IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/22

UNAM-Genomics-Mexico team

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Working on LovTAP synthesized plasmid from Mr. Gene:Gel extraction & ligation I'm going to prepare another sample of LovTAP from Mr.Gene that was transformed for gel extraction procedure and ligation, using the plasmid isolated from colony 9 instead of the one obtained from colony 1 because that plasmid sample is run out. LovTAP Restriction enzyme reactions:Preparation for Gel extraction and Ligation Ligation for promoters: 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. Results LovTAP plasmid Colony 9 Restriction enzyme reactions:Preparation for Gel extraction and Ligation In order to know if the restriction enzyme reactions were correctly done, I ran a gel. In the next figure from lane 9 to 12, the samples of LovTAP digested were loaded. Even though the gel quality is not good, it seems that the plasmid from Mr. Gene that harbors LovTAP was digested correctly, considering the three bands observed and the size of each one. After this result, I decided to continue with the procedure of LovTAP extraction from gel using these digested samples . LovTAP Colony 9 Restriction enzyme reactions. Lane1:Ladder. Lane9: LovTAP 9 EcoRI/PstI. Lane10: LovTAP 9 EcoRI/PstI.Lane11: LovTAP 9 XbaI/PstI.Lane12: LovTAP 9 XbaI/PstI. The other lanes are samples from other experiments