A general method for preparing chromatin containing intact DNA.
A simple and general method is described for preparing chromatin from eukaryotic cells using isotonic conditions. First, cells are encapsulated in agarose microbeads and then lysed using Triton X-100 in the presence of a chelating agent and a physiological concentration of salt. Most cytoplasmic proteins and RNA diffuse rapidly out through pores in the beads to leave encapsulated chromatin which is nevertheless completely accessible to enzymes and other probes. This chromatin can be manipulated freely without aggregation in a variety of different salt and detergent concentrations. It also contains intact DNA since removal of the histones releases superhelical DNA. Conditions are described for incubating this chromatin at 37 degrees C in the presence of Mg2+ ions without any nicking of the DNA. We illustrate the usefulness of this chromatin in investigations on the attachment of nascent RNA to the nucleoskeleton, the accessibility of the ribosomal locus to EcoRI and the properties of the endogenous RNA polymerase II. This type of chromatin preparation should prove useful for both structural and functional studies.
D002467:Cell Nucleus / Q000648:ultrastructure; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure*; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D003902:Detergents; D006367:HeLa Cells / Q000648:ultrastructure; D006801:Humans; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D017830:Octoxynol; D011092:Polyethylene Glycols; D012334:RNA, Neoplasm / Q000032:analysis; D014158:Transcription, Genetic
D002843:Chromatin; D004273:DNA, Neoplasm; D003902:Detergents; D012334:RNA, Neoplasm; D011092:Polyethylene Glycols; D017830:Octoxynol