Chromatin Immunoprecipitation: Difference between revisions
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2. Add formaldehyde to a final concentration of 1% directly to the media of adherent cells growing on tissue culture plates, swirl gently, and | 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. | incubate at room temperature for 10 minutes. | ||
* If using 10cm dishes add 270ul of 37% formaldehyde | |||
3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M | 3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix. | ||
* If using 10cm dishes add 0.5mL of the 2.5M glycine stock solution | |||
4. Remove media from plates and wash cells with equal volume cold (4°C) 1X PBS. | 4. Remove media from plates and wash cells with equal volume cold (4°C) 1X PBS. | ||
* 10mL for 10cm dish | |||
5. Aspirate the PBS and add 2 | 5. Aspirate the PBS and add 2.5 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. | 6. Scrape the cells off the plate with a cell scraper and transfer into 15-ml conical tubes on ice. | ||