09/08/10
- ✓ RT-PCR: repeat mCherry for Nanostring-RNA cDNA set
- ✓ p16 induction time course: set up KAH126-1, 154-2, 132-8, and FTRx in 10 cm plates (1:10 split, plain medium)
- ✓ Cell culture: split confluent back-up stocks
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 (1:500 cDNA dln.)
| Reagent |
1-20, MM (x21)
|
| cDNA |
---
|
| 10 μM primers |
21.0 (mCh f1/r1)
|
| 2x GoTaq Green |
210.0
|
| dH2O |
136.5
|
| |
20.0 μL/rxn
|
--> Add 2.5 μL cDNA (1:1000)
--> 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 amplification again. Try again with both mCh1 and mCh2 primers and use less template DNA
Trial 2
> Templates + Primers:
- Nanostring (Ns.) sample set (1-20) + mCherry f1/r1 (1.0μL of 1:1000 cDNA dln.)
- Nanostring (Ns.) sample set (1-20) + mCherry f2/r2 (1.0μL of 1:1000 cDNA dln.)
| Reagent |
1-20, MM (x21)
|
| cDNA |
---
|
| 10 μM primers |
21.0 (mCh f1/r1)
|
| 2x GoTaq Green |
210.0
|
| dH2O |
189.0
|
| |
20.0 μL/rxn
|
--> Add 1.0 μL cDNA (1:1000)
--> 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: Success! Both work. Use mCh1 for figure.
|
Project name
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