162
edits
Changes
→Sonication of Tissue
# Resuspend fixed cells in 50-150uL lysis buffer(depending on amount of tissue) and incubate for 10 min on ice.
# Dilute to 500-1.5mL cold 1x TE (this is based on the amount of lysis buffer you used).
# 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 (this is around 60% amplitude--this but it ranges depending on how much tissue so play around with amplitude for your sample to get near 5W), 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. # 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. # Snap-freeze the sample in liquid nitrogen and store at -80°C, or do not freeze and continue with the immunoprecipitation steps below.
===Immunoprecipitation===