Team:Cambridge/LabBook/Week5

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Revision as of 16:34, 24 August 2010

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:

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).

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. Experiment: Preparation of Photobacterium Broth (Theo and Anja)

35.2g broth promoter was dissolved in 500ml water by warming/boiling. Broth was filter sterilised.

35. Experiment: Ampoule of Photobacterium (extracted by Theo and Peter)

Ampoule was broken, 500μl LB broth added and 100μl added to each of 5 falcons containing broth.

Theo added 5g agar to 250ml broth and placed for autoclaving.

After autoclaving the medium was turbid. It was poured into 9 empty, sterile dishes and left to set on the bench.

36. Experiment: Renewed colonies of TOP10/pHK555 & pHK724 (Will)

• Inoculated 2x5ml LB with 1 colony for each
• Streaked out cells on Amp + Chl plate (*)
• Incubated both at 30°C overnight.

Results:

• 1 LB glow very faintly, other not at all
• Poor growth from streaking, but strong glow

36. Experiment: Transformation of TOP10 competent cells with luxR from registry (Bill)

• Followed transformation protocol from 02/08/10
• Transformed with BBa_I0462 from distribution from distribution Kit Plate 1 Well 80
• Plated out on 2 ampicillin plates the transformation, (amp is resistance marker of plasmid)
• Also cultured in 2 LB + Amp plates

Results (Hannah):