Dna Profiling of Captive Roseate Spoonbill (Ajaia Ajaja) Populations As a Mechanism of Determining Lineage in Colonial Nesting Birds. Page: 37
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was transferred to tube #3. After the sample was mixed gently, a 30 [l aliquot was
transferred to tube #4 and the serial dilution was continued through tube #9. The 30 [l
aliquot removed from tube #9 was discarded so that tube #10 remained a 30 [l uncut
control (no enzyme added). All 10 tubes were placed in a 370C water bath for the
restriction endonuclease digestion to occur. After 1 hour, the enzyme was heat denatured
by placing the tubes at 700C for 15 minutes.
Seven microliters of 5X agarose gel electrophoresis loading buffer (25% glycerol,
0.5% sodium dodecyl sulfate, 0.1% bromophenol blue, 0.1% xylene cyanol, 50 mM
EDTA) was added to each 30 [l sample. A thin 1% agarose base was prepared in an
agarose gel tray. After the agarose solidified, a thicker 0.3% agarose solution was
layered on top and allowed to solidify at 40C. The ten 37 [l samples were then loaded
into wells. A DNA sizing ladder (lambda DNA cut with HindIII) was added and the gel
electrophoresed in IX TBE buffer at 30 V and 40C for 14 hours. Visualization of the
DNA was accomplished by submerging the gel in a solution of EtBr (0.5 [g/ml) for 10
minutes followed by a 10 minute wash in deionized water and placement on a UV
transilluminator.
Gel analysis of the cut DNA samples revealed that the Sau3AI serial dilution
experiment resulted in the desired partial digestions of the Spoonbill DNA. Based upon
these results, a preparative digestion of Spoonbill DNA was immediately begun. The
preparative digestion cut 10 4g of DNA in each serially-diluted tube. One hundred
eighty microliters of Spoonbill genomic DNA (0.5 [g/l) was added to 2.250 ml TE, pH
8.0 and 270 [l 10X Sau3AI buffer for a total of 2.7 ml. Six hundred microliters was
added to microcentrifuge tube #1 and 300 [l was added to 7 additional microcentrifuge37
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Sawyer, Gregory M. Dna Profiling of Captive Roseate Spoonbill (Ajaia Ajaja) Populations As a Mechanism of Determining Lineage in Colonial Nesting Birds., dissertation, May 2002; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc3117/m1/53/: accessed April 20, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .