"
Exeter/23 July 2013
From 2013.igem.org
Unfortunately, some of our liquid cultures from yesterday appear to have not worked.
Also, the liquid cultures for OmpR and the OmpR promoter were mislabelled, so we're uncertain which is which. However, when we run "OmpR A" on a gel, it should be very obvious which is which, as OmpR is 917bp and the OmpR promoter is 108bp.
| Part | Did it work? | Source |
|---|---|---|
| NP1 (CcaS + Terminator) | ✗ | Transformation plate |
| NP2 (promoter, RBS, FixJ + Terminator | ✗ | Transformation plate |
| NP3 (yellow pigment + Terminator) | ✗ | Transformation plate |
| NP4 (magenta pigment + Terminator) | ✗ | Transformation plate |
| NP7 (YF1 + Terminator) | ✓ | Transformation plate |
| NP8 (FixJ + Terminator) | ✓ | Transformation plate |
| Ligation control | ✗ | Transformation plate |
| Lambda inverter system | ✗ | Streak plate in fridge |
| T7 promoter | ✗ | Streak plate in fridge |
| Promoter + RBS (BBa_K608002) | ✓ | Streak plate in fridge |
| RBS (BBa_B0034) | ✓ | Streak plate in fridge |
| OmpR "A" | ✓ | Streak plate in fridge |
| OmpR "B" | ✗ | Streak plate in fridge |
| RFP control | ✓ | Transformation plate |
| RBS + cph8 (BBa_K592018) | ✓ | Transformation plate |
The working liquid cultures (NP7, NP8, Promoter + RBS, RBS, OmpR "A" and RBS + cph8) were MiniPrepped, then NanoDropped, then had their concentrations increased using the SureClean protocol from 17/7/13. Each culture was replicated twice.
| Part | NanoDrop data (ng/ul) | NanoDrop data after SureClean protocol (ng/ul) |
|---|---|---|
| Promoter and RBS #1 | 3.5 | 107.3 |
| Promoter and RBS #2 | 2.1 | 107.3 |
| NP8 #1 | 4.5 | 113.3 |
| NP8 #2 | 5.9 | 40.1 |
| RBS (BBa_B0034) #1 | 14.0 | 64.8 |
| RBS (BBa_B0034) #2 | 2.2 | Would not pellet, SureClean failed |
| NP7 #1 | 35.5 | SureClean not required |
| NP7 #2 | 29.3 | SureClean not required |
| Ompr "A" #1 | 2.1 | 110.5 |
| OmpR "A" #2 | 2.0 | 19.5 |
| RBS and cph8 #1 | 3.5 | 197.1 |
| RBS and cph8 #2 | 12.5 | Would not pellet, SureClean failed |
Digest
To ensure that our genes of interest are present, we took a small amount of each DNA solution, digested them with EcoRI and PstI to remove them from the plasmids, then ran them on a gel.
Each eppendorf contains:
- 12ul distilled water
- 2ul 10X FastDigest Buffer w/ Green Dye
- 0.5ul EcoRI
- 0.5ul PstI
- 5ul DNA
The samples digested were:
- RBS and cph8 #1
- RBS (BBa_B0034) #1
- OmpR "A" #1
- NP8 #1
- NP7 #1
- Promoter and RBS (BBa_K608002) #1
We also ran a digest of our original RBS + cph8 BioBrick (BBa_K322124), cutting one sample with EcoRI and PstI and another with EcoRI and SpeI. This is because we think there may be a PstI cut-site within the BioBrick, as when we sent it of for sequencing, the data that came back seemed much, much shorter than we were expecting.
All of our digests were run against a 1kb GeneRuler DNA ladder.
| Lane | Contents |
|---|---|
| 1 | Ladder |
| 2 | RBS and cph8, cut with EcoRI and SpeI |
| 3 | RBS and cph8, cut with EcoRI and SpeI |
| 4 | Example |
| 5 | Example |
| 6 | Example |
| 7 | Example |
| 8 | Example |
| 9 | Example |
| 10 | Example |
