Tprofessional standard thermocycler

Genomic DNA was extracted from the voucher specimens using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co. Ltd., Shanghai, China), according to the manufacturer’s instructions. Primer pairs ITS5/ITS4 (White et al. 1990 (link)), LR0R/LR5 (Vilgalys and Hester 1990 (link)), and atp6-2/atp6-3 (Kretzer and Bruns 1999 (link)) were used for amplifying ITS, nrLSU and atp6, respectively. PCR reactions was performed in a total volume of 25 μl containing 0.5 μl template DNA, 11 μl distilled water, 0.5 μl of each primer and 12.5 μl 2 × PCR mix (DreamTaq^tm Green PCR Master Mix, Fermentas). Amplification reactions were performed in a Tprofessional Standard Thermocycler (Biometra, Göttingen, Germany) under the following conditions: 95°C for 4 min; then 35 cycles of denaturation at 94°C for 60s, annealing at 53°C for 60s, and extension at 72°C for 60s; with a final extension at 72°C for 8 min. The PCR products were electrophoresed on 1% agarose gels and sequencing was performed on an ABI Prism® 3730 Genetic Analyser (PE Applied Biosystems, Foster, CA, USA) at the Beijing Genomic Institute (BGI) using the same PCR primers. The raw sequences were assembled and checked with SeqMan implemented in Lasergene v7.1 (DNASTAR Inc., USA). The newly generated sequences in this study were submitted to GenBank.

The genomic DNA was extracted from the voucher specimens using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Primer pairs LR0R/LR7 [29 (link)], tef1F/tef1R or tef-1Fcanth/tef-1Rcanth [1 (link),30 (link)] and RPB2-5FCanth/RPB2-7cRCanth [1 (link)] were used to amplify the LSU, tef1, and rpb2 genes, respectively. The PCR reactions were performed in a total volume of 25 μL containing 0.5 μL template DNA, 11 μL distilled water, 0.5 μL of each primer, and 12.5 μL PCR mix [DreamTaqtm Green PCR Master Mix (2×), Fermentas, USA]. The amplification reactions were performed in a Tprofessional Standard Thermocycler (Biometra, Göttingen, Germany) under the following conditions: 95 °C for 4 min; then 35 cycles of denaturation at 94 °C for 60 s, annealing at 53 °C (LSU) /50 °C (tef1) /52 °C (rpb2) for 60 s, and extension at 72 °C for 60 s; with a final extension at 72 °C for 8 min. The PCR products were electrophoresed on a 1% agarose gel with known standard DNA markers and the sequencing was performed on an ABI Prism^® 3730 Genetic Analyzer (PE Applied Biosystems, Foster, CA, USA) at the Beijing Genomic Institute using the same primers. The raw sequences were assembled with SeqMan implemented in Lasergene v7.1 (DNASTAR, Madison, USA). The newly generated sequences in this study were submitted to GenBank.