| Summary: | Maintenance and Update Frequency: notPlanned Statement: - Sample collection - Three separate sampling events were conducted between 1989 and 2000, and a total of 15 individual colonies were sampled. Five colonies (Beagle Island, North Fisherman Island, Buller Island, Six Mile Island and Kangaroo Island) were sampled on all three occasions. Breeding colonies were visited towards the end of the breeding season, which was calculated by extrapolation from previously published breeding data (Gales et al. 1994). Estimates of pup production for the sampled colonies are taken from Chapter 2 of the thesis. Tissue samples were taken from newly born pups of a single breeding season to ensure the sampling of individuals from their birth colony, and to minimise the sampling of related individuals. Females produce only one pup per season and so each pup represents a single maternal line. Live pups were targeted as the primary source, although freshly dead pups were sampled where available. Exceptions to this occurred on Dangerous Reef and The Pages, where for a number of reasons only dead pups were sampled. Pups were captured by hand, and a small piece of tissue removed from the tip of the middle digit of the right hind flipper. Tissue was preserved in a solution of 95% EtOH and 100mM EDTA to help stabilise nucleic acids. Total genomic DNA was extracted as in Gemmell & Akiyama (1994), and thirteen microsatellite loci designed for other pinniped species were screened to determine the degree of cross species suitability and polymorphism of markers. Statement: - Screening of microsatellite loci - DNA extraction techniques were described in Chapter 3 of the thesis. Approximately 20-50ng of template DNA was added to the PCR reaction mixture, which was derived from Gemmell et al. (1997) using direct incorporation of a radio-labelled nucleotide. Individual PCR reaction consisted of 1ul X10 reaction buffer, 2.5mM MgCl2, 0.25 uM of both forward and reverse primers, 0.25U Taq, 5mM of three dNTP's (ATP, GTP, TTP) and 0.5mM of ...
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