09/20/10
- ✓ RT-PCR: full EZH2 kd set
- ✓ ChIP PCR: Input vs. myc IP (partial set)
- ✓ Cell culture: expand HepG2 cells; split HEK Gal4EED into selective maintenance (2.0 μg/mL puro) and non-selective media
RT-PCR
> EZH kd set
--> Templates
- n.t. kd 1 8-day, 9/18/09
- EZH2 kd 1 8-day, 9/18/09
- n.t. kd 2 8-day, 9/18/09
- EZH2 kd 2 8-day, 9/18/09
- n.t. kd 4-day, 11/03/09
- EZH2 kd 4-day, 11/03/09
- n.t. kd 8-day, 11/08/09
- EZH2 kd 8-day, 11/08/09
--> Primers
- p16INK 7C (94 bp) (8 rxns)
- MMP12 31A (127 bp) (8 rxns)
- THPO 33A (103 bp) (8 rxns)
- TNF 34A (119 bp) (8 rxns)
- GAPDH 21A (100 bp), 1:1000 cDNA (8 rxns)
- EZH2 22A (71 bp) (8 rxns)
Reagent |
Single rxn
|
cDNA |
0.5
|
10 μM primers |
1.0
|
2x GoTaq Green |
10.0
|
dH2O |
8.5
|
|
20.0 μL
|
--> PCR (96-well)
- 95°C/ 3 min.
- [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
- 72°C/ 3 min.
- 4°C/ ∞
> Conclusions:
- p16INK: responds slightly, but still very repressed (probably due to DNA methylation)
- MMP12: appears to respond to EZH2 kd. Need to quantitate using Image J.
- THPO: may respond somewhat, pretty active overall.
- TNF: appears to respond to EZH2 kd. Need to quantitate using Image J.
- 8-day knockdown trial #2: Need to repeat using more nt kd cDNA (loading control signal too low for good comparison)
- EZH2: Need to repeat with less template DNA; amplification not within linear range
ChIP PCR
> Optimize template concentration
> See 6/10/10 for successful primer test
--> Templates
- 126-1 Input
- 126-1 α-myc IP
- 130-4 Input
- 130-4 α-myc IP
- 132-8 Input
- 132-8 α-myc IP
- 126-1 Input (1:50)
- 126-1 α-myc IP (1:50)
- 130-4 Input (1:50)
- 130-4 α-myc IP (1:50)
- 132-8 Input (1:50)
- 132-8 α-myc IP (1:50)
--> Primers
- INKARF D (135 bp) (12 rxns)
- GAPDH A (103 bp) (12 rxns)
Reagent |
Single rxn
|
cDNA |
0.5 (1:1 cDNA)
|
10 μM primers |
1.0
|
2x GoTaq Green |
10.0
|
dH2O |
8.5
|
|
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: GAPDH negative control comes down with 126-1 (double x-linked), slightly less with 132-8 (double x-linked) but not with 130-4 (single x-linked). As previously suspected, DMA plus formaldehyde may cause over-crosslinking. Continue only with single x-linked samples from now on.
|