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

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ChIPQuantitative PCR (ChIP-qPCR)

(Protocol summary only for purposes of this preview site)

It is critical to determine if the ChIP actually enriched the DNA sequences that are associated with the target protein. If there are known genomic binding sites, primers can be designed for quantitative PCR (qPCR) to determine if the known sites are specifically enriched by immunoprecipitation compared with the input chromatin and with the chromatin that has been mock-immunoprecipitated with preimmune serum. If there are no known sites but candidate target genes are available, one can consider designing qPCR primers along the length of potential regulatory regions such as promoters and conserved noncoding sequences within intergenic and genic regions. Because real-time PCR can be performed in either a 96- or 384-well format in a minimal reaction volume and primers can be synthesized with minimal cost, ChIP-qPCR is an attractive strategy to interrogate target genes and potential regulatory regions across a large number of experimental conditions and different cell types. If candidate target genes or potential sites are not available, ChIP-chip (Protocol 5) or ChIP-seq (Protocol 6) should be considered.


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