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 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
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.