was amplify the CHD genes with the P2 and P8 PCR primers, the procedure for this is listed below. This last part of this lab, running the PCR products out on a polyacrylamide electrophoresis gel, Dr Allen performed for us. In theory, female geese should show two DNA bands and males should only show one.

Procedure
goose.gif (1297 bytes) Pipet 20 :l of protease into the bottom of a 1.5 ml microcentrifuge tube.

goose.gif (1297 bytes) Add 200 :l ample to the microcentrifuge tube, 10 :l of blood and 190 :l of PBS.

goose.gif (1297 bytes) Add 200 :l of buffer AL to the sample and mix by pulse-vortexing for about 15 sec.

goose.gif (1297 bytes) Incubate at 56E C for 10 min.

goose.gif (1297 bytes) Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.

goose.gif (1297 bytes) Add 200 :l of ethanol to the sample, and mix again by pulse-vortexing for 15 sec. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.

goose.gif (1297 bytes) Carefully apply the mixture from step 6 to the QIAamp spin column in a 2ml collection tube without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. After centrifuging, place the QIAamp spin column in a clean 2ml collection tube and discard the tube containing the filtrate.

goose.gif (1297 bytes) Carefully open the QIAamp spin column and add 500 :l of buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000 x g (8000rpm) for 1 min. After centrifuging, place the QIAamp spin column in a clean 2 ml collection tube and discard the collection tube containing the filtrate.

goose.gif (1297 bytes) Carefully open the QIAamp spin column and add 500 :l of buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed, 20,000 x g (14,000 rpm) for 3 min.

goose.gif (1297 bytes) Place the QIAamp spin column in a clean 1.5 ml microcentrifuge tube and discard the collection tube containing the filtrate. Carefully open the QIAamp spin column and add 200 :l of buffer AE or distilled water. Incubate at room temperature for 1 min and then centrifuge at 600 x g (8000 rpm) for 1 min.

Results

According to the results of the gel above geese three, for, and five should be males because they only show one band. Geese one and two should be female because they show two bands.

 

ZOOLOGY 310 GUIDE TO DNA SEXING OF GEESE