e36 Nucleic Acids Research, 2005, Vol. 33, No. 4 conditions: 94 C for 15 s, 60 C for 30 s and 72 C for 1 min, for 30 cycles. After completion of PCR, 2 ml DpnI was added to each 25 ml reaction and incubated for 2 h at 37 C to remove any plasmid template. The DpnI-digested reaction mix was run on a 1% agarose gel over night, and the PCR product was puriﬁed and eluted in 50 ml ddH2O. An aliquot of 2.5 ml was used for each experiment. For making the Nras G12D substi- tution the following primers were used for the ﬁrst step, intro- ducing galK. Homology to Nras sequence is in italics and the sequence recognizing em7-galK is underlined: Nras galK F: 50-TTTTTGCTGGTGTGAAATGACTGAGTACAAACTGGTGG- TGGTTGGAGCAGCCTGTTGACAATTAATCATCCGCA-30; Nras galK R: 50-CAAAGTGGTTCTGGATTAGCTGGATCG- TCAAGGCGCTTTTCCCAACACCATCAGCACTGTCCTGC- TCCTT-30. For making 50, 75 and 100 kb deletions in the RP23-341F12 BAC, the following primers were used (homo- logy to BAC sequence is in italics and the sequence recog- nizing em7-galK is underlined): galK F 341F12 start: 50-ACTCCCACTGGAAGCTTTTTACAAAACATGTGTTGCT- GACATGTTGACAGCCTGTTGACAATTAATCATCGGCA- 30; galK R 341F12 50 kb: 50-ACCCAAACCAAACAACAT- CCAAACCAAAAACACAGACAAAACCAAATATGTCAGCAC- TGTCCTGCTCCTT-30; galK R 341F12 75 kb: 50-ACACTAAGC- CAAACTCCTTGCCTGGGCTATTTCTCTTTGTTTTTCCAAATTCAGC- ACTGTCCTGCTCCTT-30; galK R 341F12 100 kb: 50-TATGT- GTCTGTGTGTGTATGTACAGTTCTTTGTTTTTGTTTTTTTT- CTTTTCAGCACTGTCCTGCTCCTT-30. For insertion of a loxP511 site in the RP23-341F12 BAC, the following primers were used for the ﬁrst step (introduction of galK). Homology to BAC sequence is in italics and the sequence recognizing em7- galK is underlined: 95 kb loxP511 galK F: 50-GGACA- GAGGCGTGACAGACGTGTGAGCTCCGTGGACAACTCTC- CCCGAAGCCTGTTGACAATTAATCATCGGCA-30; 95 kb loxP511 galK R: 50-GACTCTGAGCAGCAACGGCTGAGCCT- CACTTGAGAGGGTCCCTGAGTCACTCAGCACTGTCCTG- CTCCTT-30. Oligos for recombineering Oligos used to replace the galK-targeting cassette were obtained from Invitrogen. dsDNA was used and the oligos (sense and antisense) annealed in vitro: 10 mg of each oligo (sense and antisense) was mixed in an eppendorf tube in a total volume of 100 ml of 1· PCR buffer (Expand High Fidelity PCR kit, Roche Applied Science) and boiled for 5 min, allowed to cool to room temperature for 30 min, ethanol pre- cipitated and resuspended in 100 ml ddH2O to a ﬁnal concen- tration of 200 ng/ml double-stranded oligo. An aliquot of 1 ml (200 ng) was used in the recombineering experiments. To introduce the G12D (G<>A) point mutation in the Nras BAC CITB 50J2, the following oligos were used for the second step (the introduced adenosine/thymidine base pair is underlined, the ﬂanking sequences are homologous to the Nras BAC sequence): G12D S: 50-TTTTTGCTGGTGT- GAAATGACTGAGTACAAACTGGTGGTGGTTGGAGC- AGATGGTGTTGGGAAAAGCGCCTTGACGATCCAGC- TAATCCAGAACCACTTT-30; G12D AS: 50-AAAGTGGT- TCTGGATTAGCTGGATCGTCAAGGCGCTTTTCCCAA- CACCATCTGCTCCAACCACCACCAGTTTGTACTCAG- TCATTTCACACCAGCAAAAA-30. To introduce a loxP511 site in the RP23-341F12 BAC, the following oligos were used PAGE 4 OF 12 for the second step (loxP511 is underlined, the ﬂanking sequences are homologous to the BAC sequence around position 95 kb): 95 kb loxP511 S: 50-ACGTGTGAGCTC- CGTGGACAACTCTCCCCGAAGATAACTTCGTATAGT- ATACATTATACGAAGTTATGTGACTCAGGGACCCTC- TCAAGTGAGGCTCAGC-30; 95 kb loxP511 AS: 50-GCTG- AGCCTCACTTGAGAGGGTCCCTGAGTCACATAACTT- CGTATAATGTATACTATACGAAGTTATCTTCGGGGA- GAGTTGTCCACGGAGCTCACACGT-30. Verification of positive recombinants 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 with- out rearrangements were analyzed by PCR using 1 ml 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: 50-CACTCATCTGCAAGGAATGCT-30; Nras test R: 50-CCTCAGTAAGCACGAACTTGT-30. 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 ml of BAC miniprep DNA into electrocompetent and arabinose-induced EL350 cells (11) 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: 50-GCGGATGAATGGCAGAAATTC-30; 95 kb LoxP511 check R: 50-TTTGCCAGACTGGTGCCTAA-30. PCR condi- tions 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. BACs Mouse BACs were obtained from Invitrogen. The RP23- 341F12 BAC (C57BL/6 DNA) was chosen based on position within the mouse Ebfaz gene using the UCSC genome browser (http://genome.ucsc.edu/). The CITB 50J2 BAC (CJ7/129/SV DNA) was identiﬁed by screening a CITB mouse BAC library (Invitrogen) with an Nras genomic probe using standard hybridization methods. The BAC clones were streaked for single colonies and characterized by PCR and restriction ana- lysis before proceeding to the recombineering experiments. Two methods were used for DNA preparation. For BAC mini- preps (1–1.5 mg), we used the following protocol: 5 ml over- night LB culture (15 ml Falcon tube) was pelleted for 5 min at 5000 r.p.m., the supernatant removed and the pellet was dissolved in 250 ml buffer P1 (miniprep kit, Qiagen) and