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Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
Reagents 1% agarose prepared in 0.5× TAE buffer; microwave 1.5-2 minutes until all agarose has dissolved, taking care not to boil overRunning buffer: 0.5× TAE bufferLoading buffer, can be purchased commercially or prepared as a 30% glycerol and 0.25% bromophenol blue solution in deionized waterRNA or DNA ladder
Inorganic Particles against Reactive Oxygen Species for Sun Protective Products
Published in Claudia Altavilla, Enrico Ciliberto, Inorganic Nanoparticles: Synthesis, Applications, and Perspectives, 2017
Lee Wilson A., Raifailovich Miriam
A more dramatic magnitude of the free radical formation has been shown by Serpone et al. (2006) to result in damage to DNA. A solution containing λ-phage DNA (48,502 bp) in a concentration of 50 μg/mL in 1 X TE buffers, and added 2 mg/mL of either nano TiO2 (rutile) or surface modified nano TiO2 was prepared. Samples were placed 3 cm below UVA, UVB, and UVC light sources. The exposure times ranged from 1 to 4 h for different wavelengths. The gel electrophoresis was prepared with 0.8% (w/v) Agarose in 1 × TAE buffer and 5 V/cm of electric field was applied for 30 min. The results are shown in Figure 13.3. The control run on the far left column containing 1 kb ladder, which shows normal separation of digested DNA fragments. The adjacent column shows λ-DNA, which was not exposed. All the intensity remains in the input because λ-DNA is too large to elute through the gel. Exposure of the λ-DNA to UVA for 4 h does not seem to affect the intensity of the signal. A significant reduction is observed after exposure for 4 h in the presence of TiO2 uncoated particles, followed by a diffuse tail. This indicates that the DNA was broken forming short fragments, which were eluted rapidly in the channel. No effect is observed in the input containing the coated TiO2 particles. Exposure to UVB radiation for 4 h produced significant breakage in the column containing DNA and uncoated TiO2 particles; no DNA remains. Similarly, exposure to UVC for 1 h dramatically destroys DNA with and without the presence of TiO2 particles. On the other hand, it is surprising that the intensity of signal from the DNA remains nearly unchanged in the column where the coated TiO2 particles were added prior to irradiation.
DNA-analysis to study the microbial diversity in recharged groundwater
Published in Jos H. Peters, Artificial Recharge of Groundwater, 2020
B. Eschweiler, B. Kilb, B. Kuhlmann, G. Preuß, E. Ziemann
DGGE was performed with the Bio-Rad D GENE System. The polyacrylamide gels (7.5%) contained a denaturing gradient from 40 to 70% (100% denaturant: 7 M urea and 40% formamide) and were run in 0.5 x TAE buffer (40 mM Tris base [pH 7.4], 20 mM sodium acetate, 1 mM EDTA). The optimum denaturant gradient was determined by performing perpendicular DGGE according to the instruction manual (Bio-Rad Lab.). Electrophoresis was performed at constant voltage (70 V) and temperature (56°C) for 16 h. After electrophoresis the gels were stained with silver nitrate according to Heukeshoven and Dernick (1985), dried and photographed.
Synthesis, characterization, and biological evaluation of 4-[(4-hydroxy-7-methyl-1,3-benzothiazol-2-yl) diazenyl]-5-methyl-2-phenyl-2,4-dihydro-3-pyrazol-3-one and its metal complexes
Published in Journal of Coordination Chemistry, 2020
Mallikarjuna Niluvanji Matada, Keshavayya Jathi, Kiptoo Geoffry, Ravi Berenkere Nagarajappa, Harmesh Chander Tandon
The cleavage of supercoiled plasmid pUC18 DNA by all the synthesized compounds was carried out by gel electrophoresis method [27, 28]. This movement is retarded when they are bound to other molecules. In a typical experiment, 200 mg of agarose was dissolved in 25 mL of Tris-acetate (TAE) buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA) by boiling. When the gel attains approximately 55 °C, it was poured into the gel cassette fitted with a comb. The gel was allowed to solidify and then carefully the comb was removed. The gel was placed in the electrophoresis chamber flooded with TAE buffer. The synthesized compounds were dissolved in freshly distilled DMSO (1 mg mL−1). The test compounds were added separately to the isolated pUC18 DNA (225 ng) and incubated for 2 h at 37 °C. After incubation, 20 µL of DNA sample (mixed with bromophenol blue dye at 1:1 ratio) was loaded carefully into the wells, along with standard DNA marker and a constant electricity of 50 V was passed for around 45 min. The gel was removed and carefully strained with ethidium bromide (ETBR) solution (10 µg mL–1) for 10–15 min. The bands were observed under UV trans-illuminator (UVP, Germany) and photographed to determine the extent of DNA-cleavage, and the results were compared with those of a standard DNA marker.
Effect of alkaline treatment on pathogens, bacterial community and antibiotic resistance genes in different sewage sludges for potential agriculture use
Published in Environmental Technology, 2020
Bruna Coelho Lopes, Elayne Cristina Machado, Hortência Franco Rodrigues, Cintia Dutra Leal, Juliana Calábria de Araújo, Antonio Teixeira de Matos
Polymerase chain reaction (PCR) assays were used to conduct broad-scale screening of the presence of genes that encode antibiotic resistance in the sludge samples. The presence of two genes that encode resistance to erythromycin (ermB, ermC), two genes that encode tetracycline resistance (tetA, tetB), two genes that encode for β-lactam resistance (blaTEM, ampC), resistance to sulfamethoxazole (sulf1, sulf2) and two genes that encode for quinolone resistance (qrnA, qrnB) were investigated. Table 1 presents the primers used for PCR amplification of ARGs (detailed before) based on previously-published paper [27]. After DNA extraction, a 25-μL PCR reaction mixture was prepared for each sample, containing 12.5 μL of Premix 2x IB (Phoneutria, Brazil), 1.5 of μL BSA (5 ng. μL-1), 7.5 of μL nuclease-free water (Ambion, Life Technology, Frankfurt, Germany), 0.25 μL of forward and reverse primer (25 μM), and 3 μL of a template DNA (concentration 30 ng μL-1). PCR products were analysed by gel electrophoresis using 2% (w/v) agarose in a 1.0X TAE buffer. DNA bands were stained with a SYBR gold solution and then visualized by an LED transilluminator.
Influence of 2,4-D residues on the soil microbial community and growth of tree species
Published in International Journal of Phytoremediation, 2020
Luciana Monteiro Aguiar, José Barbosa dos Santos, Gabriela Madureira Barroso, Marcelo Luiz de Laia, Janaína Ferreira Gonçalves, Vitor Antunes Martins da Costa, Lílian Almeida Brito
The genetic profile of distinct groups of microorganisms was determined using PCR-Multiplex (Singh et al. 2006). PCR comprised 1X reaction buffer, 2mM MgCl2, 200 lM dNTPs of each deoxynucleotide, primers [200 μM 63f (10 μM), 200 nM 1087R (10 μM), 400 nM ITS1F (20 μM), 400 nM ITS4 (20 μM), 400 nM Ar3f (20 μM), 400 nM Ar927 (20 μM)], 2.5 U Taq polymesare enzyme, 2 μL template DNA and ultra-pure water to complete 50 μL of reaction. The DNA was amplified in a Biorad automatic cycler (MyCycler™ Thermal Cycler System) programed for 5 min followed by 30 cycles of denaturation (30 sec at 95 °C), annealing (1 min at 55 °C) and extension (1 min at 72 °C) and one final extension step (10 min at 72 °C W). The amplified product (2 μL aliquot) was evaluated by 1.5% agarose gel electrophoresis in 1X TAE buffer stained with Etho Bromide and analyzed under UV light. The amplified products were purified with the QIAquick PCR purification kit protocol reagent kit (QIAGEN), according to the manufacturer's guidelines. The purification product was subjected to enzymatic digestion using the restriction enzyme MspI (Promega, Madison, WI, USA). The digestion was conducted in a thermocycler at 37 °C for 3 h, followed by a 15 min enzyme inactivation period at 95 °C.