Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Preparation of Cross-Linked Chromatin for ChIP

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The quality and usefulness of chromatin depends on the efficiency of cross-linking and on the accessibility of epitopes to cognate antibodies. To minimize problems, several independent preparations of chromatin should be made and tested side-by-side using a well-characterized antibody (TAF1 or histone H3 acetylation) in standard ChIP assays. The number of sonication cycles must be determined empirically. When sonication is being performed the first time for a given cell type and cross-linking condition, a small chromatin sample (5 L) after each sonication cycle should be analyzed by agarose gel electrophoresis to determine the optimal number of cycles. For ChIP-chip, it is not essential that a majority of chromatin fragments are 500 bp; an average fragment size ranging 12 kb is sufficient. However, for ChIP-seq, the average chromatin fragment should be <500 bp. Alternative methods to fragment chromatin include (1) digestion with micrococcal nuclease, which digests DNA between nucleosomes (Richard-Foy and Hager 1987), and (2) restriction enzymes that can be used in the ChIP-loop method to solubilize chromatin in isolated nuclei (Simonis et al. 2007).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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