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 | =Image Explanation= | ||
The DNA is electroporated into the cell, then a plasmid coding for recombinase elements used for recombination. Cells that succeed in recombination | |||
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 | 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 [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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| current | 19:33, 10 February 2013 | | 1,184 × 1,829 (229 KB) | Evan J. Weaver (talk | contribs) |
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