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Team:Lyon-INSA-ENS/Realisation/Week4Fr
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Revision as of 19:01, 10 August 2011
Semaine 4
Du Lundi 4 juillet au Vendredi 8 juillet 2011
Lundi
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.
Start of a 5mL culture of NM522 cells.
Tuesday
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.
Sequencing results obtained. Mutagenesis of the plasmids with the least errors by quick change method to remove unwanted restriction sites (EcoRI or PstI).
Wednesday
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.
Thursday
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.
Friday
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.
