Dna extraction, , PCR

Lei, Lei, Luangharn, Thatsanee, Yu, Feng-Ming & Zhao, Qi, 2025, Two new species of Pseudohydnum (Auriculariales, Basidiomycota) from the Qinghai-Tibet Plateau in China, Phytotaxa 690 (2), pp. 243-254 : 244

DNA extraction, PCR , and sequencing

Genomic DNA was extracted from dried samples using the TriliefTM Plant Genomic DNA Kit (Tsingke Biological Technology Co., LTD, Beijing, China), following the manufacturer’s protocol. The primer pairs LR0R/LR7 ( Vilgalys & Hester 1990) and ITS5/ITS4 ( Gardes & Bruns 1993) were used to amplify the large subunit of the nuclear ribosomal region (LSU) and the internal transcribed spacers 1 and 2, including the 5.8S rDNA (ITS), respectively. The PCR amplification reactions were performed in a total volume of 25 μL, consisting of 2 μL of template DNA , 21 μL of 1.1× T3 Super PCR buffer (Tsingke Biological Technology Co., LTD, Beijing, China), and 1 μL of each primer. The PCR conditions were set as follows: initial denaturation at 95 ℃ for 5 min, followed by 35 cycles of denaturation at 95 ℃ for 25 s, annealing at 54 ℃ for 15 s (for ITS) and 50 ℃ for 30 s (for LSU), elongation at 72 ℃ for 30 s, and a final extension at 72 ℃ for 7 min. All PCR products were verified using 1% agarose electrophoresis gel, purified, and sequenced by Tsingke Biological Technology (Beijing, China).