Difference between revisions of "File:Homologous Recombination Deletion method one.png"

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=Image Explanation=
 
=Image Explanation=
  
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.
+
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 codes for [http://en.wikipedia.org/wiki/Chloramphenicol chloramphenicol] resistance, S indicates an [http://en.wikipedia.org/wiki/Meganuclease_I-SceI 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 were isolated.
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The DNA is [http://en.wikipedia.org/wiki/Electroporation electroporated] into the cell, then a plasmid is inserted coding for recombinase elements used for recombination.  Cells that succeed in recombination were 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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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 [http://en.wikipedia.org/wiki/Homologous_recombination#DSBR_pathway DSB recombinational repair] was used to ligate the pieces together.

Latest revision as of 18:50, 10 February 2013

Image Explanation

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 codes for chloramphenicol resistance, S indicates an I-ScsI cleavage site.

The DNA is electroporated into the cell, then a plasmid is inserted coding for recombinase elements used for recombination. Cells that succeed in recombination were 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 pieces together.

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