Molecular systematics of Baird's pocket gopher (Geomys breviceps) Page: 5
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(QIAGEN, Valencia, California) following their protocol for animal tissue. DNA concentration
was measured for each sample using a NanoDropTM ND-1000 UV-Vos Spectrophotometer
(NanoDrop Technologies, Wilmington, DE) and portion of each extraction was diluted with
sterile water to a concentration of 50 ng/ L.
Polymerase chain reaction (PCR; Saiki et al. 1986, 1988) was performed to amplify the
entire cytochrome-b gene with the following parameters: 1 cycle of 94C (2 min); 33 cycles of
94C (45 s) denaturing, 56C (1 min) annealing, and 72C (1 min, 10 s) extension; followed by 1
cycle of 72C (10 min). Amplification reactions contained 0.5 L of diluted extraction product,
1.25 L of 5X PCR buffer, 2.5 mM MgCl2, 0.6 M primer concentrations, 1.25 U of Taq DNA
polymerase, and sterile diH20 to bring the total volume to 25 L. The primers used to amplify
the cytochrome-b gene were L14735 (5'-TGAAAAACCATCGTTGTTAATTCAACT-3') and
H15906 (5'-CATCTCCGGTTTACAAGACCTAAGTAAT-3'; Elrod et al. 2000). The double-
stranded amplicons were purified using Promega Wizard@ PCR Clean-up kit (Promega,
Madison, Wisconsin) or ExoSAP-IT For PCR Product Clean-Up (USB, Cleveland, Ohio).
Sequences were obtained for both directions using dye-labeled chain terminators and 1.0
p1 of amplified product. In addition to the above primers, LGL 765
(5'-GAAAAACCAYCGTTGTWATTCAACT-3') and LGL 766
(5'-GTTTAATTAGAATYTYAGCTTTGGG-3'; Harlin-Cognato et al. 2006) were used in
sequencing reactions. Sequencing parameters were 95C (20 s) denaturing; 50C (20 s)
annealing, and 60C (4 min) extension for 30 cycles. Reactions were ethanol-precipitated before
being analyzed with a Beckman-Coulter CEQTM 8000 Automated Genetic Analysis System
(Beckman-Coulter, Inc., Fullerton, California). Single-stranded sequence fragments were
aligned and proofed using BioEdit v5.0.6 software (Hall 1999).
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Bodine, Deanna Martinez. Molecular systematics of Baird's pocket gopher (Geomys breviceps), thesis, August 2010; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc30438/m1/9/: accessed March 28, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .