e36 Nucleic Acids Research, 2005, Vol. 33, No. 4 PAGE 10 OF 12 Figure 6. Same experiment as in Figure 5 with modifications as indicated at the top of each panel. (A) SpeI digest of 10 minipreps from a control experiment without heat-induction and without the loxP511 dsDNA oligo. (B) SpeI digest of 10 minipreps from a control experiment without heat-induction but with the loxP511 dsDNA oligo. (C) SpeI digest of 10 minipreps from a control experiment with heat-induction but without the loxP511 dsDNA oligo. (D) SpeI digest of 10 minipreps from an experiment with heat-induction and with the loxP511 dsDNA oligo (comparable with Figure 5B). Clones with the parental digestion pattern indicating DOG resistance due to homologous recombination (clones 33 and 35, circles) are only seen in (D). DOG resistance in all other clones likely occurred due to internal deletions of the BACs. (E) SpeI restriction analysis of BAC miniprep DNA from clones 33 and 35 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 the clones analyzed, confirming the correct insertion of loxP511 in clones 33 and 35 (compare with Figure 5C). deletions in both the absence and presence of oligo, indicating that the deletions are not oligo induced. We therefore conclude that these are spontaneous deletions that occur at low level during normal BAC replication in the DH10B background. Non-deleted colonies with the parental SpeI ﬁngerprint were only observed in the Red-induced sample that received oligo (Figure 6D, clones 33 and 35). These clones were sub- sequently electroporated into EL350 Cre-expressing cells to conﬁrm they contained the introduced loxP511 site. Two col- onies from each original clone were tested by SpeI digestion of the BAC miniprep DNA. As shown in Figure 6E, each colony has undergone Cre-mediated deletion, conﬁrming that these