Hieff first strand cdna synthesis super mix for rt pcr kit

The bacteria was cultured in 3 ml TSB in tubes (15 ml) with shaking (200 rpm) at 37°C and harvested at stationary phase (OD₆₀₀ ≈2). The Mini-Beadbeater (Biospec Products) was used for cell disruption at maximum speed for 30 s. After incubation on ice for 5 min, the suspension was centrifuged. The supernatant was used for RNA extraction according to the manufacturer’s instructions (Qiagen). Totally, 1 μg of total RNA was used for DNA removal using a TURBO DNA-freeTM kit (Ambion). The HieffTM first Strand cDNA Synthesis Super Mix for RT-PCR kit (Yeasen Bio, China) was used for cDNA synthesis. The following real-time PCR was performed by SYBR-Green PCR reagents (Roche) using cDNA as a template on a 7500 Sequence Detector (Applied Biosystems). Primers used for RT-PCR are listed in Supplementary Table S2. All RT-PCR experiments were performed using gyrB as an internal control.

Cells in 3 ml TSB cultures were harvested at the stationary phase (OD₆₀₀ value of 2). Cells were disrupted by shaking with a Mini-Beadbeater (Biospec Products) at maximum speed for 30 s. Tubes were then incubated on ice for 5 min. Then, the suspension was centrifuged. Total RNA isolation from the supernatant was performed according to the manufacturer's instructions (Qiagen). After treatment using a TURBO DNA-freeTM kit (Ambion), ~1 μg of total RNA was reverse-transcribed with a HieffTM first Strand cDNA Synthesis Super Mix for RT-PCR kit (Yeasen Bio). The cDNA was used as a template for real-time PCR using SYBR-green PCR reagents (Roche). Reactions were performed in a MicroAmp Optical 96-well reaction plate using a 7,500 Sequence Detector (Applied Biosystems). Primers used are listed in Supplementary Table 2. All RT-PCR experiments were performed in triplicate, with gyrase B (gyrB) used as an internal control.