Multiplex pcr kit

We selected a total of 18 microsatellite markers (Table S1, data
unpublished), initially isolated from the Indo-Pacific humpback dolphin
Sousa chinensis, for this study. These loci were all
‘perfect-type’ and long tandem repeat motifs (e.g., tetra- or pentanucleotide),
and demonstrated high polymorphism in Thailand S. chinensispopulations (data unpublished). Microsatellites were allocated into 6 multiplex
PCR panels using software MPprimer (Shen et al.,
2010), based on annealing temperature, complementarity of primer
pairs, and allele size range. The 5’ end of each forward primer was labeled with
a fluorescent dye (6-FAM, VIC or NED). The total PCR volume (20 μL) consisted of
approximately 50 ng of genomic DNA, 1×Multiplex PCR Kit (Takara, Dalian, China),
the optimal dosage (Table
S1) of each forward and reverse primer, and ddH₂O added to
make up the final volume. PCR conditions involved an initial denaturation step
at 94 °C for 3 min, followed by 32 cycles of 94 °C for 30 s, the specific
annealing temperature (Table S1) for 90 s, extension at 72 °C for 60 s, and a final
extension for 30 min at 60 °C. PCR products were run on an ABI 3730XL automated
DNA sequencer (Applied Biosystems) using GeneScan LIZ 500 as the internal size
standard. Allele sizes were automatically scored with GeneMapper version 4.1
(Applied Biosystems) and manually checked.

The 5′ end of each forward primer was labeled with a fluorescent dye (6‐FAM, VIC or NED). The total PCR volume (20 μl) consisted of approximately 50 ng of genomic DNA, 1×Multiplex PCR Kit (Takara), 0.2 μM of each primer (forward and reverse), and ddH₂O added to make up the final volume (Table 2). PCR conditions involved an initial denaturation step at 94°C for 3 min, followed by 32 cycles of 94°C for 30 s, the specific annealing temperature (Table 2) for 90 s, extension at 72°C for 60 s, and a final extension for 30 min at 60°C. Fragment analysis was performed on the PCR products on an ABI 3730XL automated DNA sequencer (Applied Biosystems), using GeneScan LIZ 500 as the internal size standard. Allele sizes were automatically scored with GeneMapper version 4.1 (Applied Biosystems) and manually checked. We used the same individual as a positive control for genotyping each locus separately in each multiplex PCR panel to ensure consistent amplification of alleles. A negative control without DNA template was also used in each PCR batch to detect possible contamination during PCR amplification. Moreover, genotypes from six individuals (>10%) were randomly retested to estimate genotyping error rates for across all 18 loci. Samples of each of the six samples were re‐amplified and resequenced.