Imperial/Wet Lab/Lab Notebook/2007-08-28
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= 28 August 2007 = | = 28 August 2007 = | ||
| + | ==In vitro Testing of pTet-LuxR-pLux-GFP at room temperature== | ||
| + | Testing of the construct was done at a low concentration of AHL 0.1nM to emulate a real biofilm. This experiment tests whether the system is sensitive enough to respond to the low amounts of AHL. | ||
| + | <br><br> | ||
| + | Protocol can be found here under [[IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_2.2|Phase 1-In vitro testing]] on the experimental design page. | ||
| + | <br><br> | ||
| + | Results: Very low levels of fluorescence was observed. | ||
| + | |||
| + | ==Construction of pT7-GFP== | ||
| + | |||
| + | #Grew cells with plasmids containing pT7-GFP and cells with vector plasmids overnight | ||
| + | #Spun down cells and minipreped | ||
| + | |||
| + | Protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#S30.2FS12_Cell_Extract#Miniprep| Miniprep] in the general protocols page | ||
| + | |||
| + | *[Vector DNA] = 137.9 ng/μl | ||
| + | *[Insert DNA] = 987.1 ng/μl | ||
| + | |||
| + | #Set up digestion to cut out insert and vector | ||
| + | #*DNA (5 μl insert/ 15 μl vector) | ||
| + | #*Nde 0.75 μl | ||
| + | #*BamH1 0.75 μl | ||
| + | #*10X BSA 2 μl | ||
| + | #*10X BamH1 Buffer 2 μl | ||
| + | #*ddH20 to make up total volume of 20 μl | ||
| + | #Digestion left at 37°C for 1 hour | ||
| + | |||
| + | ==Vesicles== | ||
| + | '''Preparations''' | ||
| + | |||
| + | Two suspensions were prepared for the next day: | ||
| + | # 125μl of POPC in 50ml of dodecane | ||
| + | # 125μl of DOPC and 125μl of Span-80 in 50ml of Mineral Oil | ||
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Latest revision as of 12:55, 26 October 2007
28 August 2007
In vitro Testing of pTet-LuxR-pLux-GFP at room temperature
Testing of the construct was done at a low concentration of AHL 0.1nM to emulate a real biofilm. This experiment tests whether the system is sensitive enough to respond to the low amounts of AHL.
Protocol can be found here under Phase 1-In vitro testing on the experimental design page.
Results: Very low levels of fluorescence was observed.
Construction of pT7-GFP
- Grew cells with plasmids containing pT7-GFP and cells with vector plasmids overnight
- Spun down cells and minipreped
Protocols can be found at Miniprep in the general protocols page
- [Vector DNA] = 137.9 ng/μl
- [Insert DNA] = 987.1 ng/μl
- Set up digestion to cut out insert and vector
- DNA (5 μl insert/ 15 μl vector)
- Nde 0.75 μl
- BamH1 0.75 μl
- 10X BSA 2 μl
- 10X BamH1 Buffer 2 μl
- ddH20 to make up total volume of 20 μl
- Digestion left at 37°C for 1 hour
Vesicles
Preparations
Two suspensions were prepared for the next day:
- 125μl of POPC in 50ml of dodecane
- 125μl of DOPC and 125μl of Span-80 in 50ml of Mineral Oil
