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Laboratory Work As stated in the trip descriptions and field methods, fish and/or tissues were stored in 70% ETOH or placed on dry ice and transferred to freezers at -200 C or -950 C until laboratory processing was feasible. We began processing and accessioning the fish in September 2001, including tissue removal and archiving, preservation of specimens, and assignment of BYU musewn accession and population numbers. Because of the high nwnber of specimens, processing and accessioning offish continued through April, 2002 (Table 3). In total, over 1100 fish were accessioned in this period. Fin clips had to be assigned a catalogue number and collection information had to be entered on a database. Whole fish were held in a -950 C freezer. Processing of the frozen whole fish samples included thawing the fish, the removal of tissues for DNA extraction, and the assignment of museum numbers for the tissues and specimens so that cross referencing with the vouchers will be possible in the future. The vouchers were fixed in formalin and then transferred to alcohol and accessioned into the collection at the Monte L. Bean Life Science Musewn at Brigham Young University. Depending upon the size of the specimen, between two to three tissue samples (approximately 1.2 g of tissue per sample) were taken. One sample tube was preserved with 95% ETOH. The other tubes were archived in an ultracold freezer. The tubes were labeled with the BYU accession nwnber. The alcohol preserved fm clips were also assigned BYU musewn catalog nwnbers for bookkeeping and accessioning purposes. Approximately half of each fin sample was used for DNA extraction, although in some cases the entire sample was utilized if either the first isolation failed or the fin samples were very small. Small fish are processed in the same way except that the tissues removed for DNA extractions were from whole fish already preserved in 70% ETOH. There were placed in a single tube containing 95% ETOH. Fish preserved in ETOH can have additional tissue removed directly from them in the future, if needed. The DNA remaining after amplification is also archived. It is stored by population nwnber, in a -200 C freezer. BYU accession numbers are included on the tubes so that the material can be accessed in the future if necessary (Table 3). We initiated isolation of DNA from speckled dace tissue in October 2001. Dace were selected as the first species for DNA procedures since they were the most commonly collected and were more numerous than the other three species. Total DNA was isolated using the PureGene isolation kit (Gentra Systems, Minn., MN). Protocols for this isolation procedure are as follows: (1) Add 600 ~l of PureGene Cell Lysis Solution to a clean 1.5 ml microcentrifuge tube; (2) Add 20-50 mg of tissue to the tube containing PureGene Cell Lysis Solution and homogenize the tissue by cutting it into small pieces; (3) Incubate the lysate at 650 C for 15-60 minutes; (4) Add 3 ~l of Proteinase K Solution (20 mg/ml) to the lysate and incubate at 550 C for 3 hours to overnight; (5) Add 3~1 of RNase A solution (4 mg/ml) to the lysate; (6) Mix the sample by inverting the tube 25 times and incubate at 370 C for 15-60 minutes; (7) Cool the sample to room temperature and add 200 ~l of Pure Gene Protein Precipitation Solution to the RNase A treated 20