IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/07

UNAM-Genomics-Mexico team

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Working on J23101 promoter: Ligation to P0451 Once the plasmid harboring the J23101 promoter was correctly dephosphatated, I have started the ligation procedure with P0451 (RBS+cI repressor). Ligation Procedure:Promoter J23101 with P0451 (RBS+cI repressor) 1. Prepare the ligation mixture taking into account the quantity of the DNA insert -P0451 (RBS+cI repressor)- and the receiver DNA -plasmid harboring the promoter J23101-. This can be check in the following gel. Gel for ligations. Lane1:Ladder. Lane2: plasmid harboring the promoter J23101 dephosphatated (SpeI/PstI). Lane3:P0451 colony 6 (XbaI/PstI). Ligation mixture Reactive Quantity DNA insert (P0451:RBS+cI repressor) 4μL DNA plasmid (plasmid harboring the promoter J23101) 2μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (8μL) 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 P0451:RBS+cI repressor plus promoter J23101, if the ligation was correctly done. Working on Lumazine synthesized from Mr.Gene: Preparation for Ligation Once the PCR reactions for Lumazine gene were successfully done, I purified both PCR mixures using the High Pure PCR Product Purification kit from Roche. The purified product will be digested for the following ligations. Lumazine Restriction enzyme reactions:Preparation for Ligation Ligation for promoters (J23101 and J23102): Restriction enzymes XbaI 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 Ligation for plasmid pSB1C3: Restriction enzymes EcoRI 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 reactions were incubated at 37°C overnight. Note:Desactivate the reaction at 80°C during 10 min. Ligation Procedure: Lumazine/LuxY with plasmid pSB1C3 and promoters J23101/J23102 1. Prepare the ligation mixture taking into account the quantity of the DNA inserts -Lumazine/LuxY - and the receiver DNA -plasmid pSB1C3 and plasmids haboring the promoters J23101/J23102-. This can be check in the following gel. Quantity loaded 3μL. Gel for Ligations: Lumazine with plasmid pSB1C3 and plasmid haboring the promoter J23101..Lane1:Ladder.Lane3:Plasmid harboring promoter J23101(SpeI/PstI)dephosphatated.Lane4:Lumazine Mr.gene PCR amplfied product(XbaI/PstI).Lane6:Plasmid pSB1C3(EcoRI/PstI).Lane7: Lumazine Mr.gene PCR amplfied product(EcoRI/PstI). Ligation mixture: Lumazine/LuxY with plasmid pSB1C3 Reactive Quantity DNA insert (Lumazine/LuxY) 4μL DNA plasmid (Plasmid pSB1C3) 2μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (11μL) Ligation mixture: Lumazine/LuxY with plasmids haboring the promoters J23101/J23102 Reactive Quantity DNA insert (Lumazine/LuxY) 6μL DNA plasmid (Plasmids harboring J23101 and J23102 promoters) 2μL Buffer 10X T4 Ligase 2μL Enzyme T4 Ligase 1μL HPLC Up to a final volume of 20 μL of the mixture (9μL) 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 LovTAP ligated to each promoter and BBa_K098991, if the ligation was correctly done.