Chromatin Immunoprecipitation: Difference between revisions
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1. Remove culture plates from the incubator and place at room temperature on the bench. | 1. Remove culture plates from the incubator and place at room temperature on the bench. | ||
2. Add formaldehyde to a final concentration of 1% directly to the media of adherent cells growing on tissue culture plates, swirl gently, and incubate at | 2. Add formaldehyde to a final concentration of 1% directly to the media of adherent cells growing on tissue culture plates, swirl gently, and | ||
incubate at room temperature for 10 minutes (If using 10cm dishes add 0.5mL of the 2.5M glycerol stock solution). | |||
3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix. | 3. Stop the cross-linking reaction by adding glycine to a final concentration of 0.125M and swirl gently to mix. | ||
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9. Resuspend each fresh or frozen pellet (containing 2 x 107 cells) on ice in 1 ml Farnham Lysis Buffer and mix gently by flicking the test tube. Briefly homogenize | 9. Resuspend each fresh or frozen pellet (containing 2 x 107 cells) on ice in 1 ml Farnham Lysis Buffer and mix gently by flicking the test tube. | ||
Briefly homogenize cells by running the cells through a 18-gauge needle ~10X. | |||
''Note: This treatment breaks the cells while keeping the nuclei mostly intact.'' | ''Note: This treatment breaks the cells while keeping the nuclei mostly intact.'' | ||
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[[Do not vortex the tubes and try to avoid bubbles. Bubbles will cause popping and loss of samples during sonication]] | [[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), | 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. | |||
13. Spin the sonicated mixture at 14,000 rpm in a microfuge for 15 minutes at 4°C and collect the supernatant. | 13. Spin the sonicated mixture at 14,000 rpm in a microfuge for 15 minutes at 4°C and collect the supernatant. | ||