Axygen dna gel extraction kit

Soil genomic DNA was extracted from 0.5 g fresh soil using a Fast DNA Spin Kit (Omega, Norcross, Georgia, United States) and stored at −20°C prior to further analysis. The quantity and quality of extracted DNA were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and agarose gel electrophoresis, respectively.
The V3–V4 variable regions of the 16S rRNA gene were amplified using the universal primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCRs were performed using the following procedure: denaturation at 98°C for 2 min, followed by 25 cycles of denaturation at 98°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final extension step at 72°C for 5 min. The amplification quality was checked and purified by 2% agarose gel electrophoresis and purified using an Axygen DNA gel Extraction Kit (Axygen Biosciences, Union City, CA, United States). Finally, purified amplicons were sequenced (2 × 300 bp) on an Illumina MiSeq platform with MiSeq Reagent Kit v3 (600 cycles) at Shanghai Personalbio Technology Co., Ltd. (Shanghai, China).

Protein marker (Biomedical Technology Co., Ltd., Beijing, China) was used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). KOD-Plus-DNA polymerase (Toyobo, Osaka, Japan) was used for all PCR reactions. An Axygen^® DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) was used for amplification following the manufacturer’s instructions. A site-directed mutation kit (Vazyme, Nanjing, China) was used for site-directed mutation. All other reagents were homemade pure reagents.