Difference between revisions of "Chromatin Immunoprecipitation"
From Bridges Lab Protocols
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[[ Category: ChIP ]] | [[ Category: ChIP ]] | ||
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Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-Wide Mapping of in Vivo Protein-DNA Interactions. Science (80- ) 316: 1497–1502, 2007. [http://dx.doi.org/10.1126/science.1141319 doi:10.1126/science.1141319] | Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-Wide Mapping of in Vivo Protein-DNA Interactions. Science (80- ) 316: 1497–1502, 2007. [http://dx.doi.org/10.1126/science.1141319 doi:10.1126/science.1141319] | ||
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| + | ==Before You Start== | ||
===Buffers and Solutions Needed=== | ===Buffers and Solutions Needed=== | ||
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===Equipment=== | ===Equipment=== | ||
* Cool microfuge and swinging bucket centrifuge down to 4C | * Cool microfuge and swinging bucket centrifuge down to 4C | ||
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| + | ==Protocol== | ||
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| + | This protocol involves preparation of the crosslinked DNA, immunoprecipitation of the DNA and analysis by qPCR. It is possible to stop and freeze the samples after each of these steps. | ||
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| + | ===Crosslinking, Lysis and Shearing of DNA=== | ||
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| + | ===Immunoprecipitation=== | ||
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| + | ===Analysis of Immunoprecipitated DNA=== | ||
Revision as of 15:27, 20 January 2016
This protocol is modified from the Myer's Lab ChIPseq protocol v011014 found here. The original citation for this methodology is:
Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-Wide Mapping of in Vivo Protein-DNA Interactions. Science (80- ) 316: 1497–1502, 2007. doi:10.1126/science.1141319
Before You Start
Buffers and Solutions Needed
- 20% Formaldehyde (from 37% formaldehyde Sigma F87750)
- 2.5M Glycine
- PBS (cold)
- Farnham Lysis Buffer (cold)
- RIPA Buffer (cold)
- Dynabeads (Invitrogen cat#)
- PBS with 5 mg/mL BSA (cold)
- [LiCl Wash Buffer]] (cold)
- TE: 10 mM Tris 7.5, 0.1 mM EDTA (cold)
- ChIP Elution Buffer
Equipment
- Cool microfuge and swinging bucket centrifuge down to 4C
Protocol
This protocol involves preparation of the crosslinked DNA, immunoprecipitation of the DNA and analysis by qPCR. It is possible to stop and freeze the samples after each of these steps.
