IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/25
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Repeated experiment:Working on J23101 promoter: Ligation to P0451/Lumazine/LuxYDue to the first attempt to ligate P0451, Lumazine and LuxY to promoter J23101 failed, I am going to repeat the experiment. Ligation Procedure:Promoter J23101 with P0451 (RBS+cI repressor)/Lumazine and LuxY1. Prepare the ligation mixture taking into account the quantity of the DNA insert -P0451 (RBS+cI repressor),LuxY and Lumazine- and the receiver DNA -plasmid harboring the promoter J23101-. This can be check in the following gel.
2. Incubate the sample at 16°C overnight. 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 LuxY, Lumazine and P0451:RBS+cI repressor plus promoter J23101, if the ligation was correctly done. Repeated experiment:Working on cI inverter construction and fusion to pSB1T3 backbone. LovTAP repressor activity:reporter systemDue to the first attempt to ligate K098991 (cI regulated promoter+RBS+GFP) and P0451(RBS+cI repressor) inside plasmid pSB1C3 failed, I am going to ligate them in plasmid pSB1T3. Ligation Procedure: P0451 + K098991 to plasmid pSB1T31. Prepare the ligation mixture taking into account the quantity of the DNA insertS -K098991 and P0451- and the receiver DNA -plasmid pSB1T3-. 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. |