Chromatin Immunoprecipitation: Difference between revisions
| Line 87: | Line 87: | ||
==== Incubate bead-antibody complex with fragmented, cross-linked chromatin==== | ==== Incubate bead-antibody complex with fragmented, cross-linked chromatin==== | ||
# Add 100 μl of antibody-coupled beads from above to each 25ug chromatin of preparation (from Sonication protocol) and incubate on a rotator for '''1 hour at room temperature''', followed by '''1 hour at 4°C'''. | # Add 100 μl of antibody-coupled beads from above to each 25ug chromatin of preparation (from Sonication protocol) and incubate on a rotator for '''1 hour at room temperature''', followed by '''1 hour at 4°C'''. | ||
# Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack. | # Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack. | ||
# Remove and discard supernatant. | # Remove and discard supernatant. ''Turn water bath to 65°C before next step''. | ||
# Wash beads 5 times with 1 mL LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator. | # Wash beads 5 times with 1 mL LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator. | ||
# 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. | # 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. | ||