Dna preparation, , PCR

DNA preparation, PCR amplification, and sequencing

Total genomic DNA was extracted from a single leg of the specimen using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich) , following the manufacturer’s protocol, and eluted in 50 µl of elution buffer. The standard 658 bp barcode region of the mitochondrial cytochrome c oxidase subunit I gene ( COI) was amplified using universal primers LCO-1490/HCO-2198 ( Folmer et al. 1994) in 20 μl reaction mixtures containing 1xDreamTaq™ reaction buffer, 0.2 mM dNTP mix, 0.5 µM of each primer, 1.0 U of DreamTaq™ DNA Polymerase (Thermo Fisher Scientific Inc., USA) and 1 μl of DNA eluate.

The following PCR cycling conditions were applied: initial denaturation at 95 °C for 2 min; followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 1 min; with a final extension step at 72 °C for 10 min. PCR products were enzymatically purified using the Exonuclease I (0.05 U/µl) and FastAP Thermosensitive Alkaline Phosphatase (0.025 U/µl) (Thermo Fisher Scientific Inc., USA). The reaction was carried out according to the following protocol: 1 h at 37 °C, followed by 20 min at 80 °C. Bidirectional sequencing was performed by Macrogen Inc. (Amsterdam, The Netherlands) using the amplification primers. The sequence obtained in this study has been deposited in the BOLD (DIANI001-25) and GenBank (PX408929) databases.