From Monday the 4th of July to Friday the 8th of July 2011

Monday

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Additional minipreps using the QuickPure protocol of the same 6 parts ( 5 replica each )
Digestion as previously

Ligation to obtain : RBS ( strong and weak ) + GFP, RBS ( strong and weak ) + YFP. This corresponds respectively to 2M+2L, 5L+2L, 2M+24E, 5L+24E assemblies.

Start of a 5mL culture of NM522 cells.

Tuesday

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Start of a 50mL culture of NM522 from the overnight preculture ( 50mL sterile LB, 250µL of preculture ).
Transformation of 5µL ( S series ) or 10 µL ( D series ) from the previous ligations into NM522 (V=15mL) using a CaCl2 chemical transformation. Positive control was done with 1µL Puc18, negative with 5µL water.

Wednesday

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Selection of individual transformed colonies and start of solid and liquid culture.

We shoot our "Cobalt Buster's Clip" which introduces, in a funny way, our team : students, instructors and advisors. We dedicate the all afternoon to this shooting.

Thursday

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Miniprep using the QuickPure protocol of the previous liquid cultures.

Digestion of the plasmids by X+P.

Electrophoresis of the digested and non digested plasmids :
2M+2L S1 : no DNA
5L+2L S1 : 800 bp insert
2M+2L S2 : no DNA
2M+2L D1 : 800 bp insert
5L+24E S2 : no insert
5L+2L D2 : 800 bp insert
2M+24E S2 : no insert
2M+24E D2 : 800 bp insert

However, due to the small size of the RBS, a flaw in our protocol ( choice of antibiotic resistances ) doesn't allow us to detect a difference between 24E or 2L plasmids compared to their ligated equivalents. Thus we can't conclude that the 800 bp inserts correspond to the ligated parts.

Friday

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