IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/18

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Working on CCaS/CCaR and LuxY synthesized plasmid from Mr.Gene
In order to test the correct lenght for CCaS/CCaR and LuxY synthesized, I am going to do a PCR reaction using the following primers:

Forward:Preffix primer Reverse:Suffix primer

After the PCR we are going to do a restriction enzyme assay to test the products.

PCR: Plasmid transformed harboring CCaS/CCaR

PCR: Plasmid transformed harboring LuxY

PCRs Results
According with the next image CcaS PCR reaction yields a product around the expected size 3204nt however the fragment amplified in the LuxY PCR reaction has a size around 2000 nt, thus not corresponding to the expected size that is 820 nt.

I’m going to repeat the PCR reaction with a shorter Elongation step.



LovTAP extraction from gel
In order to extract LovTAP from the gel ready for ligation with J23 promoters and plasmid pSB1C3, I'm going to do two replicates of enzyme restriction reactions using XbaI and PstI to cut LovTAP plasmid isolated from colony 1, as well I will cut the same plasmid using EcoRI and PstI.

LovTAP colony 1.Restriction enzyme assay:Preparation for Ligation with J23 promoters and plasmid pSB1C3


 * Ligation for promoters: Restriction enzymes XbaI and PstI.


 * Ligation for plasmid pSB1C3: Restriction enzymes EcoRI and PstI.

Results:LovTAP colony 1.Preparation for Ligation with J23 promoters and plasmid pSB1C3
The plasmid of LovTAP from colony 1 was successfully digested in both restriction enzyme reactions.


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