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Team:Lyon-INSA-ENS/Realisation/Week4
From 2011.igem.org
Week 4
From Monday the 4th of July to Friday the 8th of July 2011
Monday
Strain construction
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
Strain construction
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.
PCR and mutagenesis of rcn, csgBA, csgEFG
Sequencing results obtained. Mutagenesis of the plasmids with the least errors by quick change method to remove unwanted restriction sites (EcoRI or PstI).
Wednesday
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.
Strain construction
Selection of individual transformed colonies and start of solid and liquid culture.
PCR and mutagenesis of rcn, csgBA, csgEFG
Digestion of PCR/mutagenesis product with DpnI to eliminate parental plasmid. Transformation of NM522 strains with plasmid. Culture and selection on ampicilline plates.
Thursday
Strain construction
Miniprep using the QuickPure protocol of the previous liquid cultures.
Ozyme 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.
PCR and mutagenesis of rcn, csgBA, csgEFG
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
PCR and mutagenesis of rcn, csgBA, csgEFG
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.
