Chromatin Immunoprecipitation: Difference between revisions
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===Crosslinking, Lysis and Shearing of DNA=== | ===Crosslinking, Lysis and Shearing of DNA=== | ||
1. Remove culture plates from the incubator and place at room temperature on the bench. | |||
2. Add formaldehyde to a final concentration of 1% directly to the media of adherent cells growing on tissue culture plates, swirl gently, and incubate at room temperature for 10 minutes. | |||
3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix. | |||
4. Remove media from plates and wash cells with equal volume cold (4°C) 1X PBS. | |||
5. Aspirate the PBS and add 5-8 ml cold (4°C) Farnham lysis buffer. | |||
6. Scrape the cells off the plate with a cell scraper and transfer into 15-ml conical tubes on ice. | |||
7. Pellet cells at 2,000 rpm for 5 minutes at 4°C. | |||
8. Place cells on ice. Carefully remove supernatant and either proceed to sonication step or snap-freeze in liquid nitrogen and store at -80°C or in liquid nitrogen. | |||
===Immunoprecipitation=== | ===Immunoprecipitation=== | ||
===Analysis of Immunoprecipitated DNA=== | ===Analysis of Immunoprecipitated DNA=== | ||