Image:Homologous Recombination Deletion method one.png

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Linear sequences of DNA were created using PCR from synthetic primers (the red stretch of DNA).  Regions A, B and C inside the target region inside the genome (green) and inside the linear PCR sequence (red) are homologous regions.  Cm^R is antibiotic resistance, S indicates an I-ScsI cleavage site.
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The DNA is electroporated into the cell, then a plasmid coding for recombinase elements used for recombination.  Cells that succeed in recombination are isolated.
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Another plasmid coding for I-SceI meganuclease was inserted into the cell to remove the Cm^R gene.  Then, a version of Rec-A mediated DSB recombinational repair was used to ligate the ends together.

Revision as of 21:34, 10 February 2013

Linear sequences of DNA were created using PCR from synthetic primers (the red stretch of DNA). Regions A, B and C inside the target region inside the genome (green) and inside the linear PCR sequence (red) are homologous regions. Cm^R is antibiotic resistance, S indicates an I-ScsI cleavage site. The DNA is electroporated into the cell, then a plasmid coding for recombinase elements used for recombination. Cells that succeed in recombination are isolated. Another plasmid coding for I-SceI meganuclease was inserted into the cell to remove the Cm^R gene. Then, a version of Rec-A mediated DSB recombinational repair was used to ligate the ends together.

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current21:33, 10 February 20131,184×1,829 (229 KB)Evan J. Weaver (Talk | contribs)

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