As the expanding of application of ES cell gene targeting technique, cloning and structural analysis of genomic DNA for phage library for homologous fragments in targeting vector are becoming more and more important. An effective strategy has been developed, termed as “separating/combining” strategy, to make restriction mapping of large genomic insert in recombinant phage vector. In this strategy, a set of subclone with commonly used plasmid vector, such as pBluescript^TM series, was generated as first step, restriction mapping of each subclone was analyzed and then, digested the whole length phage DNA and analyzed the restriction site combined with the mapping of subclones. An accurate restriction mapping of a large insert of a phage clone, which contain mouse coagulation factor IX gene, was generated successfully with this strategy.