04/10/10
- ✓ Assemblies: re-digest vectors and KAH96 from yesterday; re-do ligations
Assemblies
--> See 4/09/10
> Digests (Fermentas FD)
--> Repeat digests from yesterday; increase time to 1 hour:
- KAH96 (E/S/ApaLI)
- KAH145 (E/X)
- KAH116 (E/X)
| Reagent |
2,3 |
1
|
30 μL/lane, 1% agarose
|
| DNA (plasmid) |
up to 25 μL |
up to 24 μL
|
| 10x buffer |
3.0 |
3.0
|
| enzyme 1 |
1.0 |
1.0
|
| enzyme 2 |
1.0 |
1.0
|
| ApaLI |
--- |
1.0
|
| dH2O |
--- |
---
|
| |
30 μL |
30 μL --> 37°C/ ~ 1 hr.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH96 (E/S) |
0.987 |
1.88 |
49.3
|
| 2. KAH145 (E/X) |
0.507 |
1.72 |
25.3
|
| 3. KAH116 (E/X) |
0.539 |
1.90 |
27.0
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) 04/10/10
|
| 1. KAH96(E/S)/2825, 33 ng + KAH145(E/X)/6755, 40 ng |
KAH146 10:1 (Pick 2)
|
| 2. KAH142(E/S)/4571, 54 ng + " |
KAH147 10:1 (Pick 2)
|
| 3. KAH145(E/X)/ 40 ng |
~5 colonies
|
| 4. KAH01(E/S)/186, 2 ng + KAH116(E/X)/3975, 20 ng |
KAH148 10:1 (Pick 2)
|
| 5. KAH02(E/S)/186, 2 ng + " |
KAH149 10:1 (Pick 2)
|
| 6. KAH03(E/S)/186, 2 ng + " |
KAH150 10:1 (Pick 2)
|
| 7. KAH04(E/S)/186, 2 ng + " |
KAH151 10:1 (Pick 2)
|
| 8. KAH06(E/S)/1170, 12 ng + " |
KAH152 10:1 (Pick 2)
|
| 9. KAH06(E/S)/1173, 12 ng + " |
KAH153 10:1 (Pick 2)
|
| 10. KAH116(E/X)/ 50 ng |
~10 colonies
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10
|
| Insert DNA |
0.7 |
2.7 |
--- |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
1.5 |
---
|
| Vector DNA |
1.6 |
1.6 |
1.6 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7
|
| 2x lgn buf (Roche) |
5.0 |
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 |
1.0
|
| dH2O |
1.7 |
--- |
2.4 |
2.8 |
2.8 |
2.8 |
2.8 |
2.8 |
1.8 |
3.3
|
| |
10 μL |
~10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL
|
--> R.T./ ~10 min.; Add to 20 μL Z-DH5α
--> 4/11/10: Success! Pick 2 colonies each.
|
Project name
