User:Karmella Haynes/Notebook/Polycomb project/2010/08/20

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08/20/10

  • ✓ ChIP/ Co-IP: KAH126-1 and 132-8



ChIP: myc-bead pull-down
> See 6/29/10
> Prep sonicated ~6 mL samples according to Qingqing's protocol
--> Add:

  • 720 μL 10% Triton X-100
  • 72 μL 10% Na-deoxycholate (DOC)
  • 500 μL TE (pH 8.0)
  • 6 μL 1000x PLAAC
  • 60 μL 100x PMSF

--> Save 4x 100 μL samples as "Input"

> Binding:

  1. KAH126-1: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  2. KAH126-1: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
  3. KAH132-8: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  4. KAH132-8: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

--> Rotate at 4°C overnight
--> Wash and elute tomorrow


8/21/10
> Wash & elute IP's (Keep everything on ice)
--> Prepare washing buffer (complete sonication buffer):

  • 6 mL Buffer III
  • 60 μL 100x PMSF
  • 6 μL 1000x PLAAC
  • 720 μL 10% Triton X-100
  • 72 μL 10% Na-deoxycholate (DOC)
  • 500 μL TE (pH 8.0)

--> Spin down beads at 3000 rpm/ 3 min./ 4°C
--> Save 50 μL supernatant. Discard remaining sup.
--> Wash beads in 500 μL wash buffer (1 min.). Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup. Repeat twice more (3 washes total)
--> Add 25 μL 4x loading dye (+100 mM DDT) and 25 μL RIPA (lysis/ protein dilution buffer) to each pellet. Heat at 100°C/ 5 min., vortex, repeat.
--> Clear supernatant by spinning at 4000 rpm/ 2 min. Transfer 10 μL sup to new tube (save at -20°C for Western later).




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