Etudes sur le mécanisme de remodelage des nucléosomes par ...

tel-00413908, version 1 - 7 Sep 2009
cutoff spin filters. Eluted nucleosomes were adjusted to buffer conditions of the restriction
digestion conditions (10 mM Tris pH7.6, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT and 100
μg/ml BSA). HaeIII was added to 2 units/μl and the reaction was allowed to proceed at 29° C.
At indicated time points aliquots were taken and the reaction was stopped by addition of 0.1%
SDS and 20 mM EDTA. DNA was extracted through phenol:chloroform, precipitated and run
on 8% denaturing PAGE. Gels were dried, autoradiographed, scanned on phosphorimager and
quantified using Multigauge software (Fuji).
III.4.4 Atomic Force Microscopy
For the AFM imaging, the nucleosomes were immobilized onto APTES-mica surfaces as
described previously. Image acquisition and analysis were done as described in chapter II.
DNA complexed length (Lc) and position (ΔL) distributions were constructed as described
(Montel et al., 2007).
Other experimental procedures were essentially similar to and as described in chapter II.
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