01/12/10
- ✓ Minipreps: KAH117(4), 118(1), 119/MV2(1)
- ✓ Cultures: re-pick KAH117/MV2 (4)
- ✓ Assemblies: KAH117, 118/MV2
- ✓ Retransform: KAH94, 95
Minipreps
> Check with E/P digests
| Reagent |
Volume
|
Expected:
1-4. KAH117/MV2 = 7758
5. KAH118/MV2 = 7930
6. KAH119/MV2 = 8002
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
5.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
6.5
|
| |
15 μL --> 37°C/ ~15 min.
|
--> Pick 4 remaining colonies from KAH117/MV2 plate
--> Re-assemble KAH117, 118/MV2
Assemblies
- KAH117/MV2: KAH117/(E/P)/7758 ✓ + MV2/(E/P)/4529 ✓
- KAH118/MV2: KAH118/(E/P)/7930 ✓ + "
> Ligations
| Ligation |
Plate results (lig : neg crtl) 01/13/10
|
| 1. KAH117(E/P)/7758, 34 ng + MV2(E/P)/4529, 10 ng |
KAH117/MV2 16 colonies (Pick 6)
|
| 2. KAH118(E/P)/7930, 34 ng + " |
KAH118/MV2 14 colonies (Pick 6)
|
| 3. MV2(E/P)/ 10 ng |
16 colonies
|
| |
1 |
2 |
3
|
| Insert DNA |
11.1 |
11.1 |
---
|
| Vector DNA |
0.4 |
0.4 |
0.4
|
| 2x lgn buf (Roche) |
12.5 |
12.5 |
12.5
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0
|
| dH2O |
--- |
--- |
11.6
|
| |
25μL |
24μL |
24μL --> R.T./ 30 min.; Add 50μL Z-DH5α
|
> Digests (Fermentas FD)
--> For cloning KAH94, 95 into new JDS luciferase vectors (miR project)
- JDS47/(BglII)/7509
- JDS48/(BglII)/7519
| Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
| DNA (plasmid) |
25.0
|
| 10x buffer |
3.0
|
| BglII |
2.0
|
| dH2O |
---
|
| |
30 μL --> 37°C/ ~1 hr
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. JDS47(BglII) |
--- |
--- |
---
|
| 2. JDS48(BglII) |
--- |
--- |
---
|
|
Project name
Main project page
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