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
- 130-4 Input (1:50) (12 rxns)
- 130-4 α-myc IP (1:50) (12 rxns)
- 130-4 α-IgG IP (1:50) (12 rxns)
--> Primers
- INKARF C (138 bp)
- INKARF D (135 bp)
- INKARF E (86 bp)
- INKARF F (100 bp)
- MMP12 A (111 bp)
- MMP12 B (114 bp)
- MMP12 C (138 bp)
- TNF A (120 bp)
- TNF B (123 bp)
- TNF C (119 bp)
- GAPDH A (103 bp)
- 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/ ∞
> 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:
- KAH126-1: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
- KAH126-1: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
- KAH132-8: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
- 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:
- KAH126-1 input
- KAH126-1 α-myc Sup
- KAH126-1 α-myc IP
- KAH126-1 α-IgG Sup
- KAH126-1 α-IgG IP
- KAH132-8 input
- KAH132-8 α-myc Sup
- KAH132-8 α-myc IP
- KAH132-8 α-IgG Sup
- 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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