Orange dna loading dye

The reaction mixtures (20 μL) containing supercoiled circular pBluescipt KS II DNA stock solution (Form I, 50 μM/base pair, ~500 ng), compounds, and Tris buffer (25 μM, pH 6.8) in Pyrex vials were incubated for 30 min at 37 °C, centrifuged, and then irradiated with UV light (312 nm, 90 W) under aerobic conditions at room temperature for 15 min. For the case of pBR322 plasmid DNA, the above mentioned procedure has been followed. After centrifugion, the samples were irradiated with UVA light (365 nm, 18 W) under aerobic conditions at room temperature for 120 min. After addition of the gel-loading buffer (6X Orange DNA Loading Dye 10 mM Tris-HCl (pH 7.6), 0.15% orange G, 0.03% xylene cyanol FF, 60% glycerol, and 60 mM EDTA, by Fermentas), the reaction mixtures were loaded on a 1% agarose gel with EB staining. The electrophoresis tank was attached to a power supply at a constant current (65 V for 1 h). The gel was visualized by 312 nm UV transilluminator and photographed by an FB-PBC-34 camera vilber lourmat. Quantitation of DNA-cleaving activities was performed by integration of the optical density as a function of the band area using the program “Image J” version 1.50.I [76 ].

PAGE was performed to identify the critical ODN/PA ratio required to conjugate all ODNs in solution to PA nanostructures. 20 μg/mL ODN1826 solution (15 μL) was mixed with varying concentrations of PA solutions (15 μL) to prepare different ODN/PA ratios (from 1:10 to 1:2500). These solutions were mixed with Orange DNA loading dye (Fermentas) and loaded onto 20% polyacrylamide gels. 10 μL of 10 bp DNA ladder (O’range ruler^TM, Fermentas) was used as marker. Gels were run at 75 V for 1 h and subsequently at 50 V for 2.5 h (in 1x TAE). Stains-all dye working solution (0.005%, w/v) was prepared freshly from stock solution (0.1% w/v) as recommended by manufacturer (Sigma Aldrich). Gels were incubated in Stains-all overnight (dark conditions and room temperature). On the next day, the destaining of gels was performed under sunlight and images were taken by a Nikon camera.

Reagent-grade chemicals (HEPES, NaCl, trizma hydrochloride, boric acid, EDTA, urea, acrylamide/bis-acrylamide 40% solution ratio 29:1, APS, TEMED), DIG, copper-catalyzed click reaction reagents (tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), sodium ascorbate, copper sulfate pentahydrate, and phosphoramidate ligation reagents (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-(2-hydroxyethyl) imidazole) were purchased from Sigma-Aldrich (St. Louis, Missouri) and used without further purifications. Loading dyes 6× Orange DNA Loading Dye and Gel Loading Buffer II were obtained from ThermoFisher^™. DNA-staining dye SYBR^® gold was provided by Invitrogen. Sheep polyclonal anti-DIG Ab was purchased from Roche Diagnostic Corporation, Germany, (cat#: 11333089001), mouse monoclonal anti-DNP Ab was purchased from Sigma-Aldrich, USA, (cat#: D8406), murine monoclonal anti-HIV Ab was purchased from Zeptometrix Corporation, USA, (cat#: 0801040) and anti-DIG Fab fragments purchased from Roche Diagnostic Corporation, Germany (cat#: 11214667001). All antibody solutions were aliquoted and stored at a concentration of 1 mg/mL either at 4 °C for immediate use or −20 °C for long-term storage. Human α-thrombin was provided by Hematologic Technologies (Essex Junction, VT). Fibrinogen from human plasma was purchased from Merck (Darmstadt, Germany).