Team:USP-UNESP-Brazil/Plasmid Plug n Play/Results

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We designed two primers, one containing the lox66 and one containing the loxP sequence, these primers amplified the ORF from the kanamycin resistance gene, flanked upstream by the loxP and downstream by the lox66, using PCR. These sites should be recognized by the Cre recombinase (from NEB company), which could circularized our linear PCR product. This is important because we don't want it to be degraded when inserted in the bacteria. This ''In vitro'' assay was a test for a posterior ''In vivo'' assay, where we expect that this process happens inside the ''E. coli'' using a Cre recombinase enzyme expressed by the same bacteria.     
We designed two primers, one containing the lox66 and one containing the loxP sequence, these primers amplified the ORF from the kanamycin resistance gene, flanked upstream by the loxP and downstream by the lox66, using PCR. These sites should be recognized by the Cre recombinase (from NEB company), which could circularized our linear PCR product. This is important because we don't want it to be degraded when inserted in the bacteria. This ''In vitro'' assay was a test for a posterior ''In vivo'' assay, where we expect that this process happens inside the ''E. coli'' using a Cre recombinase enzyme expressed by the same bacteria.     
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Our experiment showed that 5U and 10U of Cre recombinase produced a reduction of linear DNA (Kanamycin resistance gene flanked with loxP and lox66) when compare to 1U of Cre recombinase and to the control DNA (No Cre recombinase added), as is showed in the figure A. It was also observed an increase of the DNA plasmid form (upper band at 2kb), as is showed in figure B. We also used a control DNA substrate supplied in the NEB recombinase kit.  
Our experiment showed that 5U and 10U of Cre recombinase produced a reduction of linear DNA (Kanamycin resistance gene flanked with loxP and lox66) when compare to 1U of Cre recombinase and to the control DNA (No Cre recombinase added), as is showed in the figure A. It was also observed an increase of the DNA plasmid form (upper band at 2kb), as is showed in figure B. We also used a control DNA substrate supplied in the NEB recombinase kit.  

Revision as of 12:37, 26 September 2012