04/03/11
- ✓ RT-PCR: test new primers
- ✓ Repressed luc reactivation: expand HEK293 23;4;9 to 12 plates total (split each current one 1:3); dox induction (4x no dox, 4x 2-day, 4x 4-day)
- ✓ Pc-TF cell cultures: split all cultures; 6-well plate dox induction (4 days) for 129-4, 154-2, 156-5, 157-1 (3 reps ea.)
RT-PCR
> Test new RT-PCR primers on KAH126-1 (20 rxns total)
> Primers:
1-4. HOXC4 50A-D (86, 87, 86, 85 bp)
5-8. IGFBP2 51A-D (117, 120, 113, 114 bp)
9-12. NFE2 52A-D (90, 88, 118, 120 bp)
13-16. NEUROG1 53A-D (105, 117, 92, 98 bp)
17-20. SOX1 54A-D (84, 80, 80, 102 bp)
> Template:
- KAH126-1 +dox (3/10/11) 1:10
| Reagent |
1 rxn |
Template mix (21x)
|
| cDNA |
0.5 |
10.5
|
| 10 μM primers |
1.0 |
---
|
| 2x GoTaq Green |
10.0 |
210.0
|
| dH2O |
8.5 |
178.5
|
| |
20.0 μL
|
--> 1 template mix: aliquot 19.0 μL
--> Add 1.0 μL primers
--> PCR (96-well)
- 95°C/ 3 min.
- [95°C/ 15 sec., 58°C/ 15 sec., 72°C/ 15 sec.] x32
- 72°C/ 3 min.
- 4°C/ ∞
--> Conclusions:
- HOXC4: qPCR test A-D
- IGFBP2: qPCR test A-D
- NFE2: failed
- NEUROG1: failed
- SOX1: failed
Next: Do qRT-PCR test with 1:10 dilution of 126-1 (+dox 3/10/11)
|
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