12/27/09
- Re-transform: KAH93/V0120; JDS42, 43
- Assemblies: KAH136; KAH137, 138 (do-over)
Assemblies
- KAH136: (1) PEST/(S/P)/3734 + EED/(X/P)/1401 ✓
- KAH137: KAH78/(E/S)/3652 ✓ + KAH134/(E/X)/4600 ✓
- KAH138: KAH79/(E/S)/3652 ✓ + "
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
5.0
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
20.0
|
|
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. PEST(S/P) |
0.197 |
2.18 |
9.9
|
> Ligations
Ligation |
Plate results (lig : neg ctrl) 12/28/09
|
1. EED(X/P)/1401, 15 ng + PEST(S/P)/3734, 20 ng |
KAH136 10:1 (Pick 2)
|
2. PEST(S/P)/ 20 ng + " |
|
3. KAH78(E/S)/3652, 24 ng + KAH134(E/X)/4600, 10 ng |
KAH137 2:1 (Pick 4)
|
4. KAH79(E/S)/3724, 24 ng + " |
KAH138 2:1 (Pick 4)
|
5. KAH134(E/X)/ 10 ng |
|
--> For KAH137 & 138, use less vector and 3:1 insert to minimize background
|
1 |
2 |
3 |
4 |
5
|
Insert DNA |
0.5 |
--- |
1.2 |
1.2 |
---
|
Vector DNA |
2.0 |
2.0 |
0.8 |
0.8 |
0.8
|
2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
1.5 |
2.0 |
2.0 |
2.0 |
3.2
|
|
10μL |
10μL |
10μL |
10μL |
10μL --> R.T./ ~15 min.; Add 25 μL Z-DH5α
|
|