Midi prep plasmid dna extraction kit

Isolates containing a subfamily of known qnrS1-containing transposons were detected by Southern blotting with two different probes: qnrS1 and bla_LAP-2, using the primers described for amplification of the qnrS1 region. Plasmid DNA from the transformants was extracted using a Qiagen Midi Prep Plasmid DNA Extraction kit, as per manufacturer's recommendations. These plasmids were then digested with EcoRI and duplicates were run on a gel. The gel was subsequently transferred to a membrane. The membrane was cut into two pieces and each was hybridized with one of the probes, qnrS1 or bla_LAP-2. If an isolate had signal with both probes binding to the same fragment, it was assigned as carrying a subfamily of a qnrS1-containing transposons.

PCR-negative isolates for the qnrS1-containing transposon were analysed for the presence of subfamilies of the known transposon. Plasmid DNA from these negative isolates was extracted using a Qiagen Midi Prep Plasmid DNA Extraction kit, as per the manufacturer's recommendations. Escherichia coli TOP10 cells (Invitrogen, Life Technologies) were transformed with isolated plasmid DNA by a Bio-Rad gene pulser, using conditions recommended by the manufacturer (Invitrogen, Life Technologies). Transformants were selected on Luria–Bertani medium supplemented with 0.03 mg ciprofloxacin l^− 1. Plasmid DNA from these transformants was extracted by the method of Kado & Liu (1981) (link), examined on an agarose gel for the presence of only one plasmid and then subjected to PCR amplification for the qnrS1 gene to ensure transformation of the appropriate plasmid.

Two strains of Escherichia Coli (E. coli) were used including DH5α and DH10β (Top10, Invitrogen, Thermo Fisher Scientific, USA). The plasmid was transformed into cells using the calcium chloride (CaCl₂) method.¹⁶ E. coli bearing the desired plasmid was cultured in lysogeny broth (LB) media for 16 hrs at 37 °C in a shaker incubator. Cells with a density of 3-4×10⁹ cells/ml with OD₆₀₀=3 were harvested and DNA was extracted by plasmid DNA extraction kit (Qiagen, Midiprep Plasmid DNA Extraction Kit).