Dna barcoding

Sardar, Sayan, Angmo, Spalzin, Stobdan, Tsering, Chauhan, Archana & Rameshkumar, A., 2025, Bees of the genus Colletes Latreille (Hymenoptera: Apoidea: Colletidae) from Ladakh, India, with description of a new species, Journal of Natural History 59 (21 - 24), pp. 1725-1741 : 1727

DNA barcoding of Colletes specimens using the mitochondrial COI marker

Specimens for DNA barcoding were collected from the field and preserved in 95% ethanol at −80°C until further use. Genomic DNA was extracted from morphologically identified specimens using the DNA easy Blood and Tissue extraction kit (Qiagen, Germany), following the manufacturer’s protocol. A polymerase chain reaction (PCR) was performed to amplify the target region of the mitochondrial COI gene using the universal primers LCO1490 and HCO2198 ( Folmer et al. 1994). Reactions were carried out using 5 µL of eluted DNA , primers, distilled water and PCR master mix containing Taq DNA polymerase to a final volume of 25 µL. The thermal regime consists of initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 45°C for 1 min, extension at 72°C for 1 min 30 sec and final extension at 72°C for 7 min. Target ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher) was used for enzymatic cleanup of the amplified PCR product. Sanger sequencing was performed using BigDye®Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Inc.) at the commercial centre (Geneombio Technologies, Pune).