Integration strategies
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| + | Sequences to be inserted in yeast (i.e. GFP and 2ORE promoter) were first cloned in plasmids containing a drug resistence gene. Then the entire cassettes, which contain both the selectable marker and the coding sequence/promoter, were amplified by PCR using oligos which bring at their 5'-end 50 nucleotides that are homologous to the precise genomic region where the cassette has to be inserted. | ||
| + | Once the PCR product has been transformed in yeast cells, it undergoes homologous recombination. Colones in which the recombination event happened in the correct way were screened by PCR as described in the figures. | ||
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Integration of GFP into Pho8 Locus | Integration of GFP into Pho8 Locus | ||
[[Image:CIRCUITO.jpg]] | [[Image:CIRCUITO.jpg]] | ||
Screening strategy | Screening strategy | ||
[[Image:TABCIRC1.jpg]] | [[Image:TABCIRC1.jpg]] | ||
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Integration of 2XORE Promoter into Pho4 Locus | Integration of 2XORE Promoter into Pho4 Locus | ||
[[Image:pho4.jpg]] | [[Image:pho4.jpg]] | ||
Screening strategy | Screening strategy | ||
| + | [[Image:Circ11.jpg]] | ||
Latest revision as of 12:20, 26 October 2007
Sequences to be inserted in yeast (i.e. GFP and 2ORE promoter) were first cloned in plasmids containing a drug resistence gene. Then the entire cassettes, which contain both the selectable marker and the coding sequence/promoter, were amplified by PCR using oligos which bring at their 5'-end 50 nucleotides that are homologous to the precise genomic region where the cassette has to be inserted. Once the PCR product has been transformed in yeast cells, it undergoes homologous recombination. Colones in which the recombination event happened in the correct way were screened by PCR as described in the figures.
Integration of GFP into Pho8 Locus
Screening strategy
Integration of 2XORE Promoter into Pho4 Locus
Screening strategy
