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Figure 5 | BMC Molecular Biology

Figure 5

From: Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

Figure 5

A counterselection strategy for engineering with ssOR. The illustration at the top shows E. coli HS996 harbouring the low copy, temperature sensitive, expression plasmid, pSC101/BAD/γβα and the 143 kb mouse genomic Mll BAC. In the first step, a PCR product of the neo and rpsL genes (50) flanked by two 50 nt homology arms sited 908 bps apart in the BAC was introduced into the BAC by selection for kanamycin resistance. In the second step, either of the two oligos listed below were used for ssOR with selection for loss of rpsL and restoration of streptomycin resistance. Both oligos had 25 nts of homology to the BAC immediately flanking the rpsL-neo cassette and included either 6 nts of an XhoI site or 34 nts of an FRT. In addition, the temperature sensitive pSC101 plasmid was eliminated by culture at 37°C. BAC DNAs prepared from 22 streptomycin resistant colonies for each experiment were analyzed by restriction enzyme digestions and in both cases, 20 were correct.

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