07/05/11
- ✓ Cultures/minipreps: KAH201, 202, 203 (3 each)
- ✓ Sequencing order: KAH201, 202, 203
- ✓ Retransform: mCherry E3000
- ✓ (Re-) Assembly: miR sensor 183-177/MV8 (as a control for Daniel's set)
- ✓ Order oligos: new mutagenesis primers for p65
Minipreps
> Check with E/P digests
| Reagent |
Volume
|
Expected:
1-3. KAH201 = 226
4-6. KAH202 = 325
7-9. KAH203 = 155
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
3.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
8.5
|
| |
15 μL --> 37°C/ ~15 min.
|
Sequencing order
--> Send all samples for Genewiz sequencing
--> Use P0001 (fwd) primer for all samples
- KAH201-1: good (keep this clone)
- KAH201-2: good
- KAH201-3: good
- KAH202-1: good (keep this clone)
- KAH202-2: good
- KAH202-3: good
- KAH203-1: no priming
- KAH203-2: good (keep this clone)
- KAH203-3: good
Note: B433 spacer is actually 42 bp (2 repeat segments, not three)
Assembly
--> Not sure about previous miR sensor construct vectors; start re-building and check with ApaLI instead of E/ApaLI
--> Note: Use KAH177/MV8 E/X digest from 1/25/11, not 5/25/11
- KAH183-177/MV8: KAH183/(E/S)/2383 ✓ + KAH177/MV8/(E/X)/3384 ✓
> Ligations
| Ligation |
Plate results (lig : neg crtl) 07/06/11
|
| 1. KAH183(E/S)/2383, 15 ng + KAH177/MV8(E/X)/7978, 25 ng |
KAH183-177/MV8 >10:1 (Pick 4)
|
| 2. vector(c/d)/ 25 ng |
|
| |
1 |
2 |
|
| Insert DNA |
1.0 |
--- |
|
| Vector DNA |
1.0 |
1.0 |
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
|
| T4 ligase (NEB) |
1.0 |
1.0 |
|
| dH2O |
2.0 |
3.0 |
|
| |
10 μL |
10 μL |
|
--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL Amp
Order oligos
--> Previous attempts to mutate the second PstI site failed. Order new primers and try both strands...
- p65 mut 2 (+ strand): TGCTGCAACTGCAaTTTGATGATGAAG
- p65 mut 3 (- strand): CTTCATCATCAAAtTGCAGTTGCAGCA
|
Project name
Main project page
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