Sephadex g 25 spin column

LV nuclear extracts were prepared as previously described with minor modifications [27 (link)]. Double-stranded oligonucleotide containing the nuclear factor-kappaB (NF-κB, consensus sequence from Promega 5′AGTTGAGGGGACTTTCCCAGGC-3′), was end-labeled with γ-32P-ATP using T4 polynucleotide kinase. Unincorporated nucleotides were removed by passing the reaction mixture through a Sephadex G-25 spin column (Amersham-Pharmacia, Sweden). Purified 32P-labeled probe (30,000 cpm) was incubated in 20 μl with 10 μg of LV nuclear extracts in a binding reaction mixture containing 50 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol (DTT), 4% glycerol, 10 mM Tris–HCl (pH 7.5) and 0.05 μg poly (dI-dC) for 30 min at room temperature. DNA–protein complexes were separated by electrophoresis through a 6% non-denaturing acrylamide:bis-acrylamide (37.5:1) gel in 0.5 × Tris-borate/EDTA for 2 h at 150 V. Gels were vacuum dried for 1 h at 80°C and exposed to X-ray film at −80°C. The bands were quantified by ImageJ software (NIH of Health, USA).

In vitro transcribed HCV (1-117) RNAs were ethanol precipitated, resuspended in RNase-free H₂O and treated with DNase I (New England Biolabs) to remove the DNA template, followed by purification with Sephadex G-25 spin columns (Cytiva) to remove unincorporated NTPs and the degraded template. The flowthrough was treated with quickCIP (New England Biolabs) to remove the 5′ triphosphate followed by heat inactivation of the phosphatase (2 min at 80 °C). Viral RNAs were labeled with T4 PNK (New England Biolabs) and γ[³²P]ATP followed by gel purification on a denaturing (8 M urea) 15% acrylamide gel run in 0.5× Tris-borate-ethylenediaminetetraacetic acid (TBE) buffer. Bands were imaged, excised, and crushed in 300 µL crush and soak buffer (0.5 M NH₄OAc, 0.2% sodium dodecyl sulfate, and 1 mM ethylenediaminetetraacetic acid [EDTA]) followed by elution at 4 °C overnight. The gel was removed with a spin-X column (Sigma Aldrich), and the RNA was precipitated using 3M NaOAc, pH 5.2 and ice-cold 100% ethanol at −20 °C for >20 min. RNAs were pelleted by centrifugation, washed with 150 µL ice-cold 75% ethanol, dried, resuspended in RNase-free H₂O, and quantified by nanodrop spectrophotometer. Extinction coefficients were calculated with the IDT online tool available at https://www.idtdna.com.