Genomic DNA was extracted using the CTAB (Cetyltrimethylammonium bromide) method, as described by Ferreira et al. [20 (link)]. Phenol/chloroform/isoamyl alcohol was used for DNA purification. DNA was ethanol precipitated and suspended in TE buffer (pH 7.0) and stored at -20 °C until use. Negative controls were performed for each extraction procedure.
Amplification of cox1 mtDNA from both male and female specimens was performed using LCO1490 and HCO2198 specific primers, described by Folmer et al. [37 (link)]. PCR was performed in 20 μl reaction mixture containing GreenGoTaq® Flexi Buffer (Promega), 5 mM of MgCl2 (Promega), 0.2 mM of each dNTP (Promega), 0.3 pM of each primer, 0.04 U/μl of GoTaq® DNA Polymerase (Promega) and 1 ng/μl of template DNA. The thermal cycler was set at 95 °C for 5 min, followed by 40 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 48 °C, extension for 45 s at 72 °C, and a final extension for 5 min at 72 °C. The amplified products of approximately 650 bp were analysed by electrophoresis in 1.5% agarose gels stained with Ethidium bromide and observed under UV light.
Amplification of cox1 mtDNA from both male and female specimens was performed using LCO1490 and HCO2198 specific primers, described by Folmer et al. [37 (link)]. PCR was performed in 20 μl reaction mixture containing GreenGoTaq® Flexi Buffer (Promega), 5 mM of MgCl2 (Promega), 0.2 mM of each dNTP (Promega), 0.3 pM of each primer, 0.04 U/μl of GoTaq® DNA Polymerase (Promega) and 1 ng/μl of template DNA. The thermal cycler was set at 95 °C for 5 min, followed by 40 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 48 °C, extension for 45 s at 72 °C, and a final extension for 5 min at 72 °C. The amplified products of approximately 650 bp were analysed by electrophoresis in 1.5% agarose gels stained with Ethidium bromide and observed under UV light.