Chromatin Immunoprecipitation: Difference between revisions
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c. Remove and discard supernatant. | c. Remove and discard supernatant. | ||
d. Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator. | d. Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator. | ||
e. Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant. | e. Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads | ||
and discard supernatant. | |||
f. Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.</nowiki> | f. Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.</nowiki> | ||
3. Reverse cross-linking and recover | 3. Reverse cross-linking and recover ChIP DNA | ||
<nowiki> a. Incubate bead pellet from step 2.f above in a 65°C water bath for 1 hour, shake or vortex | |||
every 15 minutes to elute the immuno-bound chromatin from the beads. | |||
b. Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes. | |||
c. Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the | |||
magnet to facilitate supernatant recovery. | |||
d. Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to | |||
complete the reversal of the formaldehyde cross-links.</nowiki> | |||
===Analysis of Immunoprecipitated DNA=== | ===Analysis of Immunoprecipitated DNA=== | ||