PAGE 9 OF 12 Nucleic Acids Research, 2005, Vol. 33, No. 4 e36 Figure 5. Insertion of a loxP511 site. (A) The location of the wild-type and mutant loxP sites in the BAC backbone are indicated along with the extra mutant loxP511 site that was introduced into the BAC genomic insert via galK counterselection. The 95 kb region deleted by Cre-mediated recombination between the two loxP511 sites is indicated, and PCR primers used to confirm the deletion are shown as small arrows. (B) SpeI restriction analysis of six miniprep clones selected for the replacement of galK with a dsDNA oligo containing the mutant loxP511 site. Clones 3, 5 and 6 (circles) had the same restriction pattern as the unmodified BAC, indicating that DOG resistance occurred due to the intended homologous recombination event. Clones 1, 2 and 4 had large deletions and were not analyzed further. (C) SpeI restriction analysis of BAC miniprep DNA from clones 3, 5 and 6 after transformation into Cre-induced EL350 cells. Two clones from each parental clone were tested. The restriction pattern shows that the 95 kb region flanked by two loxP511 sites is deleted from all clones analyzed, confirming the correct insertion of loxP511 in clones 3, 5 and 6. (D) PCR analysis of the six clones from (C) with one primer mapping to the BAC backbone and the other to a position distal to the inserted loxP511 site. are activated. Alternatively, the deletions could be induced by the large amount of oligos (200 ng) used in these experiments or they could simply represent spontaneous deletions that occur at low level during normal BAC replication and that are found by DOG selection. To distinguish between these possibilities, we repeated the DOG selection step used to insert the loxP511 oligo into BAC RP23-341F12 DNA using four differently treated bacteria samples: non-induced bacteria without the 100 bp loxP511 oligo (Figure 6A), non-induced bacteria with the oligo (Figure 6B), induced bacteria without the oligo (Figure 6C) and induced bacteria with the oligo (Figure 6D). The number of resistant colonies obtained from the four experiments varied by <10-fold (data not shown). Ten colonies from each electroporation were then analyzed by SpeI digestion of BAC miniprep DNA and com- pared with unmodiﬁed RP23-341F12 BAC DNA (Figure 6A– D). Unwanted galK region deletions were observed in all 10 colonies for both of the non-induced samples (Figure 6A and B). These deletions are therefore unlikely to be Red-mediated since these samples were not heat-induced. We also observed