North2south chemiluminescence hybridization and detection kit

The upstream regions of the toxP, cbuA0029, and groEL genes were first selected for PCR amplification. PCR products (1 μg) of desired templates were 3′ end‐labeled using a Pierce biotin 3′ End DNA Labeling Kit (Thermo Scientific). The resulting probe reaction mixtures were electrophoresed on a 0.8% agarose gel for 30 min at 100 V and then gel purified with a NucleoSpin Gel and PCR Clean‐up kit (Takara Bio USA). The EMSA binding reaction, consisting of 2.5% glycerol, 5 mM MgCl2, 50 mM KCl, 1 nM biotin‐labeled DNA, and varying concentrations of either ToxP or AntitoxP/ToxP in 1X Binding Buffer (LightShift Chemiluminescence kit; Thermo Scientific), was assembled and incubated at room temperature for 30 min. A nondenaturing loading dye (0.25% bromophenol blue) was added, and the resulting RNA mixtures were resolved on a 10% polyacrylamide gel for 2 h at 100 V. DNA/protein complexes were transferred to a Hybond‐N+ positively charged nylon membrane (Amersham Pharmacia Biotech) using an electroblot transfer system (Bio‐Rad) and cross‐linked with short‐wave UV light in a GS gene linker UV chamber (Bio‐Rad). A North2South chemiluminescence hybridization and detection kit (Thermo Scientific) was used to detect resulting bands. The blot was imaged on a UVP ChemStudio PLUS Imager (Analytik Jena).

Total RNA (30 mg) was denatured at 95°C for 5 min and then separated with a 12% denatured polyacrylamide–7 M urea gel (100 V, 1.5 h) in 1× Tris-borate-EDTA (TBE) and transferred onto a nylon membrane in 0.5× TBE. The product was then immobilized by UV cross-linking and blotted with the biotin-labeled oligonucleotide probes. RNA-DNA hybridization detection using a North2South chemiluminescence hybridization and detection kit (Thermo Scientific) was performed to detect crRNAs.