07/27/11
- ✓ Assemblies: KAH226, 227; 219-224 into pSB31 w/ adapter
Assemblies
> Assemble KAH226, 227 as usual
> Blunt cloning for pSB31 didn't work well. Use EcoRI/NcoI-cut vector, add SpeI-EcoRI-NcoI adapter to E/S insert. Two EcoRI sites will allow quick screening of minipreps.
- KAH226: (1) KAH225/(E/S)/1494 + KAH011/(E/X)/64 ✓
- KAH227: (2) Gluc/(E/S)/558 + (3) STOP/(E/X)/13
- KAH219/pSB31: KAH219/(E/S)/2422 ✓ + pSB31 BB adapter 1/(S/NcoI)/18 + (4) pSB31/(E/NcoI)/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 ✓ + "
> Digests (Fermentas FD)
--> Specific notes
| Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
| 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. KAH225 (E/S) |
0.867 |
1.99 |
43.4
|
| 2. Gluc (E/S) |
0.280 |
2.45 |
14.0
|
| 3. STOP (E/X) |
0.214 |
2.25 |
10.7
|
| 4. pSB31 (E/NcoI) |
0.931 |
1.97 |
46.6
|
--> pSB31 BB adapter 1 (IDT) = 850 ng/μL (Dilute to 8.5 ng/μL before use)
> Ligations
| Ligation |
Plate results (lig : neg crtl) 07/28/11
|
| 1. KAH225(E/S)/1494, 18 ng + KAH011(E/X)/~3264, 20 ng |
KAH226 >10:1 (Pick 2)
|
| 2. KAH011(E/X)/ 20 ng |
|
| 3. Gluc(E/S)/558, 7 ng + STOP(E/X)/~3213, 20 ng |
KAH226 >10:1 (Pick 2)
|
| 4. STOP(E/X)/ 20 ng |
|
| 5. KAH219(E/S)/2422, 19 ng + pSB31 BB adapter 1(S/NcoI)/18, 0.1 ng + pSB31(E/NcoI)/5127, 20 ng |
KAH219/pSB31 1:1
|
| 6. KAH220(E/S)/1345, 11 ng + " |
KAH220/pSB31 1:1
|
| 7. KAH221(E/S)/1645, 13 ng + " |
KAH221/pSB31 1:1
|
| 8. KAH222(E/S)/2614, 20 ng + " |
KAH222/pSB31 1:1
|
| 9. KAH223(E/S)/1537, 12 ng + " |
KAH223/pSB31 1:1
|
| 10. KAH224(E/S)/1837, 14 ng + " |
KAH224/pSB31 1:1
|
| 11. pSB31(E/NcoI)/ 20 ng |
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
|
| Insert DNA |
0.4 |
--- |
0.5 |
--- |
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 |
0.5 |
0.5 |
2.0 |
2.0 |
0.4 |
0.4 |
0.4 |
0.4 |
0.4 |
0.4 |
0.4
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
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 |
1.0 |
1.0 |
1.0
|
| dH2O |
3.1 |
3.5 |
1.5 |
2.0 |
1.7 |
2.7 |
2.6 |
2.1 |
2.1 |
2.5 |
3.6
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
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
--> pSB31 ligations failed. Too much background on neg ctrl. Dephosphorylate pSB31(E/NcoI) and try again.
|
Project name
Main project page
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