Chromatin Immunoprecipitation: Difference between revisions
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1. Remove culture plates from the incubator and place at room temperature on the bench. | 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. | 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 (If using 10cm dishes add 0.5mL of the 2.5M glycerol stock solution). | ||
3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix. | 3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix. | ||
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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. | ||
5. Aspirate the PBS and add | 5. Aspirate the PBS and add 2-3 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. | ||