"
Team:Cambridge/LabBook/Week5
From 2010.igem.org
Monday
31. Experiment: Diagnostic gel to test for luciferase insertion (Theo & Anja)
Took overnight culture of TOP10 transformed with plasmid (potentially) containing promoter + rbs and luciferase
Plasmid DNA purification
following "QIAprep Spin Miniprep Kit using a Microcentrifuge" protocol
Gel electrophoresis
E-gel EX Agarose 1% mounted on transilluminator Nanodrop readings:
- Plasmid with promoter + rbs + luciferase: 18.5ng/μl => 5μl gives 100ng
- Plasmid with promoter + rbs only: 64.9ng/μl => 2μl gives 100ng
Gel loading mixture:
| promoter + rbs + luciferase | promoter + rbs only | |
|---|---|---|
| 6X orange loading Dye | 3μl | 3μl |
| plasmid DNA | 5μl | 2μl |
| deionised H2O | 12μl | 12μl |
Supercoiled DNA ladder loading mixture
- 1μl Supercoiled DNA ladder (NEB)
- 3μl 6X orange loading Dye
- 16μl deionised H2O
Gel was loaded following scheme below:
| Supercoiled DNA ladder | promoter + rbs only | promoter + rbs + luciferase |
| 20μl | 20μl | 20μl |
Empty wells were filled with 200μl deionised H2O. Gell was run and a clear band around 2kb was visible for promoter + rbs, a very faint band was observed around 4kb for promoter + rbs + luciferase.
Gel electrophoresis
was repeated with increased promoter + rbs + luciferase plasmid concentration
Gel loading mixture for promoter + rbs + luciferase
(others as above)
- 3μl 6X orange LD
- 15μl plasmid DNA
- 2μl deionised H2O
Gel was run and the same bands were observed as above, with the ~4kb promoter + rbs + luciferase more clearly than before
32. Experiment: In vitro assay for luciferase activity (Theo & Anja)
50μl of TOP10 cells transformed with plasmid containing promoter + rbs + luc were transferred to an Eppendorf tube and subjected to three rounds of freeze (-80°C) and thaw (37°C) treatement. 50μl of 2mg/ml luciferin were added tot he lysed bacteria and luminescence was tested using a CCD camera. The experiment was performed twice in a row and both times luminescence was observed with the CCD camera (though not by eye in the dark room).
33. Experiment: In vitro assay for luciferase activity using CHBT as substrate (Theo and Anja)
2ml of TOP10 with promoter, rbs and luc were spun at 13000rpm for 5 mins. Supernatant was discarded and cell pellet was resuspended in 500μl fresh LB to which 1 drop of chloroform was added. The cells were subjected to three rounds of freeze (-80°C) and thaw (37°C) treatment.
EXPERIMENT WAS CANCELLED!
34. Preparation of Photobacterium Broth (Theo and Anja)
35.2g broth promoter was dissolved in 500ml water by warming/boiling. Broth was filter sterilised.
35. Ampoule of Photobacterium (extracted by Theo and Peter)
