Chromatin Immunoprecipitation: Difference between revisions
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* 10mL for 10cm dish | * 10mL for 10cm dish | ||
5. Aspirate the PBS and add 2.5 ml cold (4°C) Farnham lysis buffer. | 5. Aspirate the PBS and add 2.5 ml cold (4°C) Farnham lysis buffer (make sure to add PI). | ||
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. | ||
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*If using the Branson Sonifier 250: Set at constant cycle, output control 3 (will give output measurement of 5) and sonicate samples 10x each for 10 sec with a 20 sec recovery period between each. | *If using the Branson Sonifier 250: Set at constant cycle, output control 3 (will give output measurement of 5) and sonicate samples 10x each for 10 sec with a 20 sec recovery period between each. | ||
13. Spin the sonicated mixture at 14,000 rpm in a microfuge for 15 minutes at 4°C and collect the supernatant and nano drop samples. | 13. Spin the sonicated mixture at 14,000 rpm in a microfuge for 15 minutes at 4°C and collect the supernatant and nano drop samples and calculate the amount needed for 25ug of chromatin. | ||
14. Snap-freeze the sample in liquid nitrogen and store at -80°C, or do not freeze and continue with the immunoprecipitation steps below. | 14. Snap-freeze the sample in liquid nitrogen and store at -80°C, or do not freeze and continue with the immunoprecipitation steps below. | ||