User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/07/07

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Transformation


  • After letting the ligation ON, I inactivated the enzyme for 10 min at 70°C.


  • I also did the respective transformations. (In parenthesis the media in which strain were plated):


1. Luciferase (green) with J23106 (ampicilin).

2. Luciferase (green) with plasmid 18 (tetracycline).

3. Luciferase (red) with J23106 (ampicilin).

4. Luciferase (red) with plasmid 18 (tetracycline).

5. Control (J23106) (ampicilin).

6. Control (plasmid 18) (tetracycline).

7. Control of transformation (BBa_I51030 aka plasmid 19)(ampicilin).

8. Control of competent cells.

  • After plating the strains, I left them incubating at 37°C.


  • NOTE: Tomorrow, Enrique Paz and me will be checking the luciferase assay with Chris Wood. Up to now, I have found a medium (LAR) expected to reveal the presence and color of luciferase when present in bacteria.


"Bacterial cells expressing the luciferase protein will be added to a tube containing the luciferase assay reagent (LAR). LAR is a solution of 1mM luciferin, which is the luciferase substrate, in 100mM sodium citrate, pH 5.5. The luciferase reaction also requires the energy of adenosine triphosphate (ATP), which is provided by the living bacterial cells in this case, and oxygen, which is incorporated from the atmosphere by vigorously shaking the tube containing all of the other components. The tubes will be viewed in a dark room to easily distinguish the colors." (reference)


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