Ds 11 spektrophotometer

For low, medium, and high assembly degrees, 10 µg of plasmid library DNA (S. cerevisiae, S. pombe or E. coli) were mixed with ~2, 4, or 8 µg of Drosophila embryo histone octamers, respectively, in 100 µl assembly buffer (10 mM Tris·HCl pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05% IGEPAL CA630 (Sigma), 0.2 µg/µl BSA). Samples were transferred to Slide-A-lyzer mini dialysis devices (MWCO 3.5 kDa; Thermo Fisher Scientific, cat no. 69550), which were placed in a 3 l beaker containing 300 ml of high salt buffer (10 mM Tris·HCl pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 14.3 mM β-mercaptoethanol), and dialyzed against a total of 3 l low salt buffer (10 mM Tris·HCl pH 7.6, 50 mM NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 1.4 mM β-mercaptoethanol) added continuously while stirring via a peristaltic pump over a time course of 16 h. β-mercaptoethanol was always added freshly. After complete transfer of low salt buffer, samples were dialyzed against 1 l low salt buffer for 1 h at room temperature. DNA concentration of the SGD chromatin preparations was estimated with a DS-11+ spektrophotometer (Denovix). SGD chromatin could be stored at 4 °C for several weeks. To estimate the extent of the assembly degree, an aliquot of the sample was subjected to MNase digestion (below) for MNase-ladder read out.

For low, medium, and high assembly degrees, 10 µg of plasmid library DNA (S. cerevisiae) was mixed with ~2, 4, or 8 µg of Drosophila embryo histone octamers, respectively, in 100 µl assembly buffer (10 mM Tris·HCl, pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 0.2 µg/µl BSA). For reconstitutions with precleaved DNA (Fig. 8), the plasmid library was digested with the respective RE and purified by phenol extraction/ethanol precipitation prior to SGD. Samples were transferred to Slide-A-lyzer mini dialysis devices, which were placed in a 3 l beaker containing 300 ml of high salt buffer (10 mM Tris·HCl pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 14.3 mM β-mercaptoethanol), and dialyzed against a total of 3 l low salt buffer (10 mM Tris·HCl pH 7.6, 50 mM NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 1.4 mM β-mercaptoethanol) added continuously via a peristaltic pump over a time course of 16 h while stirring. β-mercaptoethanol was added freshly to all buffers. After complete transfer of low salt buffer, samples were dialyzed against 1 l low salt buffer for 1 h at room temperature. DNA concentration of the SGD chromatin preparations was estimated with a DS-11+ spektrophotometer (Denovix) and could be stored at 4 °C for several weeks. To estimate the extent of the assembly degree, an aliquot of the sample was subjected to MNase digestion (as described below) for MNase-ladder read out.