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HLA-DR and -DQ Typing by DNA-RFLP Analysis
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
Conditions are described for use with the Bio-Rad Wide Mini-Sub Cell (gel tray size 15 × 10 cm). Pour a 0.7% gel as follows: add 0.63 g high gelling temperature agarose (Sigma type V) to 90 ml 1x TAE buffer (lx TAE buffer = 40 mM Tris-acetate, 1 mM EDTA27). Boil to dissolve the agarose, using a magnetic stirring hotplate. Cool to 60°C, add 4.5 μl 10 mg/ml ethidium bromide (final conc 0.5 μg/ml), mix, and pour the gel. Allow the gel to set for 1 h at ambient temperature.Electrophorese samples for 1 h at 70 v in lx TAE buffer containing 0.5 μg/ml ethidium bromide. The level of buffer should be 2 to 3 mm above the gel surface.
Identification of HPV types 6 and 11 in skin tags using PCR
Published in Cut Adeya Adella, Stem Cell Oncology, 2018
J. Karayana, N.K. Jusuf, I.B. Putra
For analysis of the PCR product, 5-7 pl amplification product were electrophoresed on 2.5% agarose gel (Bioron®) in Tris Acetate EDTA (TAE) buffer with 1 ul ethidium bromide (Invitrogen®). Gel was visualised on an ultraviolet transilluminator; visualising the 258-361 bp fragment was interpreted as a positive result for HPV type 6 and visualising the 356 bp fragment was interpreted as a positive result for HPV type 11. Precautions to avoid cross-contamination and false-positive results were taken in every assay.
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
Procedure Prepare gel box by placing gel carrier perpendicular (with gaskets on box walls) in the gel box and placing sample comb in position to make loading wells.Pour agarose to fill gel box without pausing.Allow agarose to cool for approximately 20–30 minutes; the agarose will become more opaque as it sets.Pull gel carrier out of gel box, and carefully remove comb.Replace carrier with wells aligned to the black lead (-); samples will run toward the red lead (+).Fill gel box to fill line with 0.5× TAE buffer.Prepare samples for loading into gel by adding 2 μl of loading buffer to 10 μl of sample. The loading buffer will make the sample easier to see, and increases the density so it settles to the bottom of the well.Carefully load samples into gel, avoiding spilling sample or puncturing the bottom of the well.Load 3–5 μl of RNA or DNA ladder into empty wells at the sides of the gel.To ensure that samples run straight in the gel, load 12 μl of deionized water mixed with loading buffer into each unused well.Attach leads to gel box and turn on power source.Make sure bubbles form in the running buffer on each side of the gel box.The gel should take about 1 hour to run for a medium-sized gel at 150 V; check progress every 15 minutes. The loading buffer dyes will indicate progress: the yellow dye will run faster and separate from the blue dye. When the yellow dye front has almost run to the end of the gel, turn off the power supply.Carefully remove gel from the buffer box and gel carrier.Place gel on the blue LED light source and visualize using the orange filter.Take a picture of your gel, noting the exposure time and other camera parameters (if applicable).
PEGylated pH-responsive peptide-mRNA nano self-assemblies enhance the pulmonary delivery efficiency and safety of aerosolized mRNA
Published in Drug Delivery, 2023
Yingying Xu, Yijing Zheng, Xuqiu Ding, Chengyan Wang, Bin Hua, Shilian Hong, Xiaoman Huang, Jiali Lin, Peng Zhang, Wei Chen
All peptides in this study (Table 1) were purchased from ChinaPeptides (Shanghai, China). CleanCap® firefly luciferase mRNA, EGFP mRNA, and cyanine-5 EGFP mRNA were purchased from TriLink Bio Technologies (San Diego, CA, USA). LipofectamineTM 2000 transfection reagent was purchased from Invitrogen (Carlsbad, USA). Luciferin potassium salt was purchased from Promega (Madison, WI, USA). Dulbecco’s modified Eagle’s medium (DMEM), 0.25% Trypsin-EDTA, penicillin/streptomycin antibiotics, and PBS (pH 7.2–7.4) were purchased from Genview (Florida, USA). Fetal Bovine Serum (FBS, South America origin) and Opti-MEM I reduced serum medium were purchased from Gibco (New York, USA). DNA Gel Loading Dye (6 ×) was purchased from Biosharp (Anhui, China). GelRed nucleic acid stain was purchased from US Everbright (California, USA). 1 × Tris-acetate EDTA (TAE) buffer was diluted from 50 × TAE buffer (Sangon Biotech, Shanghai, China) using distilled water. A549 and HepG2 cells were purchased from China Center for Type Culture Collection (CCTCC, Shanghai, China). Other reagents were obtained from Sigma-Aldrich (Saint Louis, MO, USA) of analytical grade or better grade.
Genotoxic, biochemical and histopathological studies to assessment the topiramate hepatorenal toxicity in mice
Published in Drug and Chemical Toxicology, 2022
Aida I. El Makawy, Dalia M. Mabrouk, Faten M. Ibrahim, Kawkab A. Ahmed
DNA breaks were measured with the alkaline comet assay in liver and kidney cells according to Eissa et al. (2018). Briefly, cells were embedded in 1% low melting point agarose on pre-coated microscopic slides with normal melting point agarose. After drying of the gels at a cold plate in dark for a few minutes, the slides were placed in a lysis buffer with pH 10 (1% Triton X-100, 2.5 M NaCl, 10 mM Tris, 0.1 M EDTA) at 4 °C for at least 2 h to lyse the cells and separate DNA from the histones. Unwinding of DNA was then performed in alkaline solution (0.3 M NaOH, 1 mM EDTA) for 40 min, on ice in darkness, followed by electrophoresis for 20 min (25 V 300 mA electrophoresis platform) in a black COMET-20 tank (Scie-Plas Ltd, Cambridge, UK) with cold water circulating under the platform. During the electrophoresis, the negatively charged DNA moves towards the anode and create ‘comets’. Neutralization was performed in 0.4 M Tris (pH 7.5) for 10 min and in H2O for 5 min. Gels were dried overnight and fixed with methanol for 5 min prior to staining of the DNA with EtBr (1 μg/ml) in TAE buffer. Images of 50 randomly selected nuclei per experimental group were captured using a fluorescence microscope (Eclipse 800, Nikon, Tokyo, Japan) and analyzed with image analysis software (Comet Assay IV, Perceptive Instruments, Suffolk, UK). Scored parameters included tail length, DNA percentage in tail and Olive tail moment (OTM).
Effects of catechin hydrate in benzo[a]pyrene-induced lung toxicity: roles of oxidative stress, apoptosis, and DNA damage
Published in Toxicology Mechanisms and Methods, 2021
Samah A. Khattab, Wafaa F. Hussien, Nermin Raafat, Eman Ahmed Alaa El-Din
DNA extraction was done using QIAGEN total DNA extraction kit (QIAamp DNA Mini kit, Qiagen, Germany), according to the Buffone and Darlington (1985) method. Where 25 mg of ground tissue sample was measured and transferred to a 1.5 ml micro-centrifuge tube. 200 µl of CL buffer (lysis buffer), 20 µl proteinase K, and 5 µl of RNase A solutions were added to each sample and mixed vigorously then the lysate was incubated at 56 °C for 30 min. Then, 200 µl of BL buffer (lysis/binding buffer) were added and incubated at 70 °C for 5 min. The sample tube was centrifuged at 13,000 rpm for 5 min to remove un-lysed tissue, where 400 µl of the supernatant was transferred into a new 1.5 mL micro-centrifuge tube, then 200 µl of absolute ethanol was added to the lysate, inverted to mix for 5–6 times, then (wash buffer A) and (wash buffer B) were used, then centrifuged. Where 70 μl of buffer CE (elution buffer) was directly added to the membrane, incubated for 1 min at room temperature, and then centrifuged for 1 min at 13,000 rpm to elute the DNA. The electrophoresis was run in a TAE buffer. Electrophoresis was done at 100 mA and 70 V for approximately 1 h using the EC 360 Submarine Gel electrophoresis system (Maxicell, EC 360 M-E-C Apparatus Cooperation, St. Petersburg, FL, USA). The DNA was visualized using ethidium bromide and photographed. DNA was evaluated according to Wlodek et al. (1991).