08/12/11
- ✓ Cultures/ minipreps: (cultures from last night); Pc-TF constructs in pSB31 (3 each, 24 total)
- ✓ Assemblies: KAH234, 235/ V0200
Minipreps
> Check with E digests
> Also check with NcoI digests for orientation (Expected sizes are for the forward orientation); p65 (KAH232, 231) has 2 NcoI sites
| Reagent |
Volume
|
Expected:
Gel 1:
Vector = 5127
1-3. KAH222 = 2614
4-6. KAH223 = 1537
7-9. KAH224 = 1837
10-12. KAH232 = 1780
13-15. KAH219 = 2422
16-18. KAH220 = 1345
19-21. KAH221 = 1645
22-24. KAH231 = 1588
Gel 2:
Vector
fwd = ~5000
rev = >5000
*Last NcoI frag + 141
1-3. KAH222 = 2108*, 636
4-6. KAH223 = 1031*, 636
7-9. KAH224 = 1331*, 636
10-12. KAH232 = 513, 99, 662*, 636
13-15. KAH219 = 2108*, 450
16-18. KAH220 = 1031*, 450
19-21. KAH221 = 1331*, 450
22-24. KAH231 = 513, 99, 662*, 450
|
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
- KAH234/V0200: (1) KAH234/(N/S)/1041 + V0200/(N/S dp)/4442 ✓
- KAH235/V0200: (2) KAH235/(N/S)/2787 + "
> Digests (Fermentas FD)
| 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. KAH234 (N/S) |
0.205 |
1.66 |
10.2
|
| 2. KAH235 (N/S) |
0.242 |
2.41 |
12.1
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) mm/dd/yy
|
| 1. KAH234(N/S)/1041, 9 ng + V0200(N/S dp)/4442, 20 ng |
KAH234/V0200 >10:1 (Pick 2)
|
| 2. KAH235(N/S)/2787, 25 ng + " |
KAH235/V0200 >10:1 (Pick 4)
|
| 3. V0200(N/S dp)/ 20 ng |
|
| |
1 |
2 |
3 |
|
| Insert DNA |
1.0 |
2.0 |
--- |
|
| Vector DNA |
0.8 |
0.8 |
0.8 |
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
|
| dH2O |
2.2 |
1.2 |
3.2 |
|
| |
10 μL |
10 μL |
10 μL |
|
|
Project name
Main project page
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