Imperial/Wet Lab/Lab Notebook/2007-08-16


16 August 2007

Preparation of Cell Extract

  1. O.D. of overnight culture was 3.417 at 9am (still less than mid-log phase of 0.D.600 = 4.5)
  2. Decided to harvest cells anyway
  3. 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

  1. Cells containing constructs pTet-GFP and pT7-GFP and pTet-LuxR-pLux-GFP were incubated overnight at 37°C
  2. Tested for viability of construct pTet-GFP in vivo. Conclusion: Works
  3. Tested for viability of construct pTet-LuxR-pLux-GFP in vivo. Conclusion: Works
  4. 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.
  1. 2ml of suspension was used to prepare an interface (according to the protocol)
  2. 200x diluted GFP solution was used to prepare the emulsion
  3. 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.
  1. Two 100ml beakers were prepared with 125μl of DOPC in 50ml of mineral oil
  2. 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
  3. A total of four beakers, two well-desiccated and two poorly-desiccated, were produced
  4. These were left overnight in a 27°C incubator