07/28/11
- ✓ Cultures/ minipreps: KAH226, 227 (2 each)
- ✓ Assemblies: retry pSB31 ligations
Minipreps
> Check with E/P digests
| Reagent |
Volume
|
Expected:
1,2. KAH226 = 1564
3,4. KAH227 = 577
|
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
--> Too much background last time. Re-try with dephosphorylated pSB31(E/NcoI)
- KAH219/pSB31: KAH219/(E/S)/2422 ✓ + pSB31 BB adapter 1/(S/NcoI)/18 + (4) pSB31/(E/NcoI dp)/5127
- KAH220/pSB31: KAH220/(E/S)/1345 ✓ + "
- KAH221/pSB31: KAH221/(E/S)/1645 ✓ + "
- KAH222/pSB31: KAH222/(E/S)/2614 ✓ + "
- KAH223/pSB31: KAH223/(E/S)/1537 ✓ + "
- KAH224/pSB31: KAH224/(E/S)/1837 ✓ + "
> Dephosphorylation (Roche)
| Reagent |
Volume
|
| DNA (clean digest) |
10.7 μL (500 ng)
|
| 10x buffer d.p. |
2.0
|
| phosphatase |
1.0
|
| dH2O |
6.3
|
| |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) 07/28/11
|
| 1. KAH219(E/S)/2422, 19 ng + pSB31 BB adapter 1(S/NcoI)/18, 0.1 ng + pSB31(E/NcoI dp)/5127, 20 ng |
KAH219/pSB31 10:1
|
| 2. KAH220(E/S)/1345, 11 ng + " |
KAH220/pSB31 10:1
|
| 3. KAH221(E/S)/1645, 13 ng + " |
KAH221/pSB31 10:1
|
| 4. KAH222(E/S)/2614, 20 ng + " |
KAH222/pSB31 10:1
|
| 5. KAH223(E/S)/1537, 12 ng + " |
KAH223/pSB31 10:1
|
| 6. KAH224(E/S)/1837, 14 ng + " |
KAH224/pSB31 10:1
|
| 7. pSB31(E/NcoI)/ 20 ng |
one colony
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
|
| Insert DNA |
1.4 |
0.4 |
0.5 |
1.0 |
1.0 |
0.6 |
---
|
| adapter DNA |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
---
|
| Vector DNA |
0.8 |
0.8 |
0.8 |
0.8 |
0.8 |
0.8 |
0.8
|
| 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 |
1.3 |
2.3 |
2.2 |
1.7 |
1.7 |
2.1 |
3.2
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
|
--> Add 30 μL DH5α Turbo; plate on 100 μg/mL Amp
|
Project name
Main project page
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