Iscript genomic dna clear cdna synthesis kit

Frozen stocks of D344RRF, C68, and TC-A before passaging (passage 0 [P0]) and after passaging (P9 and P13) were used to inoculate an overnight BHI culture. On the next morning, the cells were diluted 1:1,000 and were grown with shaking at 37°C to an optical density at 600 nm (OD₆₀₀) of 0.2. At that point, cultures were treated with either ampicillin, vancomycin, or no antibiotic to a final concentration of half the MIC value and were grown to an OD₆₀₀ of 0.6 (about 4 h) with shaking. Cells were broken open with glass beads (Lysing Matrix B; MP Biomedical) using a mini-BeadBeater (BioSpec), and the RNA was purified using a Qiagen RNeasy minikit. cDNA was synthesized using a Bio-Rad iScript genomic DNA Clear cDNA synthesis kit. Quantitative PCR was carried out using a Bio-Rad iTaq universal probes kit in a multiplexed reaction in a CFX98 real-time PCR cycler. Relative gene expression was calculated using the ΔΔC_q quantification cycle (C_q) method and normalized to the expression of 16S rRNA (25 (link)). To compare expression levels, we did a one-way analysis of variance using Prism (v7) software (GraphPad Software Inc.). The primers and probes used for the experiment are listed in Table S4 in the supplemental material.