Team:Cambridge/LabBook/Week5

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 electrphoersis

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:

Supercoiled DNA ladder loading mixture

• 1μl Supercoiled DNA ladder (NEB)
• 3μl 6X orange loading Dye
• 16μl deionised H₂O

Gel was loaded following scheme below:

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).