162
edits
Changes
→Crosslinking, Lysis and Shearing of DNA
* 10mL for 10cm dish
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.
*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 samplesand 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.