Step 3: Linking genomic regions. (A & B) Diagram showing the linkage of BACLink Trap GM into BACLink Dmp1/Ibsp SP. BACLink Trap GM was digested with I-PPO1 exposing homology arms 3 (Trap homology arm) and 5 (endogenous homology arm to both GM and SP vectors) and electroporated into bacteria containing BACLink Dmp1/Ibsp SP containing the Red recombinase system. (B) The newly linked vector changes from SP resistant to GM resistant. BL-TRAP and BL-DMP1 demarcate primer sites that flank the linked region and were used as a primary screen. (C & D) Verification of linked BAC construct (OOO-1) compared to the individually generated GM-Trap-ECFP and SP-Dmp1-mCherry/Ibsp-Topaz. (C) Mlu1 and Age1 diagnostic restriction digest shows a banding pattern consistent with its original BAC clone counterparts. (D) Diagnostic PCR amplification of the linked genomic region and individual fluorescent protein reporters confirming the existence of all three fluorescent reporter genes in the linked BAC DNA construct.