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Figure 5 | BMC Genomics

Figure 5

From: Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

Figure 5

Lox66/lox71-mediated DNA integration and cloning of insertion mutation. (A) Integration of a bacterial plasmid DNA into the insertion mutation of a S. pombe insertion mutant. The plasmid pLox66 bearing the lox66 sequence can recombine with lox71 on the integrated insertion vector in S. pombe in the presence of Cre recombinase. After pLox66 integration, pLox66, the insertion vector and nearby S. pombe genomic DNA can be excised by restriction digestion and cloned in E. coli. The pUC origin allows pLox66 to be amplified and maintained in E. coli and KanR/G418R gene (kanMX) allow selection of the plasmid in E. coli (kanamycin resistance) and S. pombe (G418 resistance). (B) Cre recombinase-dependent integration of pLox66 in S. pombe. The pLox66 DNA was transformed to S. pombe insertion mutant strains 18_M24 or that expressed or did not express Cre recombinase (pREP81-Cre or pREP81). Transformed cells were replica plated to solid media with G418 to test G418 resistance and stable integration of pLox66. (C) Stable integration of pLox66 in 18_M24 was tested by PCR using primers on the insertion vector (A and D in panel A) and pLox66 (B and C in panel A). A truncated sck1+ gene fragment was co-amplified in each reaction as a positive control. Five independent colonies of each transformation were tested. (D) PCR products of 18_M24 with pREP81-Cre were sequenced to examine the recombined wild type loxP and lox66/71 hybrid sequences. The colored boxes in the electropherograms highlight the base differences in the individual lox71 and lox66 sites while the black boxes indicate the wild type loxP sequences.

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