09/07/10
- ✓ RT-PCR: Pc-ATF Nanostring set, mCherry; test HHEX primers on KAH129-4
- ✓ Sequencing order: KAH160-170/KAH015(FUW) for Casey (trouble expressing some constructs; check via sequencing)
- ✓ Order oligos: new gene subset (see 9/05/10)
RT-PCR
> Use cDNA from Nanostring samples
> Same PCR conditions as p16 (see 8/26/10)
> Templates + Primers:
- Nanostring (Ns.) sample set (1-20) + mCherry f1/r1 (1:100 cDNA dln.)
- Ns. KAH129-4 -/+ dox + HHEX 28A
- Ns. KAH129-4 -/+ dox + HHEX 28B
| Reagent |
1-20, MM (x20) |
11-14, MM (x5)
|
| cDNA |
--- |
---
|
| 10 μM primers |
20.0 (mCh f1/r1) |
---
|
| 2x GoTaq Green |
200.0 |
50.0
|
| dH2O |
80.0 |
42.5
|
| |
20.0 μL |
|
--> Add 5.0 μL cDNA (1:1000) to 1-20
--> Add 0.5 μL cDNA (1:1) and 1.0 μL primers to 11-14
--> 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/ ∞
(*Same as p16INK PCR, 8/26/10)
> Conclusions: Non-specific products for mCherry (re-do with 2 uL template); HHEX gave no signal
Order oligos
|
Project name
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