05/04/10
- ✓ Minipreps: FlpE (4)
- ✓ RT-PCR: Pc-ATF 4-day dox GAPDH controls
- ✓ Hygro kill curve: check cells
- ✓ Thaw cells: KAH126-1, 132-4, 132-6 (spin down in 5 mL FBS/ts; seed in 20% FBS/ts medium)
- ✓ FlpE: streak plates using Bruno's glycerol stock (20 and 100 μg/mL Amp plates) Note: FlpE is maintained in Invitrogen TOP10 cells
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.
--> Conclusion: Failed again. Ask Bruno for glycerol stock. Perhaps FlpE is kept in non DH5α strain...?
RT-PCR
> Continued from 5/03/10: semi-qRT-PCR
--> Use 1:1000 dilution for GAPDH primers (10,000 might be too low)
--> cDNA templates
- KAH126-1
- KAH126-1(+dox)
- KAH127-4
- KAH127-4(+dox)
- KAH128-8
- KAH128-8(+dox)
- KAH129-4
- KAH129-4(+dox)
- KAH130-2
- KAH130-2(+dox)
- KAH131-9
- KAH131-9(+dox)
- KAH132-8
- KAH132-8(+dox)
- KAH133-1
- KAH133-1(+dox)
- FTRx
- FTRx(+dox)
--> Primers; cDNA dilution
- GAPDH(21A); 1:1,000
- GAPDH(21B); 1:1,000
Reagent |
Volume |
Primer mix (x20) |
cDNA mix (x3)
|
15 μL/lane; 2% agarose
|
cDNA |
0.5 |
--- |
1.5 (3x 0.5)
|
10μM primers |
1.0 |
20.0 (20x 1.0) |
---
|
2x GoTaq Green |
10.0 |
200.0 (20x 10.0) |
---
|
dH2O |
8.5 |
80.0 (20x 4.0) |
13.5 (3x 4.5)
|
|
20.0 μL
|
|
--> GAPDH(21B) works well. Quantitate results using ImageJ.
Hygro kill curve
> Result: 200 μg/mL shows 100% cell death for Flp-in T-REx, KAH126-1, KAH130-2, and KAH132-8
> Take note for making hygro+ stables later
|