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Lysate; (8) Vortex the sample vigorously for 20 seconds to mix the PureGene Protein Precipitation Solution uniformly with the lysate; (9) Centrifuge at 13,000 to 16,000 x g for 3 minutes to form precipitated proteins into a tight pellet; (10) Pour the supernatant containing the DNA (leaving behind the protein pellet) into a clean 1.5 ml tube containing 600 J.ll of 100% Isopropanol; (11) Mix the sample by inverting the tube gently 50 times, which may cause the DNA to form a visible white clump in the solution; (12) Centrifuge at 13,000 to 16,000 x g for 3 minutes, the DNA may be visible as a small white pellet; (13) Pour off the supernatant and drain the tube on clean absorbent paper, then add 600 J.ll of70% ethanol and invert the tube several times to wash the DNA pellet; (14) Centrifuge at 13,000 to 16,000 x g for 60 seconds, carefully pour off the ethanol while watching the pellet (if visible) as it may be loose; (15) Allow the sample to air dry for 30 minutes; (16) Add 300 J.ll of PureGene DNA Hydration Solution; (17) Allow the DNA to re-hydrate at room temperature overnight, or heat the sample at 650 C for one hour, tapping the tube periodically to aid in dispersing the DNA into solution. Polymerase chain reaction (PCR) was performed on isolated DNA using a 38-cycle protocol: 40 seconds at 950 C, one minute at 500 C, and 2 minutes at 720 C. Primers used for amplification of the cytochrome B (cytB) and NADH dehydrogenase 2 subunit (ND2) genes in trout and which were designed in our lab were'tested on speckled dace DNA in October 2001. The cytB primers, 1425 and 1426, generate a mtDNA fragment of 1200 base pairs and the ND2 primers, BYU11 and BYU12, generate a 1572 base pair fragment. Collectively these two PCR products contain one-sixth of the mitochondrial genome. However, these primers failed to consistently amplify dace DNA, hence cytB primers developed specifically for cyprinid species (provided by T.R. Schmidt to T.E. Dowling; see Dowling and Naylor, 1997) were ordered in October 2001: LA-a: 5'-GTGACTTGAAAAACCACCGTTG-3', HD-a: 5'-GGGTTGTTTGATCCTGTTTCGT-3', LD-a: 5 '-CCA TTCGTCA TCGCCGGTGC-3', HA-a: 5 '-CAACGA TCTCCGGTTT ACAAGAC- 3'. Amplification of cytB was again initiated following receipt of the above primers. Primers for an additional mitochondrial gene were ordered in February 2002: ND2 primers (Broughton and Gold, 2000): ND2B-L (5'-AAGCTTTCGGGCCCATACCC-3'), ND2E-H (5'- TTCTACTTAAAGCTTTGAAGGC-3'), HD2G-L (5'-CACAACAATAATCCTTGCCGC-3'). Amplification protocols were adjusted to 30 cycles of one minute at 940 C, one minute at 500 C, and 1.5 minutes at 720 C. This region amplified and was sequenced. However at the same time initial isolations (all from speckled dace) were taken from a broad range of sample sites to check degree of variation in the cytB gene both within individual populations and between populations in the same as well as different drainages. These included populations in the Yampa (1. Snake and Coal View), White (102 Rd and Meeker City Park), Colorado (Rifle), Gunnison, Dolores (San Miguel at Uravan and Groundhog), and San Juan. It became apparent that the sequence data from Cytochrome B alone contained enough phylogenetic information for separating mitochondrial lineages and the additional ND2 sequence was redundant. We therefore discontinued further efforts with the ND2 region of the mitochondrial genome. All of the collected fish were processed and DNA was extracted and amplified However, sequences were not obtained from all of the specimens. For example, 35 dace were collected from the Little Snake River, DNA was isolated from all 35, PCR amplified the cytB gene of24, and 22 of the 24 were successfully sequenced. The disparity in some cases between number isolated and number amplified (e.g. 7 of 34 dace from White River at 102 Rd amplified) is 24