Step 1: Subclone genes of interest. (A) Diagram showing the subcloning of defined genomic DNA regions from BAC clones into BACLinking vectors. RP23-395m11 containing the genes Dmp1 and Ibsp was subcloned into BACLink SP and RP23-41205 containing the Trap gene was subcloned into BACLink GM. Homology arm pairs are indicated by 1 & 2 for Dmp1/Ibsp and 3 & 4 for Trap. (B-D) Confirmation of subcloned genomic DNA fragments. (B&C) Diagnostic restriction digests of subcloned GM-Trap and SP-Dmp1/Ibsp were carried out using I-PPO1, Age1, and Mlu1. (B) For GM-Trap, I-PPO1 releases the whole insert ~37 KB + partial vector sequences. Two I-PPO1 sites flank the belo origin of replication which shows up as a ~4.5 KB DNA fragment. Restriction digestion with Age1 yielded DNA fragment sizes 20.3 KB, 12.1 KB+partial vector sequences, and 4.7+partial vector sequences. Restriction digestion with Mlu1 yielded DNA fragment sizes ~24 KB, 7.3 KB+ vector backbone, and 5.8 KB. DNA sequence is not available for BACLink-GM, however, we estimate the vector backbone to be ~6.5 KB. (C) For SP-Dmp1/Ibsp I-PPO1 released the whole insert ~145 KB (Belo replication origin not shown). Restriction digestion with Age1 yielded DNA fragment sizes 96.7 KB+partial vector sequences, 46.4+partial vector sequences and 2.4 KB (not shown). Restriction digestion with Mlu1 yielded DNA fragment sizes of 70.7 KB, 63 KB+partial vector sequences, 7.8 KB + partial vector sequences, and 4.1 KB (not shown). DNA sequence information for BACLink SP is not available, but we estimate the vector backbone to be similar to BACLink GM (~6.5 KB). (D) Diagnostic PCR was carried out with primers that flanked the homology arms. PCR amplification of BAC ends gave predicted sizes for homology arms 3 and 4, which was >1 KB. PCR amplification of Arm1 is larger than normal because this region also includes arm 3 (>2 KB in size). PCR amplification of arm 2 downstream of Ibsp did produce a product larger than expected, but does indicate that recombination did not occur too far downstream of our target site.