Team:Cambridge/LabBook/Week9
From 2010.igem.org
Week 9: Monday 6th September - Sunday 12th September
Contents |
Monday
86. Expt: Extract CDABEG from pJS555
- Using 2 primers
- prefix start of O
- suffix end of G
- Hope that it will glow in its own right without luxR+I
- Need to put under a new promoter
87. Expt: Gibson transformation (cont. from p.70)
3 colonies on one plate were not pink. These were streaked out on new Chl plated and put into liquid cultures along with 2 of the red colonies.
Next check for the right sized fragment with colony PCR and try to induce with arabinose.
88. Expt: Plate Reader G28
Well | Arabinose conc. | |
A1 | 0 | x1,9 |
A2 | 1µM | |
A3 | 5µM | |
A4 | 10µM | |
A5 | 100µM | |
A6 | Blank | |
A7 | pSB1C3 | |
A8 | Blank | x8,16 |
Reads every 10 mins
89. Expt: Extracting YFP, CFP, Luc tetR, G28 via miniprep (Paul)
Nanodrop readings (ng/µl) | ||
YFP | 33.3, 55.4 | |
CFP | 17.9, 20.4 | |
Luc tetR | 33.7 | |
G28 | 125.3, 96.9 | |
G28? | 17.9 |
90. Expt: Colony PCR to extract the thioesterase gene from E. coli K12 (Paul)
We used TOP10 cells, a substrain of DH10B, which is a substrain of K12.
We did a colony PCR to isolate the gene:
3 replicates with: | 1 negative control: | |
2x Phusion Mastermix | 10µl | 10µl |
Template DNA | 1µl | 0 |
Primer 1 | 1µl | 1µl |
Primer 2 | 1µl | 1µl |
Nuclease-free H20 | 7µl | 8µl |
The negative control was to check for primer/dimer and contamination.
PCR protocol:
- Initial denaturation: 5min @ 98°C
- Touchdown: 16 cycles
- Denaturation: 10s @ 98°C
- Annealing: 20s @ 65°C to 57°C
- Elongation: 15s @ 72°C
- 30 cycles
- Denaturation: 10s @ 98°C
- Annealing: 20s @ 50°C
- Elongation: 15s @ 72°C
- End and hold at 10°C
- We then ran result on a gel with SyberSafe at 6x lodaing Dye and Hyperladder IV
Lane: 1 | 2 | 3 | 4 | 5 |
Ladder | Tube 1 | Tube 2 | Tube 3 | Negative Control |
Failed: Something obtained in control...
Nothing in other lanes
We repeated without touchdown PCR:
- Denaturation 5min @ 98°C
- 35 Cycles
- Denaturation: 12s @ 98°C
- Annealing: 20s @ 58.5°C
- Elongation: 15s @ 72°C
- Hold at 10°C
Failed again: Product in negative control observed again...
Tuesday
91. Expt: Further testing
µl°C