Sybr pcr master mix

C. difficile cultures were grown in TY medium, and 6 ml of cells were harvested at different time points by centrifugation at 3000g for 30 min at 4°C. Total RNA was extracted from the harvested cells following the protocol described previously (El Meouche et al., 2013 (link)). Total RNA was treated with DNase (Turbo; Ambion) for 2 hours at 37°C. The cDNA was synthesized using 5μg of total RNA at 42°C for 2 h using avian myeloblastosis virus (AMV) reverse transcriptase (Promega). A 20 µL reaction containing 10 ng or 10 pg (for 16S rRNA) of cDNA, 400 nM gene-specific primers, and 12.75 µL of SYBR PCR master mix (BioRad) was used to perform real-time quantitative PCR using iQPCR instrument (BioRad). Amplification and detection were performed as described previously (El Meouche et al., 2013 (link)). The quantity of cDNA of a gene in each sample was normalized to the quantity of C. difficile 16S rRNA gene, and the ratio of normalized target concentrations (threshold cycle [2^−ΔΔCt] method) (Saujet et al., 2011 (link); El Meouche et al., 2013 (link)) gives the relative change in gene expression. The cDNA prepared was also used as a template to perform reverse transcriptase PCR to detect pdcB transcripts using forward primers ORG925, ORG926, ORG921, ORG922, along with the reverse primer ORG 853 (Table S2).