**4.1 Fragment isolation using sequence-specific endonucleases**

The simplest and most straightforward method involves (i) digestion by special endonucleases and (ii) subsequent isolation/purification of the engineered BAC. The use of two endonucleases facilitates the recognition of extremely infrequent sequences. They are: I-*Ppo*I for the 23-base sequence ATGACTCTCTTAA/GGTAGCCAAA, and I-*Sce*I for the 18 base sequence TAGGGATAA/CAGGGTAAT (Itaya, 2009). Indeed, two I-*Ppo*I recognition sequences are created in the *B. subtilis* genome to cut the BAC part out of the host genome (Figs. 5b and 6a). The linear DNA produced by I-*Ppo*I digestion is readily isolated from agarose gels resolved by pulsed-field gel electrophoresis. The handling of giant DNA longer than several hundred kbp has been difficult because of its fragility in test tubes. However, its use for microinjection into, for example, fertilized mouse eggs requires pure and undamaged DNA. We implemented and tested various steps to isolate undamaged giant DNA maintained in liquids. Our improved protocol that involves 2 gel electrophoresis runs and collection on a dialysis membrane made it possible to concentrate undamaged giant DNA. The successful purification of up to 220 kb mouse genomic DNA carrying reporter genes facilitated the creation of transgenic mice (manuscript in preparation). Because our method is technically simple it will be of great value in future studies.

Fig. 7. Three methods to retrieve BAC-DNA from BGM.

