Nucleosome Assembly and Characterization

Nucleosomes were assembled using the salt dialysis technique essentially as described previously (27 (link)). A typical assembly reaction (20 μl) contained 250 ng Cy3-labeled DNA (methylated or azidohexynylated) and 250 ng Cy5-labelled DNA (non-modified), 200 ng/μl BSA, 125 ng/μl competitor DNA and different amounts of calf thymus histone octamers. After dialysis 2μl of the assembly reaction was mixed with 8 μl Ex80 buffer (20 mM Tris-HCl pH 7.6, 1 mM MgCl₂, 0.5 mM EGTA, 10% glycerol, 0.05% NP-50, 200 ng/μl BSA, 80 mM KCl) and with Purple Loading Dye without SDS (NEB). Samples were resolved on a 5% polyacrylamide native gel in 0.4× TBE. Gels were analyzed using Typhoon FLA-9500 (GE Healthcare) and MultiGauge v.3.0 (Fujifilm) software.