Sephacryl s 200 gel filtration

Nucleosomes were reconstituted as previously described (Dyer et al., 2004 (link)). Recombinant Xenopus histones were expressed in E. coli BL21(DE3) pLysS and purified from inclusion bodies via Sephacryl S-200 gel filtration (GE Healthcare). Stoichiometric amounts of each core histone were incubated together under high salt conditions (2 M NaCl) and the resulting histone octamer purified using a Superdex 200 gel filtration column (GE Healthcare). 216 bp DNA carrying the nucleosome-positioning 601 sequence was PCR amplified from the pGEM-3Z 601 plasmid and purified using a Resource Q anion exchange column (GE Healthcare). Purified DNA, in slight excess to octamers, was mixed together in 2 M NaCl and diluted stepwise with 10 mM Tris (pH 7.5) to reach a final concentration of 100 mM NaCl. The reconstituted nucleosomes were then analysed on a 0.8% Tris-borate agarose gel, and concentrated using a 5000 MWCO spin concentrator (GE Healthcare).

Nucleosomes were reconstituted as described previously (Dyer et al., 2004 (link)). Recombinant Xenopus histones were expressed in E. coli BL21(DE3) pLysS and purified from inclusion bodies via Sephacryl S-200 gel filtration (GE Healthcare). Stoichiometric amounts of each core histone were incubated together under high salt conditions (2 M NaCl) and the resulting histone octamer purified using a Superdex 200 gel filtration column (GE Healthcare). Purified 147bp DNA carrying the 601 nucleosome-positioning sequence was a kind gift from the Brockdorff lab. Purified DNA, in slight excess to octamers, was mixed together in 2 M NaCl and diluted stepwise with 10 mM Tris (pH 7.5) to reach a final concentration of 100 mM NaCl. The reconstituted nucleosomes were analyzed on a 0.8% Tris-borate agarose gel and concentrated using a 30,000 MWCO spin concentrator (GE Healthcare).