Fluorescent Oligonucleotide Binding Assay

A fluorescently labeled oligonucleotide corresponding to a region of λ DNA (S4A Fig in S1 File, Integrated DNA Technologies, Coralville, IA) was reacted with either WT gp5 or Δ28 gp5 in the presence or absence of dNTPs. Reactions contained 100 nM oligonucleotide and 0 or 1 mM dNTPs in 1x CutSmart buffer (50 mM potassium acetate, 20 mM Tris acetate, 100 μg/mL bovine serum albumin, 10 mM magnesium acetate, pH 7.9) in a volume of 50 μL. Prior to addition of polymerase, 5 μL of the reaction was removed and combined with 5 μL of quench buffer (20 mM Tris-HCl pH 7.5, 100 mM EDTA, 1% v/v Triton X-100). Reactions were initiated by the addition of polymerase to 20 U mL⁻¹, incubated at 37 °C and aliquots were removed and quenched at indicated time points. Quenched samples were diluted 10-fold in nfH2O and labeled products resolved using an Applied Biosystems 3730xl instrument [38 (link)].