Dna Profiling of Captive Roseate Spoonbill (Ajaia Ajaja) Populations As a Mechanism of Determining Lineage in Colonial Nesting Birds. Page: 30
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1.7 ml 1 M Tris-HCI
0.170 ml 1 M MgCl2
0.021 ml 2-mercaptoethanol
7.1 ml Total volume
16 [l primer (1 ug)
13 [l bovine serum albumin (BSA) at 10 mg/ml
100 [l Total volume of primer buffer mix
25 [l ddH20
44 pl Preliminary total volume for probe mixture
This mixture was then denatured by heating it to 950C for 3 minutes, followed by chilling
on ice for 5 minutes. Five microliters of a-labeled 32P-dCTP, 3000 Ci/mmol, and 1 [l
Klenow fragment were then added. This complete mixture was incubated at room
temperature (22-250C) for 1 hour. After this time the probe was passed through a
SephadexTM G-50 column (see Size exclusion chromatography) to remove
unincorporated nucleotides.
Quantification of each probe's specific activity was performed by the use of a
BeckmanTM LS7000 Liquid Scintillation System. Routinely, a 1 I1 aliquot of the probe
was placed into a dry 0.6 ml centrifuge tube and analyzed by the scintillation counter to
obtain an estimate of the disintegrations per minute/microliter (dpm/Il) probe. The
dpm/al was considered to roughly equal the counts per minute/microliter (cpm/l)
available from each prepared probe.
Spun-column chromatography for probe or large fragment cleanup30
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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/m1/46/: accessed April 24, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .