Selected article for: "mm hepes and reaction mixture"
Author: Adedeji, Adeyemi O.; Marchand, Bruno; te Velthuis, Aartjan J. W.; Snijder, Eric J.; Weiss, Susan; Eoff, Robert L.; Singh, Kamalendra; Sarafianos, Stefan G.
Title: Mechanism of Nucleic Acid Unwinding by SARS-CoV Helicase
  • Document date: 2012_5_15
    • ID: 1ssh296a_38
    Snippet: To determine if GST-nsp13could form a complex with nsp12-H 6 in the presence of nucleic acid substrate, we performed gel mobility shift assay. We measured the binding of 60/40-mer (20ss:40ds) DNA substrates (Fig. S1 ), at specific concentrations of GST-nsp13 and varying concentrations of nsp12H 6 in a reaction mixture containing 20 mM HEPES pH 7.5, 20 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/ml BSA and 5% glycerol at 30uC for 10 minutes. The conce.....
    Document: To determine if GST-nsp13could form a complex with nsp12-H 6 in the presence of nucleic acid substrate, we performed gel mobility shift assay. We measured the binding of 60/40-mer (20ss:40ds) DNA substrates (Fig. S1 ), at specific concentrations of GST-nsp13 and varying concentrations of nsp12H 6 in a reaction mixture containing 20 mM HEPES pH 7.5, 20 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/ml BSA and 5% glycerol at 30uC for 10 minutes. The concentration for the 59-Cy3-labeled dsDNA substrates was 5 nM. Samples were electrophoresed at 100 V for 1.5 h at 4uC on a 5% non-denaturing polyacrylamide gel, using 89 mM Tris borate pH 8.2. Gels were scanned in a Phosphor-Imager (FLA 5000, FujiFilm).

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