03/24/10
- ✓ Minipreps: KAH143 (4)
- ✓ Assembly: KAH144
- ✓ Sequencing order: KAH86, 96, 143
Minipreps
> Check with E/P digest
| Reagent |
Volume
|
Expected:
1-4. KAH143 = 1853
|
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
- KAH144: KAH104/(E/S)/540 ✓ + (1) KAH142/(E/X)/~5053
> 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. KAH143 (E/X) |
0.125 |
1.97 |
6.3
|
--> KAH104(E/S) = 25.8 ng/μL
> Ligations
| Ligation |
Plate results (lig : neg crtl) 03/25/10
|
| 1. KAH104(E/S)/540, 5.3 ng + KAH143(E/X)/~5053, 25 ng |
new BioBrick 5:1 (Pick 4)
|
| 2. KAH143(E/X)/ 25 ng |
~100 colonies
|
| |
1 |
2 |
|
| Insert DNA |
0.5 |
--- |
|
| Vector DNA |
4.0 |
4.0 |
|
| 2x lgn buf (Roche) |
5.5 |
5.5 |
|
| T4 ligase (NEB) |
1.0 |
1.0 |
|
| dH2O |
--- |
0.5 |
|
| |
11 μL |
11 μL |
|
--> R.T./ ~10 min., Add to 50 μL Z-DH5α
Sequencing order (same day, placed @ 8pm today, results Friday)
- KAH86 P0001: missing HSVtkTATA-K promoter
- KAH86 P0002: good
- KAH96 P0001: missing HSVtkTATA-K promoter
- KAH96 P0002: good
- KAH143 P0001: good
- KAH143 (check NLSstop) P0002: good (NLS stop2 confirmed)
|
Project name
Main project page
Previous entry Next entry
