Genejet genomic dna purification

All CoPS strains were subcultured on TSA with 5% sheep blood at 37°C overnight. Bacterial DNA was extracted following the instructions from the kit (GeneJET Genomic DNA purification; Thermo Fisher Scientific, USA). A pair of primers was designed to amplify the 1,369 bp region of the 16S rDNA of S. schleiferi subsp. coagulans. Primer-1 (5′–GAACGGACAAGGAGCTTGCTCCTTTGAA-3′) and primer-2 (5′–TTACAAACTCTCGTGGTGTGAA-3′) correspond to the nucleotide residues 61–88 and 1,407–1,429, respectively. The PCR was performed in a 20 μl reaction volume. Each reaction mixture contained 1 μl (17–240 ng) of the sample DNA solution, 10 μl of 1X MasterMix (2XPCR MasterMix; abm, Canada), 1 μl (0.5 μM) of each primer, and completed with 7 μl of ultra-pure water. The amplification reaction was carried out in a standard thermal cycler (GenAmp Biometra; Gmbb Analytik Jena AG, Germany), using the following program: 1 minute at 94°C, followed by 30 cycles of 1 minute at 94°C, 1 minute at 57°C and 2 minutes at 72°C. The program was completed with an additional 2 minutes at 72°C. After the amplification, 5 μl of the reaction mixture was analyzed by electrophoresis on a 2% agarose gel in Tris-borate-EDTA buffer at 90 V for 90 minutes. A charge buffer Safe-Green was used with all the samples and a 100 bp ladder of 1.5 kb.