Du Lundi 4 juillet au Vendredi 8 juillet 2011

Lundi

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Minipreps supplémentaires des mêmes six parts que le vendredi précédents en utilisant le protocole QuickPure (5 répliques de chaque).
Digestion comme précédemment

Ligation pour obtenir : RBS (fort et faible) + GFP, RBS (fort et faible) + YFPCela correspond respectivement aux assemblages 2M+2L, 5L+2L, 2M+24E et 5L+24E.

Lancement d'une culture 5mL de NM522.

Mardi

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Lancement de cultures 50mL de NM522 à partir de la culture de la nuit ( 50mL LB stérile, 250µL preculture )
Transformation de 5µL ( série S ) ou 10 µL ( série D ) des ligations précédentes dans NM522 ( V=15mL ) via le protocole de transformation chimique au CaCl2. Le contrôle positif a été effectué avec 1µL pUC18, le négatif avec 5µL d'eau.

Obtention des résultats du séquençage. Mutagénèse des plasmides comportant le moins d'erreurs par la méthode quick change afin d'enlever des sites de restriction indésirables (EcoRI et PstI).

Mercredi

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

Digestion of PCR/mutagenesis product with DpnI to eliminate parental plasmid. Transformation of NM522 strains with plasmid. Culture and selection on ampicilline plates.

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.

Jeudi

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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 : we have observed either no DNA, no insert, or a plasmid with a correct size 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.

Selection of clones on plate and liquid culture for miniprep. Digestion of purified PCR product in Tango buffer to have a better efficiency. Transformation of NM522 with purified plasmids.

Vendredi

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Miniprep of clones with mutated plasmids. Restriction to check the removal of unwanted restriction sites (EcoRI or PstI). Send of plasmids with the expected profile to sequencing.