01/18/10
- ✓ Cultures: KAH94, 95/JDS49 (not 47!) (6 each); KAH94, 95/JDS48 (6 each)
- ✓ Minipreps: KAH118/MV2 (4)
- ✓ Assemblies: KAH139, 140, 141
Minipreps
> Check with E/P digests
Reagent |
Volume
|
Expected: MV2 = 4529 1-4. KAH118/MV2 = 7930
|
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.
|
--> None have correct size for MV2 vector. Pick 12 more colonies.
Assemblies
- KAH139: HPK-Kstop(KAH103)/(E/S)/546 + (1) KAH137/(E/X)/~5593
- KAH140: HPK-K(KAH104)/(E/S)/540 + "
- KAH141: HPK-K(KAH104)/(E/S)/540 + (2) KAH138/(E/X)/~5181
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
up to 25 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH137(E/X) |
0.526 |
1.79 |
26.3
|
2. KAH138(E/X) |
0.858 |
1.82 |
42.9
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 01/19/10
|
1. KAH103(E/S)/546, 6 ng + KAH137(E/X)/5593, 30 ng |
KAH139 >10:1 (Pick 2)
|
2. KAH104(E/S)/540, 6 ng + " |
KAH140 >10:1 (Pick 2)
|
3. KAH137(E/X)/ 30 ng + " |
|
4. KAH104(E/S)/540, 17 ng + KAH138(E/X)/1981, 30 ng |
KAH141 >10:1 (Pick 2)
|
5. KAH138(E/X)/ 30 ng + " |
|
|
1 |
2 |
3 |
4 |
5
|
Insert DNA |
0.9 |
0.9 |
--- |
1.3 |
---
|
Vector DNA |
1.1 |
1.1 |
1.1 |
0.7 |
0.7
|
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 |
2.0 |
2.0 |
2.9 |
2.0 |
3.3
|
|
10μL |
10μL |
10μL |
10μL |
10μL --> R.T./ ~15 min.; Add 20 μL Z-DH5α
|
|