Team:Cambridge/LabBook/Week5

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Contents

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!