09/19/10
- ✓ RT-PCR: EZH2 kd cDNA, NS candidate genes; re-do MMP12, THPO, TNF in NS cDNA set (1:10 dilutions)
RT-PCR
> EZH kd set
--> Templates
- n.t. siRNA 4-day, 11/03/09
- EZH2 kd 4-day, 11/03/09
- n.t. siRNA 8-day,
11/03/09 (?)
- EZH2 kd 8-day,
11/03/09 (?)
--> Primers
- p16INK 7C (94 bp) (4 rxns)
- MMP12 31A (127 bp) (4 rxns)
- THPO 33A (103 bp) (4 rxns)
- TNF 34A (119 bp) (4 rxns)
> NS cDNA set
--> Templates (1:10 dilutions)
--> Primers
- MMP12 31A (127 bp) (16 rxns)
- THPO 33A (103 bp) (16 rxns)
- TNF 34A (119 bp) (16 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:
- EZH2 kd: looks like genes are de-repressed after EZH2 kd, but p16INK is very weak. Do PCR again on full set of EZH2 kd's
- Pc-ATF repeats: choose best trial for figure (vs. previous trial with more template)
|