Dna extraction, , PCR

Zhou, Yifan, Liu, Jingwen, Syed, Asad, Liu, Jian-Kui & Maharachchikumbura, Sajeewa S. N., 2025, Drumopama girisa: A rare hyphomycete from bamboo, resolved within Xylariales through morphological and molecular data, Phytotaxa 696 (1), pp. 71-82 : 72

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from fungal mycelium using the TreliefTM Plant Genomic DNA Kit (TSINGKE Biotech, Shanghai, China) following the manufacturer’s protocol. Four partial loci, ITS5/ITS4 for the nuclear ribosomal internal transcribed spacer (ITS) ( Vilgalys & Hester 1990), LR0R/LR5 for the partial nuclear ribosomal large subunit rRNA (LSU) ( Vilgalys & Hester 1990), RPB2-5F/RPB2-7cR for the partial second-largest subunit of RNA polymerase II (RPB2) ( Liu et al. 1999), Bt-2a/Bt-2b for the partial beta-tubulin (TUB) ( Glass & Donaldson 1995). The polymerase chain reaction (PCR) was carried out in a 25 μL reaction volume containing 12.5 μL PCR Master Mix (Sangon Biotech, Shanghai, China), 9.5 μL double-distilled water (ddH 2 O), 1μL of DNA and 1 μL of each primer. The amplification condition for all four loci was consisted of initial denaturation at 94 °C for 3 min; followed by 35 cycles of 30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C, and a final extension period of 10 min at 72 °C. The PCR products were analyzed by electrophoresis in 1 % agarose gels. The PCR products were submitted to Beijing Tsingke Biological Engineering Technology and Services Co. Ltd (Beijing, China).