DNA comet assay was performed using the CometAssay kit (4250-050-K, Trevigen) according to the manufacturer's protocol. Briefly, 25 μl of the sample cell suspension at 2 × 105 cells/ml were combined with 225 μl of low-melting agarose (LMAgarose) (1:10 ratio, v/v) and 50 μl of this mixture was quickly spread onto the provided comet slides (Trevigen). Following solidification at 4°C, slides were immersed in cold lysis solution for 1 h at 4°C then placed in freshly prepared alkaline unwinding solution (20 mM NaOH, 1 mM EDTA) for 20 min at RT. Electrophoresis of unwound DNA was performed at 21 V for 30 min. Slides were then washed twice with dH2O for 5 min, dehydrated with 70% ethanol for 5 min, dried, and stained with SYBR Gold staining solution (Thermo Fisher) for 30 min. Comets were imaged at 10x magnification with an Eclipse Ts2R-FL inverted fluorescence microscope (Nikon) equipped with a Nikon DSQi2 digital camera and analyzed using OpenComet on Fiji and Prism (GraphPad). Up to at least 100 individual nuclei were evaluated per group.
Sybr gold staining solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYBR Gold staining solution is a fluorescent dye used to detect and quantify nucleic acids, including DNA and RNA, in various applications such as gel electrophoresis and microarray analysis. It binds to nucleic acids and emits a bright green fluorescence when excited by an appropriate light source.
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Lab products found in correlation
4 protocols using sybr gold staining solution
1
DNA Comet Assay for Genotoxicity
2
Alkaline Comet Assay for DNA Damage
3
Alkaline Comet Assay for DNA Damage
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Alkaline comet assays were performed using the CometAssay kit from Amsbio (4250-050-K) according to the manufacturer’s protocol. Alkaline electrophoresis was performed in a BioRad Mini-Sub Cell GT system (20 volts, 250-300 mA for 30 min), and the comets were stained with SYBR Gold staining solution (Thermo Fisher, 1: 10,000 in TE buffer). The percentage of DNA in the tail was measured using ImageJ software with Comet Assay plugin (https://www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/comet-assay ).
4
DV2 RdRp Enzyme Activity Assay
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The enzyme activity of the DV RdRp recombinant protein was determined by gel electrophoresis–based RNA analysis. To prepare DV serotype 2 RdRp substrate, ssRNA labeled with Cy5 was prepared (5′-Cy5-UUUUUUUUUUUUUACUAACAACU-3′; Bioneer, Korea) in TE (Tris-EDTA) buffer solution. The DV2 RdRp enzyme (3.5 pmol) was mixed with 3 pmol of RNA substrate, 0.1 mM ATP, and 0.5 mM CTP/UTP/GTP in the 60-μl RdRp buffer (50 mM Hepes, 10 mM NaCl in pH 7.5) and incubated for 45 min at 37°C. Each sample was transferred to 20% native polyacrylamide gel electrophoresis (PAGE) gel, and electrophoresis was performed at 100 V for 20 to 30 min. Gels were stained with SYBR gold staining solution (Thermo Fisher Scientific, USA) and then analyzed using ChemiDoc (Bio-Rad, USA). The size of the resulting gel bands was determined compared to the small interfering RNA ladder marker (Takara Bio, Japan).
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