The DNA of isolated strains showing phenotypic resistance to beta-lactams and carbapenems was extracted using “Zymo DNA Mini Kit” extraction kits according to the manufacturer’s instructions. PCR was used to detect beta-lactam resistance genes (blaTEM, blaSHV, blaOXA-1, blaCTXM-1, blaCTXM-9, and blaCTXM-15) and carbapenem resistance genes (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48) following the PCR conditions described by Dallenne et al. (33 (link)), Memariani et al. (34 (link)), and Cerezales et al. (35 (link)). All primer sequences are listed in Table 1 . DNA fragments were analyzed by electrophoresis on a 2% agarose gel for 30 min at 100 V using BioRad Horizontal Electrophoresis System. The migration was imaged using the Biorad Gel Doc EQ imaging system, followed by interpretation of the results based on comparison of the migration of the fragments to the marker sizes (100 bp).
Horizontal electrophoresis system
Manufactured by Bio-Rad
Sourced in United States
The Horizontal electrophoresis system is a lab equipment used to separate biomolecules, such as DNA, RNA, or proteins, based on their size and charge. It provides a consistent and controlled environment for the electrophoretic separation process.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Gel doc eq imaging system, by Bio-Rad (1 mentions)
Baseline zero dnase, by Illumina (1 mentions)
Te buffer, by Merck Group (1 mentions)
Ultraviolet spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Beadbug homogenizer, by Benchmark Scientific (1 mentions)
Beadbug tubes, by Merck Group (1 mentions)
5 protocols using horizontal electrophoresis system
1
Molecular Detection of Beta-lactam and Carbapenem Resistance
2
Isolation and Purification of In Vitro Transcribed RNA
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The most active variants identified during the screening were in vitro transcribed as before but in a larger reaction volume (500 µL) and for 3 h at 37°C. DNA was then removed by adding 10 units of Baseline Zero DNase (Epicentre) and the corresponding buffer at the recommended working concentration. After an extra hour at 37°C, proteins were eliminated by phenol extraction and RNA recovered by ethanol precipitation and centrifuged for 20 min at 20,000g. Dry pellets were suspended in 20% glycerol, 0.02% xylene cyanol, 0.02% bromophenol blue, 1× TBE and 8 M urea, and the solution loaded on a 12% acrylamide/bisacrylamide (19/1), 8 M urea denaturing gels. The position of the RNA was determined by UV-shadowing and the corresponding piece of gel excised and transferred into a dialysis tube (MWCO = 3500, Spectrum Labs) with TE buffer (Sigma-Aldrich). Dialysis tubes were closed, placed in a horizontal electrophoresis system (Bio-Rad) and subjected to a 100V DC field for 1 h. Residual gel fragments were removed from aqueous phases using 0.45 µM microfuge filters (VWR). RNA was then recovered by ethanol precipitation, dry pellets dissolved in DEPC-treated water and quantified with a Nanodrop Spectrophotometer (Thermo Scientific).
3
Agarose Gel Electrophoresis of PCR Products
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PCR products were run on a 3% agarose gel in 1×TAE buffer. The horizontal electrophoresis system and power supply were obtained from Bio-Rad (Hercules, CA). The potential was set at 75 V.
4
Plasmid Extraction and Visualization
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The plasmid pattern of the recombinant strain was evaluated for confirming the transformation process. The Plasmid was extracted from fresh cultured strains using FavorPrep™ Plasmid Extraction Mini Kit (Pintung, Taiwan) according to the manufacturer’s instructions. Horizontal electrophoresis system (Bio-Rad USA) was used to visualization of plasmid pattern by agarose gel electrophoresis. In addition, plasmid DNA quality was measured by an ultraviolet spectrophotometer (Nano drop Technologies, Inc., Wilmington, DE, USA) at 260 and 280 nm.
5
Adipose Tissue RNA Extraction and Analysis
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The extraction and purification of RNA from adipose tissue were carried out using the E.Z.N.A Total RNA Kit II (Omega, Bio-tek, Inc., Norcross, GA, USA) following the manufacturer’s recommendations with 200 mg of adipose tissue, adding 20 μL of 2-mercaptoethanol (Sigma-Aldrich M6250, St Louis, MO, USA) to inhibit DNases and RNases. A BeadBug homogenizer (Benchmark Scientific, Inc., Sayreville, NJ, USA) was used to disintegrate the tissue into BeadBug tubes with 1 mm zirconium beads (Sigma-Aldrich Z763780, St. Louis, MO, USA). RNA concentration (OD-260) and purity (OD-260/OD-280) were determined by depositing 1 µL of RNA on the UV-Vis NanoDrop spectrophotometer (Thermo Scientific ND1000, Wilmington, DE, USA).
To verify the integrity of the RNA, 1% agarose (BioRad 1613102, Hercules, CA, USA) gel electrophoresis was performed using a BioRad horizontal electrophoresis system (BioRad 1704467, Hercules, CA, USA).
To verify the integrity of the RNA, 1% agarose (BioRad 1613102, Hercules, CA, USA) gel electrophoresis was performed using a BioRad horizontal electrophoresis system (BioRad 1704467, Hercules, CA, USA).
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