Dna extraction

Ercan, Nuri, 2025, First report on the molecular characterization and phylogenetic analysis of Fannia sp. (Diptera: Fanniidae) identified in Türkiye, Revista Brasileira de Entomologia (e 20250017) 69 (2) : 2

DNA extraction and PCR amplification

Following morphological identification, two to three legs were removed from each specimen using sterile lancets and placed in microcentrifuge tubes for DNA extraction . The leg samples were individually ground in a microcentrifuge tube under liquid nitrogen using a mortar and pestle. Genomic DNA was extracted using a commercial kit (PureLink Genomic DNA Mini Kit, Invitrogen, Carlsbad, CA ) according to the manufacturer’s instructions. Extracted gDNA was eluted in 50 µl of elution buffer and stored at − 20 °C. The concentration of gDNA isolates was determined by Qubit Fluorometric Quantitation (Thermo Fisher Scientific, USA) prior to PCR amplification.Two molecular markers were amplified independently for each sample: a partial fragment of the COI gene was amplified with the universal primers (LCO-1490/HCO-2198) described by Folmer et al. (1994) with the annealing temperature set to 50 °C, and the nuclear ITS2 region was amplified with the primers ITS2-F2814 and ITS2-R3295 ( Song et al., 2008). Thermal cycling conditions were as follows: initial denaturation at 95°C for 4 min, followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. Approximately 20 ng of gDNA was used per reaction. PCR products were electrophoresed on 1.5% agarose gel and visualized using a gel documentation system. Amplicons from positive samples were purified using GeneJet Gel Extraction Kit (Thermo Scientific, USA) according to manufacturer’s instructions, and sequenced in both directions with amplification primers (BMLabosis, Ankara, Türkiye). All gDNA isolates were screened for the presence of the endosymbiotic bacterium Wolbachia with the wsp 81F and wsp 691R primers ( Braig et al., 1998).