Superscript 3 platinum sybr green one step qpcr mix

K. pneumoniae strains were grown to mid-log-phase (OD₆₀₀ = 0.8 to 1) and harvested by centrifugation (18,000 × g, 5 min) and resuspended in RNA Protect Bacteria Reagent (Qiagen). Total RNA was extracted using the RNeasy minikit, digested twice with on-the-column DNase I digestion, as per the manufacturer’s guidelines. Samples were quantified using the FLUOStar Omega Spectrophotometer.
Quantitative real-time PCR was performed on the QuantStudio 7 real-time PCR system (Thermo Fisher Scientific) using the SuperScript III Platinum SYBR Green one-step qPCR Mix according to the manufacturer’s instructions. Oligonucleotides used for qRT-PCR are listed in Table S2. The gene rpoD was used to normalize gene expression. The data represent at least three biological replicates.

K. pneumoniae strains were grown in Zn(II)-limited or Zn(II)-supplemented media at 37°C with aeration to mid-log phase (OD_{600 =} 0.6-0.8) and harvested by centrifugation at 18,000 × g for 5 min at room temperature. Cells were resuspended in RNA Protect Bacteria Reagent (Qiagen) and total RNA extracted using the RNeasy Mini Kit with two rounds of on-column DNase I digestion, as per manufacturer’s instructions, with final concentrations determined by spectrophotometry. Quantitative real-time-PCR (qRT-PCR) was performed on the QuantStudio 7 Real-Time PCR System using the SuperScript III Platinum SYBR Green One-Step qPCR Mix (ThermoFisher Scientific) using rpoD to normalize gene expression, essentially as described previously (Maunders et al., 2022 (link)). Oligonucleotides used are listed in Supplementary Table 2.