In the G12D Nras experiment, the selected Gal− clones were analyzed by SpeI digestion of BAC miniprep DNA using unmodified CITB 50J2 BAC DNA as a control. Clones without rearrangements were analyzed by PCR using 1 μl BAC miniprep DNA as the template. The PCR products were gel purified and sequenced using the same primers as were used for PCR. Primers flanking the targeted mutation were: Nras test F: 5′-CACTCATCTGCAAGGAATGCT-3′; Nras test R: 5′-CCTCAGTAAGCACGAACTTGT-3′. PCR conditions were 94°C for 15 s, 60°C for 30 s and 72°C for 30 s, for 30 cycles. Modifications of the RP23-341F12 BAC (50, 75 and 100 kb deletions and the introduction of a loxP511 site) were tested by SpeI restriction analysis of BAC miniprep DNA and compared with unmodified 341F12 BAC DNA. In the loxP511 experiment, clones 3, 5 and 6 were further tested for correct insertion of the loxP511 site by transforming 1 μl of BAC miniprep DNA into electrocompetent and arabinose-induced EL350 cells (11 (link)) and plating on LB plates with chloramphenicol. Two colonies from each starting clone were tested by SpeI digestion of BAC miniprep DNA for the 95 kb Cre-mediated deletion. Finally, the Cre-recombined clones were tested by PCR with one primer mapping to the end of the pBACe3.6 BAC backbone and the other mapping to a position 95 kb away on the wild-type BAC. The primers (Invitrogen) used for this analysis were: 95 kb loxP511 check F: 5′-GCGGATGAATGGCAGAAATTC-3′; 95 kb LoxP511 check R: 5′-TTTGCCAGACTGGTGCCTAA-3′. PCR conditions were 94°C for 15 s, 60°C for 30 s and 72°C for 30 s, for 30 cycles. The resulting PCR bands were gel purified and confirmed by sequencing using the same primers as were used for the PCR amplification. The follow-up experiment for testing the source of the observed BAC deletions was done as described above.