Cytochrome b Gene Amplification and Sequencing

The primers used to amplify the cytb gene are listed in Table S1. Where amplification could not be achieved with the primary PCR primers, a secondary nested PCR was performed using internal primers L14749 and H14896 as described by (Nicolas et al., 2012 (link)). PCR was performed in a 25 µL reaction volume containing 2 µL of DNA template, 0.2 µM of each primer, 0.05 U of MyTaq polymerase and 5 µL of 5X MyTaq buffer (Bioline, UK). Amplification was performed in an Eppendorf Master cycler pro 384 (Eppendorf, USA) with an initial denaturation step of 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min followed by a final extension step at 72 °C for 10 min. Amplicons were visualized on a 2% agarose gel (Thermo Fisher Scientific, CA, USA) stained with GelRed (Biotium, Australia). PCR products from positive samples were purified using AMPure XP beads (Beckman Coulter, CA, USA). The forward and reverse primers were used for cycle sequencing by the Big Dye Terminator Cycle Sequencing Kit v 3.1 (Applied Biosystems, CA, USA). Reaction products were then purified using CleanSEQ beads (Beckman Coulter, CA, USA) and sequenced on a 3130 Genetic Analyzer (Applied Biosystems CA, USA).