Plant, Animal & Microbe Genomes X Conference Plant, Animal & Microbe Genomes X ConferenceJanuary 12-16, 2002
Town & Country Convention Center
San Diego, CA
Poster: Large Insert Libraries
The BAC vector system based on F plasmid allows stable maintenance of large genomic DNA fragments as single copy plasmids. The disadvantage of low DNA yield from single copy BACs is a bottleneck to many high throughput processes such as BAC end sequencing, fingerprinting and shotgun library construction. Epicentre's CopyControl system containing iBAC cloning vector and TransforMaxEC300araC/trfA Electrocompetent E.coli cells, presents a novel technology allowing the amplification of BAC copy number in excess of 10 fold, enough for several high throughput processes from a single DNA prep. The iBAC vector is a derivative of pBeloBAC11 and EPICENTRE's pIndigoBAC-5 and contains both a single copy origin of replication and the inducible oriV multiple copy origin of replication. This vector allows conditional induction of the plasmid into multiple copies. TransforMaxEC300araC/trfA E.coli contains trfA gene that works in trans with oriV to control amplification. The BAC clones constructed in iBAC vector stay as a single copy and can be induced to multiple copies when required by adding arabinose to a subculture. It results in production of more BAC plasmid DNA with less chromosomal contamination. We report here, the first HindIII BAC library made in our CopyControl HindIII iBAC vector with an average insert size of 130kb. The clones are stably maintained in TransforMaxEC300araC/trfA cells and showed amplification of DNA to 10-15 fold higher when treated with arabinose. We also report an improved approach for construction of BAC library that will allow rapid sizing of BAC inserts and shorter ligation time.