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Exeter/24 July 2013
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| + | The gel shows that some of our digestions appear to have worked (our yellow pigment in lane 6 has a clear band at about ~700bp, where would expect) but others appear not to have worked, or have bands that don't match our expected genes. | ||
We also ran a seperate gel of: | We also ran a seperate gel of: | ||
Revision as of 15:15, 28 July 2013
Contents |
Results of liquid cultures
Some of our liquid cultures didn't work; the cells died in the antibiotic. Mildly frustrating. The failed cultures were:
- OmpR (BBa_K098011)
- Lambda inverter system (BBa_Q04510)
Minipreps using the Qiagen kit
We miniprepped:
- CcaR
- Promoter, RBS x 3 replicates
- CcaS
- B0034
- Magenta
- OmpR promoter
- OmpR
- YF1
- Lambda inverta
- Fix J
- Yellow
- Fix J promoter
Glycerol stocks
We made glycerol stocks of what is above, then stored them at -80oC
For future reference, to get glycerol stocks back as plates, take a loop and streak on gel. Has to be taken from frozen glycerol stocks, which are immediately returned to the freezer.
We then made a gel for digests using ethidium bromide.
NanoDrop from miniprep
| Part | Concentration (ng/ul) |
|---|---|
| CcaR | 324.6 |
| CcaS | 264.4 |
| Yellow | 64.3 |
| Magenta | 102.7 |
| RBS | 48.9 |
| Promoter, RBS, #1 | 21.5 |
| Promoter, RBS, #2 | 22.6 |
| Promoter, RBS, #3 | 22.0 |
| Fix L | 31.9 |
| Fix J | 35.4 |
| YF1 | 27.1 |
| OmpF | 28.2 |
No SureClean was needed.
Digestion
We then cut the genes out of the plasmid using Xbal and Pstl. Prepare to run on a gel.
Each eppendorf contains:
- 12ul purite water
- 2ul 10X FastDigest Buffer w.Green
- 0.5ul Xbal
- 0.5ul Pstl
- 5ul DNA
In gel
| Lane | Content |
|---|---|
| 1 | Ladder (1kb Gene Ruler) |
| 2 | Promoter, RBS, #1 |
| 3 | B0034 |
| 4 | CcaS |
| 5 | Fix J promoter (a.k.a Fix L) |
| 6 | Yellow |
| 7 | Promoter, RBS, #2 |
| 8 | Fix J |
| 9 | OmpR promoter (a.k.a OmpF) |
| 10 | Promoter, RBS, #3 |
| 11 | Magenta |
| 12 | CcaR |
| 13 | YF1 |
| 14 | Ladder (1kb Gene Ruler) |
The gel shows that some of our digestions appear to have worked (our yellow pigment in lane 6 has a clear band at about ~700bp, where would expect) but others appear not to have worked, or have bands that don't match our expected genes.
We also ran a seperate gel of:
- cph8 #3
- cph8 #4
- cph8 #5
- cph8 #6
- cph8 #7
Cut with Xbal and Pstl.
The lanes were run in the order above, flanked by a 1kb Gene ruler.
