IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/20
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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 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: SpeI and PstI.
The restriction reactions were incubated at 37°C overnight.
Note: Inactivate the enzymes at 80°C during 10 min.