Declaration Dr. Thomas H. Pringle - Buffalo Field Campaign

carried out with one last modification. The final elution of DNA was repeated by passing
the eluate through the column once more. This last step was modified in an attempt to
recover as much DNA as possible. The tubes were centrifuged for one minute at 16,100
rcf. Negative extraction controls, containing sterile distilled water, were used along with
each set of fecal extractions to monitor for possible cross contamination.
Microsatellite PCR and genotyping error rates
We screened a set of 36 microsatellite loci for use with fecal DNA samples,
which were previously used in other genetic studies of bison (Halbert (2003). Only loci
that were previously found to have a minimum heterozygosity of 0.50 in GYA bison
were used to provide high power for future population genetic studies. We identified four
dinucleotide loci with allele ranges less than 200 bp, BM 711(157-163 bp; NED),
BM2113 (123-143 bp; NED), BMS1001 (101-109 bp; 6- FAM), and BMS2258 (123-144
bp; VIC) for use in a single multiplex PCR to estimate genotyping error rate (Appendix
1).
Multiplex PCR was carried out in 10 μl volumes containing 2 μl sterile HPLC
H2O, 5 μl QIAGEN Multiplex PCR Mix, 1 μl QIAGEN Q-Solution, 1 μl 10x primers
(Table 1), and 1 μl template DNA. PCR was performed in an MJ Research PTC-200
Peltier Thermal Cycler using the following touchdown profile: 95º C for 5 min, followed
by one cycle of 94º C for 30 s, 58º C for 1 min 30 s, and 72º C for 20 s. For the
subsequent 19 cycles, all conditions remained the same except that the annealing
temperature decreased by 0.5 degrees per cycle. This was followed by 26 cycles of 94º C
for 30 s, 48º C for 1 min 30 s, and 72º C for 20 s. The profile concluded with a single
extension of 72º C for 10 min.
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