Dna Profiling of Captive Roseate Spoonbill (Ajaia Ajaja) Populations As a Mechanism of Determining Lineage in Colonial Nesting Birds. Page: 78
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MgCl2, 10 mM dithiothreitol), 0.5 [l 10 mM ATP, and 0.5 [l (1 U/l) T4 DNA Ligase
(GIBCO BRLR). The ligation mixture was incubated at 13.50C for 21 hours.
The cloned DNA (lambda arms with ligated DNA inserts) was packaged by use of
the MaxPlaxTM Lambda Packaging Extract. A 25 [l volume of packaging extract was
removed from the -700C freezer, thawed at room temperature and mixed with the cloned
DNA (5.5 [l) in a 0.6 ml microcentrifuge tube. The tube was then incubated at 300C for
90 minutes. An additional 25 [l thawed extract was added to the reaction tube and mixed
well without introducing bubbles. The tube was again incubated at 300C for 90 minutes.
Five hundred microliters of phage dilution buffer (10 mM Tris-HC1, pH 8.3, 100 mM
NaC1, 10 mM MgCl2) was then added and mixed by gentle vortexing. Twenty-five
microliters of chloroform was added, mixed well by gentle vortexing, and the tube was
stored at 40C.
Each Spoonbill genomic ZAP Express* library was titered and tested for
background (lambda DNA with no inserts) by first mixing 100 [l of the diluted (10-2)
packaged phage with 200 [l XL1-Blue MRF' (at an OD600 of 0.5) and incubating the
mixture at 370C for 15 minutes with periodic gentle shaking to allow the phage to attach
to the cells. After this attachment period, 15 [l of 0.5 M IPTG (in ddH20), 50 [l of 250
mg/ml X-gal (in dimethylformamide) and 3 ml of 480C NZY top agar (5 g NaC1, 2 g
MgSO4-7H20, 5 g yeast extract, 10 g casein hydrolysate, deionized H20 to 1 liter, pH 7.5,
and 0.7% (w/v) agarose) were added to the phage/cell mixture and immediately poured
onto a pre-warmed NZY agar plate. The plates were allowed to solidify for 10-15
minutes and were then inverted and placed into an incubator at 370C for 12-16 hours.
The recombinant lambda ZAP ExpressR plaques appeared as cleared zones amongst a78
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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/m1/94/: accessed April 26, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .