The USDA-ARS, Shafter Research and Extension center, Shafter, CA has a requirement for a shotgun library construction of BAC clones which will consist of 7.5x coverage sequencing of 20 BAC clones which will be used in a genetics research and breeding program.  The entire unit will consist of shotgun library, plate and array for 20 BAC's and the sequences for 20 BAC's.  Entire project will consist of the construction of a small-insert library of 20 BAC clones (estimated insert size of 137kb), sequence to a desired level of coverage.

Small-insert DNA fragments will be generated by extracting BAC DNA from BAC clones and subjecting to hydroshearing (random fragmentation).  Fragments between 3 and 5 kb will be size selected by gel electrophoresis, subject to end-repair, cloned into the hi-copy plasmid based cloning vector   pBlueskriptIIKSII+ and then electorprorated into E. coli DH108 cells.  Transformants will be selected on LB plates containing carbenicillan, X-Gal and IPTG.  White recombinant colonies will be picked robotically using the Genetix Q-bot and stored as individual clones in Genetix 96-well microtiter plates as glycerol stocks at -80°C.  Clones will be miniprepped and DNA sequencing will be performed with BigDye version 3.1 in both the forward and reverse directions using the universal priming sites on the vector.  DNA will be accumulated on an ABI 3730 xl DNA analyzer. 

A RFQ is not available for this requirement.  The USDA ARS, intends on negotiation with Clemson University Genomics Institute (CUGI) on a sole source basis.  See numbered note 22