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Methods and Protocols for In Vitro Animal Nanotoxicity Evaluation: A Detailed Review
Published in Vineet Kumar, Nandita Dasgupta, Shivendu Ranjan, Nanotoxicology, 2018
Venkatraman Manickam, Leema George, Amiti Tanny, Rajeeva Lochana, Ranjith Kumar Velusamy, M. Mathan Kumar, Bhavapriya Rajendran, Ramasamy Tamizhselvi
Comet assay is a single cell gel electrophoresis assay which is performed to check the breaks in the DNA strands in eukaryotic cells. In genotoxic evaluation of foreign materials, comet assay is commonly used, and with respect to nanomaterials, it has been extensively used to screen the genotoxicity from titanium dioxide nanoparticles, silver nanoparticles, and magnetic nanoparticles (Figure 12.15). During inflammatory and oxidative stress, as a balancing measure it is common to see cellular cytotoxicity from apoptotic death. DNA laddering assay is an agarose gel electrophoresis based visualization technique in which DNA fragments resulting from apoptosis are visualized. In apoptosis, caspase-activated DNase (CAD) cleaves DNA at inter-nucleosomal linker regions. Resulting DNA fragments of 180–185 bps and higher multiples of them are considered to be a key event during apoptosis. These fragmented DNA can be separated and visualized by using agarose gel electrophoresis, resulting in a characteristic “ladder” pattern.
Basic Molecular Cloning of DNA and RNA
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
The plasmid map can be used in several ways. For screening, a small amount of plasmid (20–100 ng) is cut with one or more enzymes, resulting in two or more fragments of specific sizes. These fragments are then separated by a technique called agarose gel electrophoresis, which is based upon two principles: the negative charge of DNA and the ability of 0.5–2% solutions of agarose to gel into a hydrogel with pore sizes that limit the diffusion of linear DNA molecules according to size. Thus, if DNA is placed into the gel in an electrolyte solution and an electric field is applied, the fragments will migrate toward the cathode as a function of size. The precise physics behind the migration has not been determined (see Advanced Topic 2.1), but linear DNA of molecular weight MW migrates at a speed proportional to log−1(MW) (Figure 2.5b). The DNA is visualized using a fluorescent intercalating dye, usually EtBr (Figure 2.5c). Commercial ladders are available that provide standardized weight markers at 100- or 1000-base-pair increments or some other calibrated values (Figure 2.5c). The positions of the fragments on the ladders can be compared with the restriction map to make sure that they are consistent. The brightness of each piece corresponds to the mass of DNA present, allowing for a rough estimate of concentration by comparing the brightness of the screened plasmid’s bands with the known amount of DNA supplied in the ladder.
Laboratory tutorials
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Agarose gel electrophoresis is the easiest and commonest way of separating and analyzing DNA. The purpose of the gel might be to look at the DNA, to quantify it, or to isolate a particular band. The DNA is visualized in the gel by addition of ethidium bromide (EtBr). This binds strongly to DNA by intercalating between the bases and is fluorescent, meaning that it absorbs invisible UV light and transmits the energy as visible orange light.
Groundwater, soil and compost, as possible sources of virulent and antibiotic-resistant Pseudomonas aeruginosa
Published in International Journal of Environmental Health Research, 2021
Edit Kaszab, Júlia Radó, Balázs Kriszt, Judit Pászti, Virág Lesinszki, Adám Szabó, Gergő Tóth, Ariane Khaledi, Sándor Szoboszlay
Haemolytic activity was determined on Columbia agar with 5% sheep blood (Chemium Ltd., Hungary) with incubation parameters recommended by the manufacturer (37°C, 22 h). Microbial DNA was isolated by UltraClean Microbial DNA isolation kit (MoBio), following the manufacturer’s instructions. Virulence genes (effector protein-encoding genes exoS and exoU, elastase producing lasB, alginate encoding algD, alkaline phosphatase producing aprA and haemolytic phospholipase encoding plcH) were amplified using PCR with reaction parameters and primers described by Ullah et al. (2017), Badamchi et al. (2017) and Ajayi et al. (2003). Results were visualized by agarose gel electrophoresis. Positive controls were P. aeruginosa ATCC 27853 for exoS, lasB, algD, aprA and P. aeruginosa KPS-3 (a nosocomial strain isolated in Hungary) for exoU and plcH genes.
Synthesis, spectral-characterization, biological and DFT studies of mixed ligand metal(II) complexes of 1,10-phenanthroline bearing 2-aminothiazole moiety
Published in Inorganic and Nano-Metal Chemistry, 2020
Chellaian Justin Dhanaraj, Dharmasingh Sobhanabai Remya
Agarose gel electrophoresis is a technique for separating as well as visualizing DNA fragments. Plasmid DNA nicking assay was performed using pUC18 plasmid DNA. A mixture of 10 μl of extract (10 μg) and plasmid DNA (10 μl) was incubated at room temperature for 30 min at 37 °C. A tube with plasmid was kept as negative control and one with 10 μl of Fenton’s reagent as a positive control. The DNA samples were undergone electrophoresis on 1% agarose gel. The fragments are broken up by charge as well as the size and then move toward the agarose gel matrix when settled into an electric field. The electric field is produced by giving potential across the electrolyte (buffer). 6 µl of 10 mg/ml of ethidium bromide with gel comb was added into the gel casting apparatus. After setting, the comb and the gel were separated. The buffer has flowed through the gel tank and the platforms were placed with the gel. The gel was filled with the samples and run at 50 V for half an hour. The marked gel was visualized using a gel documentation system.
Investigation of catalytic, anti-bacterial, anti-oxidant, and DNA cleavage properties of bimetallic and trimetallic magnetic nanoalloys base on cupper
Published in Inorganic and Nano-Metal Chemistry, 2018
Kaveh Parvanak Boroujeni, Ahmad Farokhnia, Mansooreh Shahrokh, Mohsen Mobini
After evaluation of antibacterial and antioxidant activities of the Cu–Ni and Cu–Ni–Ag NPs, we also examined DNA cleavage property of these NPs. The cleavage of plasmid DNA was revealed by gel electrophoresis. In agarose gel electrophoresis method DNA molecules migrate in the gel texture based on their charge, size, and topology. Figure 10. shows gel electrophoretic pattern of DNA after incubation with distinct concentrations of Cu–Ni and Cu–Ni–Ag NPs in contrast to plasmid DNA mixed with water as the negative control. After exposure of plasmid DNA to different concentrations of the nanomaterial suspensions no destructive effects were observed on DNA.