Primers used for PCR are listed in Table 1 . The genotyping approach for this study was used previously in several studies [17 (link)18 ]. Four separate PCR reactions were performed using different sets of primers in each sample. Each 20 µL reaction contained 2 µL DNA mixed with 1 µL of each allele-specific primer (10 pmol), 10 µL master mix (2X), and 7 µL nuclease free water. PCR was performed in a thermal cycler (Applied Biosystems, Wilmington, DE, USA). The cycling conditions followed an initial denaturation at 95℃ for 3 min, followed by 35 cycles of denaturation at 95℃ for 40 s, annealing at 56℃ for 40 s, and elongation at 72℃ for 40 s, followed with a final elongation at 72℃ for 5 min. PCR products were separated by electrophoresis on 3% agarose gels containing ethidium bromide at 40 mV for 20 min followed by 70 mV for 30–40 min. A 100 base pair (bp) DNA ladder was used as a molecular marker (Invitrogen, Wilmington, DE, USA). The gel was visualized using a G-BOX UV transilluminator (Syngene, Cambridge, UK).
G box uv transilluminator
Manufactured by Syngene
Sourced in United Kingdom
The G-BOX UV transilluminator is a laboratory instrument designed for the visualization and documentation of DNA and RNA samples separated by gel electrophoresis. It utilizes UV light to excite fluorescent dyes within the nucleic acid samples, allowing researchers to observe and capture images of the separated bands.
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3 protocols using g box uv transilluminator
1
PCR Genotyping of Genetic Variants
2
Discontinuous RNase E Activity Assay
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Discontinuous RNase E assays were carried out essentially as described previously [28 (link)]. Briefly, control reaction mixtures (30 μl) contained: 5 nM purified E. coli RNase E NTD; 1 μM 5′ phosphorylated, 3′ fluorescein amidite (FAM)-labelled p-UUUACAGUAUUUG-FAM (5′-p-RNA13-FAM-3′) RNA substrate (Dharmacon); 25 mM Tris-HCl (pH 8); 100 mM NaCl; 15 mM MgCl2; 1 mM DTT; 37.5 mg/ml Ficoll 70; and 5% (v/v) dimethyl sulfoxide (DMSO). Screening reaction mixtures were identical to the control except that they were supplemented with 0.625, 1.25, 2.5, 5 or 10 mM of the respective small molecule (Sigma), as indicated. All reactions were incubated at 28 °C for 45 min and terminated by the addition of 0.5 vol of quench buffer (95% (v/v) formamide, 20 mM EDTA). The reaction products were resolved by denaturing 7.5 M urea 20% polyacrylamide gel electrophoresis (PAGE) and gels were visualised using a G:Box UV transilluminator (Syngene).
3
Analyzing DNA Fragmentation in HeLa Cells
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HeLa cells in 10 cm dishes were scratched and pelleted for 5 min at 220 g. After PBS washing the pellet was dissolved in DNA laddering buffer (5 mm Tris, pH 7.4; 20 mm EDTA; 0.5% Triton X-100) on ice to lyse the cells. To pellet the chromatin lysates were centrifuged at 4 °C and 10,000 g for 15 min. The supernatant containing DNA fragments was phenol/chloroform purified and re-extracted by chloroform addition. The DNA fragments containing phase was ethanol-precipitated and ethanol-washed pellets were dissolved in DNase- and RNase-free water (Gibco) for 30 min at RT. To remove RNA contents RNase A (10 mg/ml, DNase free) was added to the samples for 30 min at RT. Samples were separated by gel electrophoresis (1.2% agarose gels containing EtBr in TAE buffer) at 45 V for 4 h and analyzed by UV transilluminator (G:BOX UV transilluminator, Syngene).
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