07/26/12
- ✓ Sequencing order: DNASU
- ✓ Minipreps digests: Francesca's plasmids and V0120
Sequencing order
Measure concentrations:
Sample |
260 |
260/280 |
ng/μL
|
FCt1 |
0.095 |
1.928 |
94.595
|
FCt2 |
0.174 |
1.903 |
173.76
|
FCt4 |
0.068 |
1.937 |
68.042
|
> DNASU @Biodesign, Results:
- FCt1 - VF2: good, P2547 confirmed
- FCt2 - VF2: bad seq, wrong primer (Francesca: part is in pGA18, not pSB1A2)
- FCt4 - VF2: good, but operator is O1, not O2
> Need to re-do FCt2 (use M13 primer) to find out if it's O2 or actually O1
Minipreps
> Note: Streaks for FCt1 and FCt2 are red; FCt4 is very pale pink
> Check Francesca's plasmids with S/P digests; should remove RBS-RFP (703 bp); see http://partsregistry.org/Part:BBa_J61002
> Check ccdB-V0120 with E/P digests
Reagent |
Volume
|
Expected: 1. FCt1 (P2547) = ~2250, 703 2. FCt2 (O2-RFP/pGA18) = ~3000, 703 3. FCt4 (P2547-O2-RFP) = ~2250, 703 4-6. V0120 = ~3200, ~1700
|
15 μL/lane; 1% agarose; Ladder
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
9.5
|
|
15 μL --> 37°C/ ~15 min.
|
|