162
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Changes
→Crosslinking, Lysis and Shearing of DNA
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).
''Note: Remember to take a portion of this resuspended pellet prior to lysing to compare unshared unsheared to sheared DNA.''
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
(60~45-50% 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.