Preparation of Cell Extract
- O.D. of overnight culture was 3.417 at 9am (still less than mid-log phase of 0.D.600 = 4.5)
- Decided to harvest cells anyway
- Prepared s30 cell extract
- Added protease inhibitors to the cell extract
- Might add RNAse inhibitors when preparing the next cell extract if this one does not work
Protocols can be found at S30/S12 Cell Extract in the general protocols page
Testing of DNA Constructs
- Cells containing constructs pTet-GFP and pT7-GFP and pTet-LuxR-pLux-GFP were incubated overnight at 37°C
- Tested for viability of construct pTet-GFP in vivo. Conclusion: Works
- Tested for viability of construct pTet-LuxR-pLux-GFP in vivo. Conclusion: Works
- Tested for viability of construct pT7-GFP in vivo. Conclusion: Uncertain.
- We realised that the E.coli cells were not yet induced to produce T7 promoter.
- 200µl IPTG was added to 600µl of the cells, and placed in a 37°C incubator overnight
- Testing for this construct will be done tommorrow
- pcI-GFP was not tested in vivo because construct was not ready.
Pilot Preparation of Vesicles
- Formation of Vesicles: The suspension prepared the day before was used to form vesicles.
- 2ml of suspension was used to prepare an interface (according to the protocol)
- 200x diluted GFP solution was used to prepare the emulsion
- Two samples were prepared:
- One following the protocol, with 2ml suspension interface and 100μl of emulsion added
- One using 2ml of emulsion to form the interface, without further addition of material
- Results: Very few vesicles were observed under a light microscope with phase contrast. The fluorescence microscope did not produce any results.
- Preparations: The desiccation and suspension stages of the vesicle preparation protocol were carried out once again.
- Two 100ml beakers were prepared with 125μl of DOPC in 50ml of mineral oil
- The suspensions were sonicated for 30mins each
- At this point, it was found that the desiccator had no vacuum, and a further two suspensions were prepared as described above
- A total of four beakers, two well-desiccated and two poorly-desiccated, were produced
- These were left overnight in a 27°C incubator