2006), KWM2, KWM12a (Hoelzel et al. 1998), EV1 (Valsecchi and Amos 1996), MK6 and MK8 (Krützen et al. 2001)). Amplification reactions contained BGB324 in vitro 50–100 ng DNA, 1 × reaction Promega Taq buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.1 μM of each primer and 1 unit of Promega Taq DNA polymerase. The thermal cycler profile for
the tetranucleotide loci and Dde66 and Dde70 consisted of initial denaturation at 94°C for 3 min followed by a touchdown profile for 5 cycles with initial denaturation at 94°C for 20 s, annealing temperature starting at 63°C and decreasing 2°C per cycle for 45 s and 72°C for 1 min, followed by 30 cycles with the same initial denaturation and final extension temperatures, but an annealing temperature of 53°C, followed by a final extension step at 72°C for 10 min.
In each cycle, for the remaining dinucleotide loci, conditions followed the original publications. All reactions included both positive and negative controls. Following amplification, samples were mixed with an internal size standard (LIZ 500) and run on an ABI 3130 Genetic Analyzer. The GeneMapper 4.1 software (Applied Biosystems, Carlsbad, CA) was used for sizing of allele fragments. The first 577 basepairs (bps) at the 5ʹ end of the mtDNA control region were sequenced in both forward and reverse directions with the primers L15926 and H00034 (Rosel et al. 1994). The PCR reaction conditions were see more as follows: 10–100 ng of DNA, 0.15 mM dNTPs, 1.5 mM MgCl2, 10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.3 μM of each primer, 1.5 U Taq polymerase per reaction. The PCR cycling profile was 35 cycles of 1.5 min at 94°C, 2 min at 45°C, 2.5 min at 72°C, followed by 3 min at 72°C. A subset of 40 samples, randomly chosen from each of the three putative populations, was further sequenced for the cytochrome b gene, in both forward and reverse directions, using conditions described in Amaral et al. 2007a. These sequences were used to investigate the taxonomic status of New Zealand common dolphins by comparing them
with 155 sequences available from different oceans (Amaral et al. 2012). The cytochrome b gene has been suggested to be more reliable than the control region in species identification in closely related groups such as delphinids (e.g., Amaral et al. 2007a). PCR Nintedanib (BIBF 1120) products were purified using a QIAquick PCR purification kit (QIAGEN Pty. Ltd.), and sequencing reactions performed using the ABI PRISM BigDye Terminator Sequencing Kit. Ethanol precipitation was performed on the sequencing products and then separated on the ABI PRISM 3730 automated DNA Analyzer. Sequences were visualized and minor edits performed using SEQUENCHER 4.1 (Gene Codes Corporations Inc.). Sequences were aligned using CLUSTAL X (Thompson et al. 1997). The program Micro-checker v. 2.2.3 (Oosterhout et al. 2004) was used to check for the presence of genotyping errors such as scoring errors due to stuttering, large allele dropout or evidence for null alleles.