Difference between revisions of "Part:BBa K1744000:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
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The first step is to amplify this part with appropriate primers. | The first step is to amplify this part with appropriate primers. | ||
These have to contain the priming sites sequence indicated on each side of the part with an additional tail (~38-80bp) corresponding to the upstream homology to the region to delete for the forward primer and to the downstream homology for the reverse primer. | These have to contain the priming sites sequence indicated on each side of the part with an additional tail (~38-80bp) corresponding to the upstream homology to the region to delete for the forward primer and to the downstream homology for the reverse primer. | ||
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Then this cassette with the right homologies is used to recombinate (through recombineering) and delete the targeted region of the plasmid/genome. After what the recombinants will be selected with ampicillin, since the gene bla is present in the cassette. | Then this cassette with the right homologies is used to recombinate (through recombineering) and delete the targeted region of the plasmid/genome. After what the recombinants will be selected with ampicillin, since the gene bla is present in the cassette. | ||
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The next step is to amplify both region on each side of the targeted sequence. | The next step is to amplify both region on each side of the targeted sequence. | ||
Then a fusion PCR of those 2 regions has to be done. | Then a fusion PCR of those 2 regions has to be done. | ||
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With the cassette obtained, another recombination is done that will delete the part BBa_K1744000 and only the cassette used to delete it will remain. To get only the recombinants of this step, the cells must be put on a medium containing arabinose (we used 1%w/v of final concentration) to trigger the killswitch in cells that wouldn't have lost the part. | With the cassette obtained, another recombination is done that will delete the part BBa_K1744000 and only the cassette used to delete it will remain. To get only the recombinants of this step, the cells must be put on a medium containing arabinose (we used 1%w/v of final concentration) to trigger the killswitch in cells that wouldn't have lost the part. | ||
Revision as of 23:21, 14 September 2015
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K1744000
BBa_K1744000 is designed to achieve clean deletion in a genome or in a plasmid.
The first step is to amplify this part with appropriate primers. These have to contain the priming sites sequence indicated on each side of the part with an additional tail (~38-80bp) corresponding to the upstream homology to the region to delete for the forward primer and to the downstream homology for the reverse primer.
<figure>
Then this cassette with the right homologies is used to recombinate (through recombineering) and delete the targeted region of the plasmid/genome. After what the recombinants will be selected with ampicillin, since the gene bla is present in the cassette. Note that during all the culture steps implicated, the cells has to be in a medium with glucose (we use 5%w/v) to keep the expression of the toxin vcrx028, that is present in the cassette, repressed.
The next step is to amplify both region on each side of the targeted sequence. Then a fusion PCR of those 2 regions has to be done.
<figure>
With the cassette obtained, another recombination is done that will delete the part BBa_K1744000 and only the cassette used to delete it will remain. To get only the recombinants of this step, the cells must be put on a medium containing arabinose (we used 1%w/v of final concentration) to trigger the killswitch in cells that wouldn't have lost the part.
After all these steps, you will get a clean deletion in your plasmid/genome.
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UNIQa84edc7320088d62-partinfo-00000000-QINU UNIQa84edc7320088d62-partinfo-00000001-QINU