03/31/10
- ✓ Minipreps: KAH86 re-built (4)
- ✓ Assembly: KAH96 re-build
- ✓ Sequencing (same-day): KAH86 plasmids
- ✓ Cultures: KAH014 (DPRE) (4)
Minipreps
> Check with E/P digests
> Compare to KAH81
| Reagent |
Volume
|
Expected:
1-4. KAH86 = 2726
5. KAH81 = 2640
|
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.
|
--> Samples 1 and 2 appear to run larger than KAH81. Check all 4 via sequencing and proceed with KAH96 assembly.
Assemblies
- KAH96: KMC01(5xGal4)/(S/P)/~3300 ✓ + (1) KAH86/(X/P)/2726
> Digests (Fermentas FD)
--> X/P/ScaI triple digest
| Reagent |
Volume |
|
30 μL/lane, 1% agarose -->
|
| DNA (plasmid) |
15.0
|
| 10x buffer |
3.0
|
| XbaI |
1.0
|
| PstI |
1.0
|
| ScaI |
1.0
|
| dH2O |
9.0
|
| |
30 μL --> 37°C/ ~15 min.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH94 (X/P) |
0.371 |
2.05 |
18.5
|
--> KMC01(S/P) = 62.3 ng/μL
> Ligations
| Ligation |
Plate results (lig : neg crtl) 04/01/10
|
| 1. KAH86(X/P)/2726, 41 ng + KMC01(S/P)/~3300, 25 ng |
KAH96 10:1 (Pick 4)
|
| 2. KMC01(S/P)/ 25 ng |
~50 colonies
|
| |
1 |
2 |
|
| Insert DNA |
2.5 |
--- |
|
| Vector DNA |
0.5 |
0.5 |
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
|
| T4 ligase (NEB) |
1.0 |
1.0 |
|
| dH2O |
1.0 |
3.5 |
|
| |
10 μL |
10 μL |
|
--> R.T./ ~10 min.; Add 50 μL Z-DH5α
Sequencing (Genewiz, same-day, 04/01/10)
--> Confirm HSVtkTATA-K
- KAH86-1 P0001: good
- KAH86-2 P0001: good
- KAH86-3 P0001: good
- KAH86-4 P0001: good
| 
Project name
Main project page
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