07/13/11
- ✓ Cultures/minipreps: KAH213-218, KAH207-209/V0200 (2 each)
- ✓ Assemblies: miR-luc sensor set with 177/MV8 vector (re-build, see 6/7/11)
Minipreps
> Check KAH213-218 with E/P
> Check KAH207-209/V0200 with N/S
| Reagent |
Volume
|
Expected:
V0120 = ~3200
1,2. KAH213 = 2834
3,4. KAH214 = 1757
5,6. KAH215 = 2057
7,8. KAH216 = 2590
9,10. KAH217 = 1513
11,12. KAH218 = 1813
V0200 = 4442
13,14. KAH207/V0200 = 1836
15,16. KAH208/V0200 = 1884
17,18. KAH209/V0200 = 1932
|
15 μL/lane; 1% agarose
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~15 min.
|
Assemblies
--> Last time (6/06/11), may have used too much insert. Use calculated amount this time
--> Use 177/MV8(E/X) 1/25/11. Was successful with 183-177/MV8 assembly (see 7/09/11)
- KAH186-177/MV8: KAH186/(E/S)/2025 ✓ + KAH177/MV8/(E/X)/7978 ✓
- KAH184-177/MV8: KAH187/(E/S)/2269 ✓ + "
- KAH187-177/MV8: KAH187/(E/S)/2269 ✓ + "
- KAH188-177/MV8: KAH188/(E/S)/2124 ✓ + "
- KAH189-177/MV8: KAH189/(E/S)/2296 ✓ + "
- KAH190-177/MV8: KAH190/(E/S)/2368 ✓ + "
- KAH191-177/MV8: KAH191/(E/S)/2223 ✓ + "
- KAH192-177/MV8: KAH192/(E/S)/2461 ✓ + "
> Ligations
| Ligation |
Plate results (lig : neg crtl) 07/14/11
|
| 1-8. KAH###(E/S)/~2200, 11 ng + KAH177/MV8(E/X)/7978, 20 ng |
Notes: >10:1 for all plates
|
| 9. KAH177(E/X)/ 20 ng |
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
|
| Insert DNA |
1.0 |
1.0 |
1.5 |
1.0 |
1.0 |
0.5 |
1.0 |
1.0 |
--- |
|
| Vector DNA |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
|
| dH2O |
2.0 |
2.0 |
1.5 |
2.0 |
2.0 |
2.5 |
2.0 |
2.0 |
3.0 |
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
|
--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL amp
|
Project name
Main project page
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