162
edits
Changes
→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===
===Analysis of Immunoprecipitated DNA===
