Intasome-Mediated Strand Transfer Assay

Reactions were carried out using intasomes purified by size exclusion chromatography. A standard reaction contained 1.25 μg of intasome and 5 μg of nucleosome in 300 μl of 240 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 4 μM ZnCl₂, 25 mM BisTris propane-HCl, pH 7.45. Strand transfer was allowed to proceed for 15 min at 37 °C, and the reaction was stopped by addition of 0.5% SDS and 25 mM EDTA. DNA products, deproteinized by digestion with 30 μg proteinase K at 37 °C for 1 h and ethanol precipitation, were separated in 4-12% TBE PAGE gels. Fluorescein-labeled DNA was detected using a Typhoon TRIO fluorescence scanner (GE Healthcare); non-labeled DNA was visualized by staining with GelRed. Strand transfer assays using naked supercoiled plasmid target DNA was done according to published procedures^{2 (link)}.