04/29/10
- ✓ RT-PCR: 4-day dox induction of Pc-ATF lines, mCh primer optimization
- ✓ Senescence assay: expand KAH126-3, -4 cell cultures to 10 cm plates (maintenance med.)
- ✓ Minipreps: FlpE (4)
RT-PCR
> Reaction 1: optimization of mCherry primers
--> cDNA templates
- KAH126-1 (1:1)
- KAH126-1 +dox (1:1)
- KAH126-1 (1:10,000)
- KAH126-1 +dox (1:10,000)
- FTRx (1:1)
- FTRx +dox (1:1)
- FTRx (1:10,000)
- FTRx +dox (1:10,000)
--> primers
- mCh f1/r1, 100 bp, #1-8
- mCh f2/r2, 100 bp, #9-16
| Reagent |
Volume |
Master mix (x10)
|
15 μL/lane; 1% agarose
|
| cDNA |
0.5 |
---
|
| 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 of each DNA mix to appro. wells
--> Add 0.5 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/ ∞
--> Result: primer pair one shows expected results at 1:10,000 dilution
--> Next PCR: full Pc-ATF sample set, plus gene target primers
Minipreps
> Check with E, P, and E/P digests
| Reagent |
1,4,7,10 |
2,5,8,11 |
3,6,9,12
|
Expected:
1. FlpE-1, E = 7160, 546
2. FlpE-1, P = 7706
3. FlpE-1, E/P = 5649, 1511, 546
4. FlpE-2, E = 7160, 546
5. FlpE-2, P = 7706
6. FlpE-2, E/P = 5649, 1511, 546
7. FlpE-3, E = 7160, 546
8. FlpE-3, P = 7706
9. FlpE-3, E/P = 5649, 1511, 546
10. FlpE-4, E = 7160, 546
11. FlpE-4, P = 7706
12. FlpE-4, E/P = 5649, 1511, 546
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 |
2.0 |
2.0
|
| 10X buffer |
1.5 |
1.5 |
1.5
|
| EcoRI |
1.0 |
--- |
1.0
|
| PstI |
--- |
1.0 |
1.0
|
| dH2O |
10.5 |
10.5 |
9.5
|
|
|
15 μL --> 37°C/ ~15 min.
--> None look correct. Next time, do a "slow" transfection (heat shock, recovery).
|
Project name
Main project page
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