Analysis of Alkaline-Labile DNA Sites

Analysis of alkaline-labile sites in genomic DNA was performed as reported earlier^{1 (link)}. Briefly, overnight cultures were diluted for exponential growth in 50 ml medium and harvested at the optical density of OD₆₀₀ = 0.6. Genomic DNA was isolated with the Qiagen Gentra Puregene Yeast/Bact Kit (Qiagen, 158567). The gDNA concentration was determined by an intercalating fluorescent dye supplied via the Qubit dsDNA broad-range kit (Invitrogen, Q32854). In a final volume of 40 µl, 10 µg of extracted gDNA were treated, shielded from light, for 2 h at 55 °C with 0.3 M KOH or 0.3 M KCl. KOH treated DNA samples were mixed with 8 µl 6× alkaline loading buffer (300 mM KOH, 6 mM EDTA, 18% (w/v) Ficoll Type 400, 0.15% (w/v) bromocresol green, 0.25% (w/v) xylene cyanol) before loading on a 1% alkaline agarose gel; the alkaline gel was casted using the 1× dilution of the 10× alkaline running buffer (0.5 mM NaOH, 10 mM EDTA). The alkaline gel was run in 1× alkaline running buffer. The KCl treated gDNA samples were loaded on a 1% TBE agarose gel with standard loading dye. Both gels were run at room temperature for 5 min at 65 V before reducing the voltage to 26 V for 18 h. The alkaline gel was neutralized in two washes of 250 ml (700 mM Tris-HCl pH 8.0, 1.5 M NaCl) for 45 min each with gentle shaking. Both gels were stained in a 1:10,000 SyBr Gold solution (ThermoFisher, S11494) (diluted in 1×TBE for the neutral gel; diluted in water for the alkaline gel) for 2 h with gentle shaking before image acquisition.