Figure 4

Efficient deletion in a BAC using ssOR. The illustration at the top shows E. coli HS996 harbouring the pBAD-γβα plasmid and a 143 kb BAC containing mouse genomic Mll DNA. This BAC had previously been modified by Red/ET recombination to carry a cassette containing the neo gene interrupted at its NcoI site by the zeocin resistance gene as depicted. A series of oligos, listed at the bottom, symmetrically centred on the Nco1 site were electroporated into the E. coli host and plated to score ssOR frequencies. In this case, ssOR will delete the approximately 800 bp zeocin gene to restore kanamycin resistance. Total colony numbers were assessed by counting chloramphenicol resistance conveyed by the BAC vector.