09/29/10
- ✓ HEK Gal4EED cells: died (neglected cells for too long, restart culture); request vial from Mirra
- ✓ ChIP-PCR: all α-myc and α-IgG IP's
ChIP PCR
> Note: dx = DMA/CH2O double x-linked; fx = CH2O x-linked
--> Templates (use 1 μL and 2 μL 1:50 dilutions of each template; 20 rxn's total)
- (8) 130-4 α-myc IP
- (10) 130-4 α-IgG IP
- (3) 126-1 dx α-myc IP
- (5) 126-1 dx α-IgG IP
- (18) 126-1 fx α-myc IP
- (20) 126-1 fx α-IgG IP
- (13) 132-8 dx α-myc IP
- (15) 132-8 dx α-IgG IP
- (23) 132-8 fx α-myc IP
- (25) 132-8 fx α-IgG IP
--> Primers
- INKARF D (135 bp) (20 rxns)
- GAPDH B (103 bp) (20 rxns)
Reagent |
1 μL template |
2 μL template
|
ChIP DNA |
1.0 |
2.0
|
10 μM primers |
1.0 |
1.0
|
2x GoTaq Green |
10.0 |
10.0
|
dH2O |
8.0 |
7.0
|
|
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:
- Double x-linked samples are very non-specific; do not use
- Single x-linked samples show mostly no pull-down for GAPDH neg ctrl.
- 126-1 appears to IP with INKARF D
- Next ChIP-PCR: compare antibody IP vs. supernatant to get ratios of enrichment before running qPCR
|