Pcr clean up gel extraction nucleospin gel

PCR products were purified using the PCR Clean-Up Gel Extraction NucleoSpin^® Gel (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. The purified PCR products were directly sequenced. Sequencing reactions were performed and the sequences were then automatically determined in a genetic analyzer at the 1st Base Company Co., Ltd., (Kembangan, Malaysia) using EF1/EF2, CAL-228F/CAL-2Rd and RPB2-5F2/RPB2-7cR primers for tef-1, cam, and rpb2, respectively.

The genomic DNA of each week-old fungal isolate cultivated on PDA at 25 °C was extracted using a DNA extraction kit (FAVORGEN, Ping-Tung, Taiwan). Polymerase chain reaction (PCR) was employed to amplify the tef-1, cam, and rpb2 genes using the primer pairs EF1/EF2 [87 (link)], CAL-228F/CAL-2Rd [88 (link)], and RPB2-5F2/RPB2-7cR [65 (link)], respectively. The three genes’ amplification programs were carried out in independent PCR reactions, consisting of an initial denaturation for 3 min at 95 °C, followed by 35 cycles of denaturation for 30 s at 95 °C, annealing steps for 50 s at 60 °C (tef-1), 30 s at 59 °C (cam) or 1 min at 52 °C (rpb2), and a final extension step for 1 min at 72 °C on a peqSTAR thermal cycler (PEQLAB Ltd., Fareham, U.K.). PCR products were checked and purified using a PCR clean-up Gel Extraction NucleoSpin^® Gel and a PCR Clean-up Kit (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. Following final purification, the PCR products were directly sequenced. Sequencing reactions were carried out and the above-mentioned PCR primers were employed to automatically determine the sequences in the Genetic Analyzer at the 1st Base Company (Kembangan, Malaysia).