Gotaq g2 flexi buffer

Specimens were selected for genetic analysis, and genomic DNA was extracted from a single individual using the Mollusc DNA Kit (Omega), following the manufacturer's protocols and with the modifications suggested in^{20 (link)}. Subsequently, the concentration of the extracted DNA was quantified, and the presence of contaminants was verified using a Nanodrop. Finally, the DNA was stored at − 20 °C.
To perform the genetic analyses, two genes were employed: the mitochondrial gene encoding the cytochrome c oxidase subunit I (COI) and the nuclear gene encoding the small subunit of the ribosome (18S). The primers used to amplify each gene fragment, along with the observed and expected fragment lengths and the hybridization temperatures during PCR amplification cycle are specified in supplementary materials Table S4. PCR amplification was conducted in a total volume of 30 μL, where included 3 μL of 5X Green GoTaq G2 Flexi Buffer, 3 μL of 25 mM MgCl₂, 0.5 μL of dNTPs (10 mmol L⁻¹), 0.5 μL of each primer (100 pmol µL⁻¹), 0.045 units of GoTaq G2 Flexi DNA Polymerase (Promega), and 5 μL of DNA from each individual. The PCR products were verified by electrophoresis on 1.2% agarose gel. Subsequently, the amplified products were sent to Macrogen for sequencing. To avoid ambiguous readings of certain nucleotides, the PCR products were sequenced in both directions (forward and reverse).

Whole animals were ground in Trizol ReagentTM using the Ultra-Turrax T25^® (IKA). RNA was extracted according to the manufacturer's instructions. The concentration of extracted RNA was estimated with the Qubit^® 3.0 Fluorometer (ThermoFisher Scientific). RNA extracts were treated by RQ1 RNase-free DNase (Promega) and used for cDNA synthesis with the RevertAid M-MuLV RT kit (ThermoFisher Scientific) according to the manufacturer's protocol. Reaction mixtures for PCR amplifications contained 0.1 μM of each primer, 0.25 mM of each desoxynucleotide triphosphate, 5 × Go Taq G2 Flexi buffer (Promega), and 5 U of GoTaq G2 Flexi DNA polymerase (Promega). The PCR program involved an initial denaturation step at 95°C for 3 min, followed by 39 cycles of 95°C for 1.5 min, 55°C for 1.5 min, and 72°C for 1.5 min, with a final elongation step at 72°C for 5 min.