07/09/11
- ✓ Miniprep digests: check 183-177/MV8 clones #2, 4 (sequence verified)
- ✓ Assemblies: KAH207, 208, 209/V0200; KAH210, 211, 212
Minipreps
> Check with ApaLI digest
| Reagent |
Volume
|
Expected:
MV8 bands = 1243, 1135, 288
1. KAH183-177/MV8 #2 = 4218*, 2772**, 711
2. KAH183-177/MV8 #4 = 4218*, 2772**, 711
*insert + 234 (vector) + 1601 (177 frag)
**last 177 frag + 1655
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
5.0 μL
|
| 10X buffer |
1.5
|
| ApaLI |
1.0
|
| dH2O |
7.5
|
| |
15 μL --> 37°C/ ~15 min.
|
Assemblies
- KAH207/V0200: (1) KAH207/(N/S)/1836 + (4) V0200/(N/S)/4442
- KAH208/V0200: (2) KAH208/(N/S)/1884 + "
- KAH209/V0200: (3) KAH209/(N/S)/1932 + "
- KAH210: (5) KAH204/(E/S)/2328 + NLS myc stop/(E/X)/64 ✓
- KAH211: (6) KAH205/(E/S)/1251 + "
- KAH212: (7) KAH206/(E/S)/1551 + "
> Digests (Fermentas FD)
| Reagent |
Volume |
|
|
| 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. KAH207 (N/S) |
0.333 |
2.27 |
16.7
|
| 2. KAH208 (N/S) |
0.484 |
2.04 |
24.2
|
| 3. KAH209 (N/S) |
0.467 |
2.11 |
23.4
|
| 4. V0200 (N/S) |
2.266 |
1.83 |
113.3 (diluted to 50 ng/μL w/ EB)
|
| 5. KAH204 (N/S) |
0.205 |
2.01 |
10.3
|
| 6. KAH205 (N/S) |
0.401 |
1.78 |
20.0
|
| 7. KAH206 (N/S) |
0.423 |
1.72 |
21.1
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) 07/10/11
|
| 1. KAH207(N/S)/1836, 21 ng + V0200(N/S)/4442, 25 ng |
KAH207/V0200 1:1 (re-do*)
|
| 2. KAH208(N/S)/1884, 21 ng + " |
KAH208/V0200 1:1 (re-do*)
|
| 3. KAH209(N/S)/1932, 22 ng + " |
KAH209/V0200 1:1 (re-do*)
|
| 4. V0200(N/S)/ 25 ng |
|
| 5. KAH204(E/S)/2328, 35 ng + NLS myc stop(E/X)/~3264, 25 ng |
KAH210 >10:1 (Pick 2)
|
| 6. KAH205(E/S)/1251, 19 ng + " |
KAH211 >10:1 (Pick 2)
|
| 7. KAH206(E/S)/1551, 24 ng + " |
KAH212 >10:1 (Pick 2)
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|
| Insert DNA |
1.5 |
1.0 |
1.0 |
--- |
3.4 |
1.0 |
1.5 |
--- |
|
| Vector DNA |
0.5 |
0.5 |
0.5 |
0.5 |
0.6 |
0.6 |
0.6 |
0.6 |
|
| 2x lgn buf (Roche) |
5.0 |
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 |
1.0 |
|
| dH2O |
2.0 |
2.5 |
2.5 |
3.5 |
--- |
2.4 |
1.9 |
3.4 |
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
|
--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL Amp
--> * Re-do V0200 ligations using dephosphorylated vector
|
Project name
Main project page
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