04/01/10
- ✓ Cultures: KAH96 rebuilt (4); KAH014 (8)
- ✓ Minipreps: KAH014(DPRE) (4)
- ✓ Cultures: KAH014 (8)
- ✓ Sequencing: KAH014; KAH144, 145 (confirmation of EED expression cassettes)
Minipreps
> Check with E/P digests
| Reagent |
Volume
|
Expected:
1-4. KAH014 = 1740
Double mut = 1740
Mut 1st Pst = 1078, 662
Mut 2nd Pst = 1663, 77
5. DPRE (no mut) = 1001, 662, 77
|
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.
|
--> KAH014-1 and 2 might be good (77 bp band?). Send for sequencing.
--> Pick 8 more clones from colony plate.
Sequencing (Genewiz, standard)
- KAH014-1 P0001: bad (w.t. PstI #1)
- KAH014-1 P0002: good (mut PstI #2)
- KAH014-2 P0001: bad (w.t. PstI #1)
- KAH014-2 P0002: good (mut PstI #2)
- KAH144 P0001: good
- KAH144 P0002: good
- KAH145 P0001: good
- KAH145 P0002: good
|
Project name
Main project page
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