12/16/09
- ✓ Digest: confirm KAH105-108 (& /MV2) via E/P digests
- ✓ Minipreps: KAH126-133/MV2 (2 ea); KAH123, 125 (6 ea)
- ✓ Assemblies: KAH117-119; KAH135, 136
Digest check
> May have mixed up and mislabeled KAH105-108 (& /MV2). Sequencing results are out of order (see | 12/09/09). Could be cause of last failed transfection experiment.
Reagent |
Volume
|
Expected: MV2 = 4529 1. KAH105 = 2794 2. KAH105/MV2 = 2794 3. KAH106 = 2788 4. KAH106/MV2 = 2788 5. KAH107 = 2966 6. KAH107/MV2 = 2966 7. KAH108 = 2960 8. KAH108/MV2 = 2960
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
1.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
10.5
|
|
15 μL --> 37°C/ ~15 min.
|
Results are consistent with the sequencing data: 1 = KAH108, 3 = KAH105, 5 = KAH106, 7 = KAH107
--> Correct labeling on the tubes
Minipreps
> Check with E/P digests
Reagent |
Volume
|
Expected: MV2 = 4529 1,2. KAH126/MV2 = 1880 3,4. KAH127/MV2 = 1880 5,6. KAH128/MV2 = 1880 7,8. KAH129/MV2 = 1880 9,10. KAH130/MV2 = 2864 11,12. KAH131/MV2 = 2867 13,14. KAH132/MV2 = 1688 15,16. KAH133/MV2 = 1532 17-22. KAH123 = 8002 23-28. KAH125 = 8246
|
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.
Assemblies
- KAH117: KAH99/(E/S)/2913 ✓ + (1) KAH93/(E/X)/8039
- KAH118: KAH100/(E/S)/3085 ✓ + "
- KAH119: KAH101/(E/S)/3157 ✓ + "
- KAH135: KAH78/(E/S)/3652 ✓ + (2) KAH134/(E/X)/4600
- KAH136: KAH78/(E/S)/3652 ✓ + "
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
10.0
|
10x buffer |
3.0
|
enzyme 1 |
2.0
|
enzyme 2 |
2.0
|
dH2O |
13.0
|
|
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH93(E/X) |
0.283 |
2.49 |
14.2
|
2. KAH134(E/X) |
0.367 |
1.94 |
18.3
|
> Dephosphorylation (Roche)
Reagent |
Volume
|
DNA (clean digest) |
up to 17 μL (250 ng)
|
10x buffer d.p. |
2.0
|
phosphatase |
1.0
|
dH2O |
---
|
|
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 12.5 ng/μL
|
> Ligations
Ligation |
Plate results (lig : neg ctrl) 12/17/09
|
1. KAH99(E/S)/2913, 29 ng + KAH93(E/X)/8039, 40 ng |
KAH117 too few col's (re-do)
|
2. KAH100(E/S)/3085, 31 ng + " |
KAH118 too few col's (re-do)
|
3. KAH101(E/S)/3157, 31 ng + " |
KAH118 too few col's (re-do)
|
4. KAH93(E/X)/ 40 ng |
|
5. KAH78(E/S)/3652, 41 ng + KAH134(E/X)/4600, 20 ng |
KAH135 too few col's (re-do)
|
6. KAH79(E/S)/3724, 41 ng + " |
KAH136 too few col's (re-do)
|
7. KAH134(E/X)/ 20 ng |
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7
|
Insert DNA |
0.5 |
0.6 |
0.8 |
--- |
2.0 |
2.0 |
---
|
Vector DNA |
3.2 |
3.2 |
3.2 |
3.2 |
1.6 |
1.6 |
1.6
|
2x lgn buf (Roche) |
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
|
dH2O |
0.3 |
0.2 |
--- |
0.8 |
0.4 |
0.4 |
2.4
|
|
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL --> R.T./ ~15 min.; Add 25 μL Z-DH5α
|
|