Turbo dnase from turbo dna free kit

For the isolation of total RNA, the previously described [26 (link)] procedure was employed. Briefly, the prophage induction was performed with 1 mM H₂O₂ or UV irradiation (at the dose of 50 J/m²). Following induction, the samples were withdrawn at indicated times and the growth of bacteria was inhibited by the addition of NaN₃ (Sigma-Aldrich) to a final concentration of 10 mM. Total RNA was isolated from 10⁹ bacterial cells by using the High Pure RNA Isolation Kit (Roche Applied Science). Bacterial genomic DNA carryover was removed by incubation with TURBO DNase from TURBO DNA-free Kit (Life Technologies) for 60 min at 37°C, according to the manufacturer's guidelines. To evaluate the quality and quantity of the isolated RNA, a NanoDrop spectrophotometer was employed, considering the absorbance ratio (which should be 1.8 ≤ A260/A280 ≤ 2.0). Moreover, band patterns of total RNA were visualized by electrophoresis. The absence of DNA from RNA samples was controlled by PCR amplification and by real-time PCR amplification (all analyzed genes were tested). RNA preparations were stored at −80°C. cDNA was obtained with Transcriptor Reverse Transcriptase and random hexamer primers (Roche Applied Science), using total RNA samples (1.25 μg) as templates. cDNA reaction mixtures were diluted 10-fold for use in real-time PCR.

For the preparation of RNA, the induction of temperate bacteriophages from E. coli strain MG1655 was performed with mitomycin C (final concentration 0.2 µg/ml) or H₂O₂ (final concentration 1 mM) as described in previous subsections. To inhibit the growth of bacteria, all samples were treated with NaN₃ (Sigma-Aldrich) to a final concentration of 10 mM. Total RNA was isolated from 1×10⁹ bacterial cells with the High Pure RNA Isolation Kit (Roche Applied Science). RNA preparations were repeatedly digested with TURBO DNase from TURBO DNA-free Kit (Life Technologies) for 60 min at 37°C, as described by the manufacturer. To evaluate the quality and quantity of total isolated RNA, we used a NanoDrop spectrophotometer, considering the ideal absorbance ratio (1.8≤A260/A280≤2.0), and visualized the band patterns of total RNA by electrophoresis. The absence of DNA from RNA samples was controlled by PCR amplification, and by real-time PCR amplification of the all tested genes. RNA preparations were stored at −80°C for use. The preparation of cDNA from the total RNA samples (1.25 µg) was performed with Transcriptor Reverse Transcriptase and random hexamer primers (Roche Applied Science), following the instructions supplied by the manufacturer. cDNA reaction mixtures were diluted 10-fold for use in real-time PCR.