Allprep bacterial dna rna protein kit

A 100 mL of TSB was inoculated with 20 mL of TSB containing CIMRSA XIII and subsequently treated with the corresponding treatments (Cipro, 1b, or 2b) at their GI₅₀s. A 25 mL of each cultured TSB was pelleted, and RNA was extracted using the AllPrep^® Bacterial DNA/RNA/Protein Kit (Qiagen, California, USA) following the manufacturer’s instructions. cDNA synthesis was prepared using the Power SYBR^™ Green RNA-to C_T^™ 1-Step Kit (Applied Biosystems, CA, USA) with the following primers: 5’-ATGGAAAAGCCGTCAAGAGA-3’ (forward primer) and 5’-AACCAATGATTGTGCAAATAGC-3’ (reverse primer) according to the manufacturer’s instructions. The resulting cDNA templates were quantitatively amplified in a Step One Real-Time PCR System (Applied Biosystems, California, USA). The cycling parameters included: initial denaturation at 95°C for 4 min, followed by 40 cycles of denaturation (95°C, 45s), annealing (57°C, 45s), extension (72°C, 55s), and final extension at 72°C for 2 min. Efflux pump genes’ relative expression was calculated using the ΔΔC_T method, in which the amount of the desired cDNA was normalized to an endogenous reference (housekeeping gene 16S rRNA) and relative to an in vitro calibrator, is given by the variable $2^{-\Delta {\Delta }_{\text{CT}}}$ , where C_T is the cycle number of the detection threshold.

To assess the effects elicited by varying concentrations of treatment compounds on total DNA, RNA, and protein levels during cell growth, a master culture of S. aureus ATCC 29213 was first inoculated at OD₆₀₀ 0.1 and allowed to grow to OD₆₀₀ 0.2 (early log phase) at 37 °C with agitation at 200 rpm. Upon reaching this stage, the cultures were divided into aliquots, where compounds and drugs were added at their corresponding 1/4 and 1/8 MICs, complete with an untreated control culture. The samples were then harvested upon reaching OD₆₀₀ 0.6 (the mid-log phase of S. aureus) at 3 mL. Other samples were harvested at volumes normalized to OD₆₀₀ 0.6, before being pelleted at 5000g for 5 min at 4 °C, and the supernatant discarded. Total DNA, RNA, and protein were extracted and purified using the AllPrep Bacterial DNA/RNA/Protein Kit (Qiagen) following the manufacturer’s guidelines. DNA and RNA levels were measured with a Qubit DNA BR Assay Kit (Invitrogen) and Qubit RNA BR Assay Kit (Invitrogen) coupled with a Qubit 4 Fluorometer (Thermo Fisher), while proteins levels were determined using an Pierce BCA Protein Assay Kit (Thermo Fisher). The experiment was performed in triplicates.

The effects of varying concentrations of treatment compounds elicited on total levels of DNA, RNA and protein during cell growth was assessed by first preparing a master culture of S. aureus ATCC 29213. First inoculated at OD₆₀₀ 0.1 and agitated at 175 rpm at 37 °C, its confluence was doubled to 0.2 (early log phase) before being divided into aliquots where compounds and control drugs were added at their respective ¼ and ⅛ MICs, complete with a drug-free control culture. Cells were harvested upon the control reaching an OD₆₀₀ of 0.6 (mid-log phase), where other samples were harvested at volumes adjusted to an OD₆₀₀ equivalent of 0.6. 3 mL cultures were pelleted at 5000 × g for 5 min at 4 °C and the supernatant discarded. The major macromolecules were extracted and purified using the AllPrep® Bacterial DNA/RNA/Protein Kit (Qiagen) following the manufacturer’s protocols, and the nucleic acid levels quantified with Qubit™ DNA BR Assay Kit (Invitrogen) and Qubit™ RNA BR Assay Kit (Invitrogen) in conjunction with a Qubit 4 Fluorometer (Thermo Fisher). Pierce BCA Protein Assay Kit (Thermo Fisher) was used to measure protein levels. The experiment was performed in triplicate.

Total RNA was purified using AllPrep Bacterial DNA/RNA/Protein Kit (QIAGEN) from cell pellets of triplicate cultures of B. dorei DSM 17855 grown in ΒΗΙ+ with or without 10 μM 3-oxoLCA for 1 hour. RNA was DNase treated, and cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Transcripts of interest were quantified by real-time PCR carried out using LightCycler 480 SYBR Green I Master (Roche Life Science). All qPCRs were normalized to 16S rRNA gene expression. Primers used are listed in Table S5.