The potential interaction of the complexes with DNA was evaluated through the assessment of the electrophoretic mobility of supercoiled ϕX174 DNA, using a previously described method [46 (link)]. To that end, a mixture was prepared containing 200 ng of supercoiled ϕX174 DNA (Promega) in 10 mM of phosphate buffer (pH 7.2) and increasing concentrations of Cisplatin, as a positive control, or the metal complexes (in a total volume of 20 μL). The mixture was incubated for 24 h at 37 °C, in the dark. As controls, samples having non-incubated plasmid and plasmid incubated with DMSO were also prepared. Then, all samples were prepared for electrophoresis through the addition of 2 μL of 10× DNA loading buffer (Applichem) and loading on an 0.8% agarose gel in TBE buffer (Thermo Fisher Scientific, Waltham, MA, USA). The gel was run at 90 V for approximately 3 h, stained using a 3× GelRed® (Biotium) solution in H2O, and the bands visualized using an AlphaImagerEP (Alpha Innotech) under UV light.
Supercoiled x174 dna
Manufactured by Promega
Supercoiled ϕX174 DNA is a plasmid-based DNA sample used as a reference material in various molecular biology applications. It provides a well-characterized and consistent source of circular, supercoiled DNA. This DNA sample is commonly used as a control or standard in experiments involving DNA analysis, quantification, or manipulation.
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2 protocols using supercoiled x174 dna
1
Evaluating DNA Interaction of Metal Complexes
2
DNA Interaction Assessed by Electrophoresis
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DNA interaction was assessed by monitoring the electrophoretic mobility of the supercoiled ϕX174 DNA, as previously described [53] (link). Briefly, each reaction mixture contained, in a final volume of 20 µL, 200 ng of supercoiled ϕX174 DNA (Promega) in 10 mM phosphate buffer (pH 7.2) with different concentrations of cisplatin, auranofin, or the metal complexes. After incubation in the dark for 24 h at 37 ºC, 2 μl of 10× DNA loading buffer (Applichem) was added to each tube and the samples were loaded on an 0.8% agarose gel in TBE buffer (Thermo Fisher Scientific). The electrophoresis was carried out for about 3 h at 90 V. Samples with non-incubated plasmid and plasmid incubated with DMSO were used as controls. The gels were then stained in a 3× GelRed® (Biotium) staining solution in H 2 O, according to the manufacturer's instructions. Bands were visualized using an AlphaImagerEP (Alpha Innotech) under UV light.
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