Genomic DNA was extracted from muscle tissues using the Qiagen DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. After DNA extraction, a 655 base pairs (bp) length fragment within the cytochrome c oxidase subunit I gene (COI hereafter) was amplified by PCR from each sample. Each reaction contained 0.5 µM of forward and reverse COI-Fish primers [32 (link)], 0.25 mM dNTPs, 2.5 mM MgCl2, 1× Buffer GoTaq® Promega, 0.15 µL of GoTaq® Polymerase (5 µ/µL), 2 µL of DNA in a final volume of 20 µL. PCR products were run in a thermal cycler (Model 2720, Applied Biosystems, Foster City, CA, USA) with the following program: initial denaturation step at 95 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, elongation at 72 °C for 30 s, and a final extension at 72 °C for 15 min.
PCR results were visualised by electrophoresis in 2% agarose gel stained with 2.5 μL SimplySafe™ dye (EURX®, Gdańsk, Poland). After amplicons purification, automated fluorescence sequencing was performed and both strands (forward and reverse) of each DNA fragment were sequenced.
PCR results were visualised by electrophoresis in 2% agarose gel stained with 2.5 μL SimplySafe™ dye (EURX®, Gdańsk, Poland). After amplicons purification, automated fluorescence sequencing was performed and both strands (forward and reverse) of each DNA fragment were sequenced.