08/17/10
- ✓ RT-PCR: KAH126-1 -/+ dox cDNA
RT-PCR
> Need to re-optimize p16INK primers, 7B produces non-specific bands in biological replicates
> KAH126-1 -/+ dox cDNA
1,2. KAH126-1 -/+ dox (6/15/10 Trizol, 5 μg RNA)
3,4. KAH126-1 -/+ dox (6/18/10 Trizol, 5 μg RNA)
5,6. KAH126-1 -/+ dox (7/26/10 RNeasy, 2 μg RNA)
7,8. FTRx -/+ rotenone (7/08/10 Trizol, 5 μg RNA)
> Primer pairs:
- p16INK 7A
- p16INK 7B
- p16INK 7C
--> See 5/04/10 for successful p16INK RT-PCR
| Reagent |
1:1 cDNA |
MM (x10) |
|
15 μL/lane; 2% agarose
|
| cDNA |
0.5 (1:1) |
---
|
| 10μM primers |
1.0 |
10.0
|
| 2x GoTaq Green |
10.0 |
100.0
|
| dH2O |
8.5 |
85.0
|
| |
20.0 μL |
|
|
|
--> Aliquot 19.5 μL ("1:1" tubes) of each DNA mix to appro. wells
--> Add 0.5 (1:1) cDNA to each well
--> 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/ ∞
--> Conclusion: p16 7C gives the most specific product. Use this primer pair for all samples.
|
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