User:Karmella Haynes/Notebook/Polycomb project/2010/09/22

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09/22/10

  • ✓ ChIP PCR: test MMP12 and TNF primers on KAH130-4 IP
  • ✓ ChIP: IP for KAH126-1, 132-8 (form. x-linked chromatin)



ChIP PCR
> KAH130-4 CH2O-x-linked chromatin --> Templates

  1. 130-4 Input (1:50) (12 rxns)
  2. 130-4 α-myc IP (1:50) (12 rxns)
  3. 130-4 α-IgG IP (1:50) (12 rxns)

--> Primers

  1. INKARF C (138 bp)
  2. INKARF D (135 bp)
  3. INKARF E (86 bp)
  4. INKARF F (100 bp)
  5. MMP12 A (111 bp)
  6. MMP12 B (114 bp)
  7. MMP12 C (138 bp)
  8. TNF A (120 bp)
  9. TNF B (123 bp)
  10. TNF C (119 bp)
  11. GAPDH A (103 bp)
  12. GAPDH B (103 bp)


Reagent Single rxn
cDNA 1.0
10 μM primers 1.0
2x GoTaq Green 10.0
dH2O 8.0
  20.0 μL


--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞


ChIP PCR 9/22/10

> Conclusions: Might be some pull-down on INKARFD; try again with full set of chromatin IP's using 1x and 2x amount of ChIP DNA



ChIP: myc-bead pull-down
> Use new CH2O-x-linked chromatin preps > See 9/15/10
> Sonicated ~6 mL samples prepped 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


> 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


9/23/10
> 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 100 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 three more times (4 washes total)
--> Add 500 μL 1% SDS/TE buffer. Incubate at 65°C/ 15 min. Vortex every 2 min.
--> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube.


> Reverse crosslinks and digest protein
--> Add 400 uL 1% SDS/TE buffer to 100 uL saved Supernatant.
--> Thaw Input aliquot for each cell line. Add 400 uL 1% SDS/TE buffer to 100 uL Input.
--> Samples:

  1. KAH126-1 input
  2. KAH126-1 α-myc Sup
  3. KAH126-1 α-myc IP
  4. KAH126-1 α-IgG Sup
  5. KAH126-1 α-IgG IP
  6. KAH132-8 input
  7. KAH132-8 α-myc Sup
  8. KAH132-8 α-myc IP
  9. KAH132-8 α-IgG Sup
  10. KAH132-8 α-IgG IP

--> Add 20 μL (20 μg) Pronase to each sample
--> Incubate at 42°C/ 1 hr. --> Incubate at 65 °C/ 2 days



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