IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/27

UNAM-Genomics-Mexico team

Main project page Previous entry      Next entry

Working on LuxYsynthesized from Mr.Gene: Preparation for Ligation Once the PCR reactions for LuxY 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: LuxY 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 Results restriction enzyme assays Quantity loaded 3μL Restriction enzyme assays. Lane1:Ladder. Lane6: LuxY PCR amplified product from Mr.Gene (EcoRI/PstI). Lane7:LuxY PCR amplified product from Mr.Gene(XbaI/PstI). The other lanes are samples from other experiments The sizes of the digested products are around the expected lenght for each part. 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 20 min. Getting more sample:Working on J23102 promoter: Ligations to P0451, LuxY and Lumazine Once the plasmid harboring the J23102 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. Dephosphatation mixture Reactive Quantity DNA 15μL Buffer 10X 3μL Dephosphatase Enzyme 1.5μL HPLC Up to a final volume of 30 μL of the mixture (10.5μL) Procedure 1.Incubate the samples at 37°C during 20 min. 2.Incubate the samples at 65°C during 10 min.