IGEM:IMPERIAL/2007/Notebook/2007-9-3: Difference between revisions
Line 60: | Line 60: | ||
|- | |- | ||
|colspan="2"|Microscope pictures of vesicles formed from 1ml DOPC-dodecane-GFP emulsion. Above left: Several vesicles under white light. Above right: The same objects (not visible), under fluorescence. The fluorescence was present and visible, but VERY faint. The camera was unable to register the fluorescence - even with 4 seconds of exposure. Gamma and saturation corrections did not reveal any colour. Below left: Another pair of vesicles. Below right: Again, the fluorescence inside these vesicles was too faint to be captured by the camera, but it was visible. | |colspan="2"|Microscope pictures of vesicles formed from 1ml DOPC-dodecane-GFP emulsion. Above left: Several vesicles under white light. Above right: The same objects (not visible), under fluorescence. The fluorescence was present and visible, but VERY faint. The camera was unable to register the fluorescence - even with 4 seconds of exposure. Gamma and saturation corrections did not reveal any colour. Below left: Another pair of vesicles. Below right: Again, the fluorescence inside these vesicles was too faint to be captured by the camera, but it was visible. | ||
|- | |||
| width="200px"| [[image:IC07_image138.jpg|200px]] | | width="200px"| [[image:IC07_image138.jpg|200px]] | ||
| width="200px"| [[image:IC07_image139.jpg|200px]] | | width="200px"| [[image:IC07_image139.jpg|200px]] | ||
|} | |} | ||
Revision as of 14:48, 3 September 2007
<calendar> name=iGEM:IMPERIAL/2007/Notebook date=2007/09/24 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>
Construction of pT7-GFP
- Minipreped the 5 overnight cultures
Protocols can be found at Miniprep in the general protocols page
- Digested 5μl of DNA with Nde1 and BamH1
- Checked the digestion on 1% agarose gel
Protocols can be found at Electrophoresis in the general protocols page
- Transformed recombinant plasmid DNA into BL21
- Plated transformed cells onto LB + Kan plates
- Left plates at 37°C overnight
Protocols can be found at Transformation in the general protocols page
Maxiprep of Biobricks
- Maxipreped 2 Biobricks
- 2. BBa_I13422 [ptet-GFP]
- 18. BBa_T9002 [plux-GFP]
Protocols can be found at Maxiprep in the general protocols page
Vesicles
Formation of Vesicles
For both the POPC/dodecane 10ml and DOPC/dodecane 10ml suspensions prepared today - without overnight incubation - the following steps were taken:
- 2ml of suspension was taken to prepare an interface according to protocol.
- 25μl of 100x diluted GFP solution was used to prepare the emulsion; stirred gently with magnetic stirrer.
NOTE: There was a mistake in labelling the containers for the two suspensions - both were mistakenly labelled as DOPC. When the mistake was spotted, it was impossible to distinguish between the POPC and DOPC containers. The labels used then were a best guess, and have been carried through the remainder of these results. So, please bear in mind that the results given could either be POPC or DOPC.
4 samples prepared:
- Sample 1: 1ml of POPC/dodecane/GFP emulsion added over interface prepared with 2ml of POPC/dodecane suspension.
- Sample 2: 1ml of DOPC/dodecane/GFP emulsion added over interface prepared with 2ml of DOPC/dodecane suspension.
- Sample 3: 2ml of POPC/dodecane/GFP emulsion, with no previously prepared interface.
- Sample 4: 2ml of DOPC/dodecane/GFP emulsion, with no previously prepared interface.
Samples 1 and 2 were centrifuged at 120x g. Samples 3 and 4 have been left overnight for sedimentation to occur.
Results
Samples 1 and 2 were collected for observation under the microscope. The following results were obtained:
Preparations
No preparations were carried out.