162
edits
Changes
→Crosslinking, Lysis and Shearing of DNA
10. Collect the crude nuclear prep by centrifuging at 2,000 rpm at 4°C for 5 minutes.
11. Resuspend pellet to 1 ml with RIPA Buffer in a '''15 mL falcon tube ''' (Do not vortex the tubes and try to avoid bubbles. Bubbles will cause popping and loss of samples during sonication).
12. Using the Sonics VibraCell Sonicator, sonicate each 1.0 ml ChIP sample on ice, in a cold room, at Power Output 5 watts 6 times for 30 seconds each
(60% amplitude), with at least 30 second cooling on ice between each 30-second sanitation. Remember to clean sonicator with water prior to use, in between samples and following use.*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.