Dna extraction, , PCR

Liu, Xiangfu, Tibpromma, Saowaluck, Gautam, Ajay K., Suwannarach, Nakarin, Elgorban, Abdallah M., Chukeatirote, Ekachai, Thilini Chethana, K. W. & Karunarathna, Samantha C., 2025, A novel species and a new host record of Trichoderma from caves in Yunnan Province, China, Phytotaxa 694 (2), pp. 119-143 : 121

DNA extraction, PCR amplification, and sequencing

Fresh mycelia were scraped from living cultures, which grew and incubated at room temperature for 15 days. Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, P.R. China) was used to extract DNA , following the manufacturer’s protocol. The extracted DNA was maintained at –20 °C for long-term storage. The polymerase chain reactions (PCR) were performed for three genes: internal transcribed spacer (ITS), RNA polymerase II subunit 2 (RPB2), and translation elongation factor 1-alpha ( TEF 1-α) gene, using the primers ITS5 and ITS4 ( White et al. 1990), fRPB2-5F and fPB2-7R ( Liu et al. 1999), and EF1 and EF2 ( O’Donnell et al. 1998), respectively. The PCR amplifications were carried out in a 25 µL reaction volume, containing 12.5 µL of 2 × Power Taq PCR Master Mix (Beijing Bomaide Biotechnology Co., Ltd., Haidian District, Beijing, China), 8.5 µL distilled-deionized water (ddH 2 O), 2 µL of DNA template, and 1 µL of each forward and reverse primer ( Tibpromma et al. 2018).

The amplification condition for ITS and TEF 1-α consisted of initial denaturation at 94 °C for 2 min, followed by 35 cycles of 30 s denaturation at 95 °C, 50 s annealing at 55 °C, and 1.5 min extension at 72 °C, and a final extension period of 10 min at 72 °C. The amplification condition for RPB2 consisted of initial denaturation at 94 °C for 2 min, followed by 35 cycles of 45 s denaturation at 95 °C, 50 s annealing at 58 °C, and 1.5 min extension at 72 °C, and a final extension period of 10 min at 72 °C. The PCR products were detected by 1% agarose gel electrophoresis stained with TS-GelRed (TSJ002, Beijing Kinco Biotechnology Co., Ltd. Kunming Branch, P.R. China). Purification and sequencing of PCR products were performed at the Tsingke Biological Engineering Technology and Services Co., Ltd (Yunnan, P.R. China).