Agarose gel

Genomic DNA of C. perfringens was extracted from overnight FTA broth culture inoculated with a single colony plate by Ultraclean Microbial DNA Isolation Kit (MoBio, Germantown, MD, USA), adhering to the manufacturer’s directives with minor modifications to attain high concentration. In brief, the bacterial suspension and lysis buffer mixed in an additional 20 μL of 20 mg/mL proteinase-K (Fisher Scientific Waltham, MA, USA). The reaction mixture was then incubated at 65 ℃ for 15 min to lyse the bacterial cell wall and to preclude DNase digestion to bacterial DNA. In order to prevent DNA degradation, a tube with a reaction mixture was placed on a flat pad horizontally and vortexed for 10 min. The serial dilution of DNA was performed with 25 μL 10 mM Tris- HCl (pH 8.0), and segregated on a 0.8% agarose gel (BD Biosciences, Franklin Lakes, NJ, USA). The quality assertion and purity of extracted DNA was determined by NanoDrop™ 2000 (Thermo Scientific Inc. Waltham, MA, USA). The absorbance ratio at 260 nm and 280 nm was employed to estimate protein contamination and the recommended purity level was at the A260/A280 ratio of 1.8 to 2.2. The DNA was stored at −20 ℃ for further genotyping analysis.

Total genomic DNA was isolated from approximately 250 mg of fecal contents using the MOBIO PowerFecal DNA Isolation Kit (Mobio, Germantown, Maryland, USA) following the manufacturer’s protocol with some modifications. After adding bead solution and lysis buffer, the mixture was heated in a water bath at 65°C for 30 minutes followed by 5 minutes of vortexing. The concentration and quality of harvested DNA were determined by NanoDrop One Microvolume UV-Vis Spectrophotometer (Fisher Scientific) and visualized on 0.8% agarose gel (BD Biosciences, San Jose, California, USA). Afterward, genomic DNA was stored at -20°C until further analysis.