Declaration Dr. Thomas H. Pringle - Buffalo Field Campaign

PCR amplification
Primers BISCR-16348F 5'-CTACAGTCTCACCGTCAACCC-3' and BISCR-
16990R 5'-GATGAGATGGCCCTGAAGAA-3' (Shapiro et al. 2004; Vogel et al. 2006)
were used to amplify a 470 bp segment of the bison mtDNA control region. PCR was
carried out in 25 μl volumes containing; 8.95 μl sterile HPLC H2O, 2.5 μl
Invitrogen © 10X PCR buffer, 1 μl dNTP’s, 0.5 μl of each primer, 2.5 μl BSA (2ng/μl),
1.25 μl MgCl (50 mM), 0.3 μl Invitrogen © Platinum Taq Polymerase (5 units/μl), and 7.5
μl of template DNA. Amplification was performed in an MJ Research PTC-200 Peltier
Thermal Cycler using the following touchdown protocol: 94º C for 5 min, followed by
one cycle of 94º C for 30 s, 60º C for 30 s, and 72º C for 30 s. For the subsequent 10
cycles, all conditions remained the same except that the annealing temperature decreased
by 0.5 degrees per cycle. This was followed by 25 cycles of 94º C for 30 s, 55º C for 30 s,
and 72º C for 30 s. The profile concluded with a single extension of 72º C for 5 min.
Sequencing
PCR products were purified using QIAquick © purification columns according to
manufacturers’ instructions with one exception; the final elution was carried out with 20
μl of Buffer EB instead of the recommended 30-50 μl to compensate for potential low
quantity template DNA . The amount of purified post-PCR product was quantified by
fluorometry to insure that 5-10 ng/μl of amplified DNA was present for sequencing on
the ABI 3100xl sequencer. Sequences were visualized, assessed for quality, and edited
using Chromas 2.31 (http://www.technelysium.com.au/chromas.html). MEGA 3.1
(Kumar et al. 2004) was used to align edited mtDNA sequences with known bison
haplotypes.
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