Sarcoma 180 tumor cells (1 × 106 cells/mL) were seeded in 12-well culture plate with 250 µL of medium RPMI-1640 (10% v/v of FCS) and were treated for 24 h with complex 1 at 1 µM, 4 µM and 20 µM. The genomic DNA (gDNA) was extracted using the universal Quick-DNATM kit (Zymo research-USA), and quantified in NanoDrop (Thermo Fisher Scientific). In order to verify gDNA degradation, 50 ng of gDNA extracted from each treatment was analyzed in an agarose gel (0.8%) stained with ethidium bromide. The electrophoresis was carried out for 2 h at 80 V and the DNA bands were visualized using a UV transilluminator Image Quant 150 (GE Healthcare) and photographed.
Imagequant 150
Manufactured by GE Healthcare
Sourced in Belgium
The ImageQuant 150 is a compact and versatile imaging system designed for quantitative analysis of a wide range of sample types, including gels, blots, and cell-based assays. It features a high-resolution CCD camera, optimized optics, and intuitive software for capturing and analyzing images.
Automatically generated - may contain errors
3 protocols using imagequant 150
1
Sarcoma 180 Cells DNA Degradation
2
Amplification of toxR Gene by PCR
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Primers specific to toxR gene were purchased from SGP Biodynamics (Sofia, Bulgaria) (Table 1 ). The reaction mixture for PCR had volume of 25 μl and contained 2 μl of the extracted DNA, 12.5 μl TopTaq Master Mix (QIAGEN, Germany), 0.2 μl of each primer and 10.1 μl of water free from nucleases. Polymerase chain reaction took place in thermocycler QB-96 (Quanta Biotech, USA) with the following programme: i) initial denaturation at 94°C for 5 min, followed by 30 cycles of ii) denaturation at 94°C for 1 min., iii) annealing at 63°C for 2 min., iv) extension at 72°C for 1.5 min., v) final extension at 72°C for 10 min. The separation of the amplified DNA fragments was done by horizontal electrophoresis in 2% agarose gel TopVision Agarose (Thermo Scientific, USA) at 100 V for 1.30 hours. The gel was stained with safe dye peqGREEN (VWR International, Belgium) and visualized and documented with UV Transilluminator (ImageQuant 150, GE Healthcare). To determine molecular weight marker Gene Ruler 100 bp DNA Ladder (Thermo Scientific, USA) was used.
3
Molecular Detection of Staphylococcus spp. and S. aureus
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Primers specific to 16S rRNA gene of Staphylococcus spp. and nuc gene of S. aureus were purchased from Eurofins Genomics (Germany) (Table 2 ). The reaction mixture for PCR had volume of 25 μl and contained 1 μl of the extracted DNA, 12.5 μl TopTaq Master Mix (QIAGEN, Germany), 0.2 μl of each primer (Eurofins Genomics, Germany) and 11.1 μl water free of nucleases. The polymerase chain reaction was performed in a thermocycler QB-96 (Quanta Biotech, USA) with the following programme: i) initial denaturation at 94°C for 5 min. followed by 30 cycles of ii) denaturation at 94°C for 1 min. iii) annealing at 55°C for 30 sec., iv) extension at 72°C for 1 min., v) final extension at 72°C for 7 min. The separation of the amplified DNA fragments was made by horizontal electrophoresis in 2% agarose gel TopVision Agarose (Thermo Scientific, USA) at 100 V for 1.30 hours. The gel was stained with peqGREEN (VWR International, Belgium) and visualized and documented with UV Transilluminator (ImageQuant 150, GE Healthcare). To determine the molecular weight Gene Ruler 100 bp DNA Ladder marker (Thermo Scientific, USA) was used.
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