Dna Profiling of Captive Roseate Spoonbill (Ajaia Ajaja) Populations As a Mechanism of Determining Lineage in Colonial Nesting Birds. Page: 40
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DNA was electrophoresed at 30 V for 14 hours and stained by EtBr. After a 1 hour
destain of the gel, and a 75 second exposure to Polaroid type 55 film, the faint smearing
pattern of DNA could be discerned on the film negative. Fractions in the size range of 9
to 23 kbp were chosen to be recovered and purified.
The sucrose was removed from the samples by affinity column chromotography.
A NENSORBTM 20 (DupontTM) nucleic acid purification cartridge was wetted by the
addition of 2 ml 100% methanol, followed by a 2 ml rinse with Reagent A (100 mM Tris-
HC1, 10 mM triethylamine, 1 mM disodium EDTA, pH 7.7). Each of the 1 ml sucrose
samples (10-14) was supplemented with 1.4 [tl Reagent A, then loaded onto the column.
Three milliliters of Reagent A was added to wash the column, followed by a 4 ml wash
with water. The elution of the DNA began with the addition of 1 ml Reagent B (50%
methanol) to the top of the column. Three fractions, of approximately 300 [Il each, were
collected into 1.5 ml microcentrifuge tubes. The DNA containing microcentrifuge tubes
were placed in a Savant Speed VacTM vacuum concentrator for 4 hours or until the
samples had dried completely.
The dried DNA samples were resuspended in TE, pH 8.0 and combined to yield a
total volume of 100 [tl. One-half of the entire sample (50 [tl) was analyzed in a
BeckmanTM DU-40 Spectrophotometer. Additionally, a 5 [tl sample was loaded on a
0.5% (1% base) agarose gel and subjected to electrophoresis at 20 V for 12 hours. The
gel was stained in EtBr and destained in water. A smear of DNA from about 7-23 kbp
should be observed. After verification of the recovery of properly sized DNA, a 25 [Il
volume was diluted to 100 [Il with ddH20 and ethanol precipitated by the addition of 11
[Il 3 M sodium acetate and 350 [Il -200C 100% ethanol. After centrifugation at 10,000 x40
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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/m1/56/: accessed May 9, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .