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Materials
You will need:
- Pre-made nutrient agar plates
- Soft Agar
- Cultures of desired bacteria to be plated
- Antibiotic resistance and other required substances (see extra notes)
- Vortex
- Polypropylene tubes
- Water bath
Extra Notes
- For our construct specifically, we also needed to use arabinose, and possibly heme in our top agar solution. (The initiation of our spatial oscillator is controlled by a pBad promoter. To be induced, the promoter needs 0.02% arabinose and possibly heme, in addition to darkness.)
- Measure/test the OD of the culture beforehand to know the required amount of bacteria to create a lawn of E. Coli.
Procedure
1. Pre-warm the premade nutrient agar plates.
2. Melt soft agar by microwaving.
3. Aliquot/pipette 2mL into each polypropylene tube.
4. Cool the melted soft agar to 45C. This can be done by placing the polypropylene tubes into a water bath.
5. Creating the solution
- Pipette the required substances
- Pipette the required amount of bacteria culture
- Vortex!
6. Pour soft agar solution onto premade agar plates. For an even lawn, tilt, tilt, tilt! It takes practice.
Purpose
To create an even lawn of E. Coli on a nutrient agar plate.
References
- Fankhauser, David B. "Agar Overlay Technique." Agar Overlay Technique. 4 May 1994. Web. 4 Aug. 2010. .
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We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!
We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)
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