IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/20

UNAM-Genomics-Mexico team

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Working on LovTAP promoters: Restrictions of Medium and Weak Promoters Once the cells were correctly transformed with the six plasmids harboring the three weak and three medium promoters, I have started the plasmid extraction procedure. Plasmid J61002 with promoter J23102: The plot shows the enzyme restriction cutting sites inside Plasmid J61002 and the location of the following elements: promoter J23102, RFP protein, AmpR-ampicillin resistance- and the origin of replication. Plasmid extraction Protocol I am using the High Pure Plasmid Isolation kit from Roche Restriction enzyme assay:Preparation for ligation After finishing the isolation of the six plasmids, I have started the restriction enzyme assay in order to remove the RBS site, the RFP gene and the doble terminator from each one to replace them with LovTAP gene. Restriction enzymes Mixture: Restriction enzymes: SpeI and PstI. Reactive Quantity Plasmid DNA 15μL Buffer 2 4μL BSA 1μL Enzymes 2μL for each one. HPLC Up to a final volume of 40 μL of the mixture The restriction reactions were incubated at 37°C overnight. Note: Inactivate the enzymes at 80°C during 10 min.