IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/06
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Working on cI inverter construction and fusion to pSB1C3 backbone. LovTAP repressor activity:reporter system
Once the parts K098991 (cI regulated promoter+RBS+GFP) and P0451(RBS+cI repressor) were correctly digested, I am going to ligate them in plasmid pSB1C3.
Ligation Procedure: P0451 + K098991 to plasmid pSB1C3
1. Prepare the ligation mixture taking into account the quantity of the DNA insertS -K098991 and P0451- and the receiver DNA -plasmid pSB1C3-. This can be check in the following gel. Quantity loaded 3μL.
3. Transform the cells. Click here for the protocol.
4. Culture the cells in the proper selective medium, in our case LB + Kanamycin.
5. Incubate the petri dishes at 37°C overnight.
6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight.
7. Analyze the colonies with Colony PCR to confirm that they contain the correct ligation
The primers that I am using are:
Forward (5'->3'): Preffix primer.
Reverse:(5'->3'): Suffix primer.
These primers would amplify the whole cI inverter, if the ligation was correctly done.
Working on J23101 promoter: Ligations to P0451, LuxY and Lumazine
Once the plasmid harboring the J23101 promoter was correctly digested with the enzymes SpeI and PstI, I have started the dephosphatation reaction in order to prepare it for ligation with P0451, LuxY and Lumazine constructions.
1.Incubate the samples at 37°C during 20 min.
2.Incubate the samples at 65°C during 10 min.