Sybr premix ex taq ex taq 2

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate buffer solution (PBS) (pH = 7.2), Diethyl Pyrocarbonate (DEPC)-treated water (Ambion), and TRIzol reagent (Invitrogen) were purchased from Gibco (Thermo Fisher Scientific, Shanghai, China); ethanol (70%), isopropyl alcohol, and Triton X-100 were bought from Sigma-Aldrich (Shanghai, China); Cell Counting Kit-8 (CCK-8) was bought from Dongren Chemical Technology (Shanghai, China); GV358-PCDH9 lentivirus and GV358-siRNA (short interfering RNA) lentivirus were designed by GeneChem (Shanghai, China); SYBR^® Premix Ex Taq™ Ex Taq ™ II and PrimeScript™ RT reagent Kit with gDNA Eraser were bought from Takara Bio Inc. (Beijing, China); water was obtained from EPED-20TF (Nanjing, China).

To mimic real conditions, the expression levels of porin genes and conjugation related genes in the mixture of donor and recipient bacteria (1:1 ratio at a ensity of 1 × 10⁸ CFU/mL) treated with sub-MIC colistin at 30°C for 12 h were investigated (10 (link)). The EASYspin bacterial RNeasy minikit was used to extract total RNA. The extracted RNA was reverse transcribed into cDNA using a PrimeScript RT reagent kit (TaKaRa, Japan). Real-time quantitative PCR (qPCR) was used to quantify gene expression using SYBR Premix Ex TaqEx Taq II (TaKaRa, Japan). The expression levels of porin forming related genes: ompF, ompC; ROS related genes: sodA, sodC; mating pair formation genes: korA, korB, trbA, traF, trbBp and traJ were determined. 16S rRNA was used as the internal control. Primer sequences are listed in Table S1. The qPCR primers were synthesized by Qinke Biotech Co., Ltd. (Nanjing, China), and qPCR was performed using a Mini Opticon real-time PCR system.