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

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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.
=Image Explanation=


The DNA is electroporated into the cell, then a plasmid coding for recombinase elements used for recombination. Cells that succeed in recombination are isolated.
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.


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.
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 [http://en.wikipedia.org/wiki/Homologous_recombination#DSBR_pathway DSB recombinational repair] was used to ligate the pieces together.

Latest revision as of 19:50, 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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