The entire process, from design of primers to production of clonal glycerol stocks, is shown. Most steps are common to both phases; steps specific to phase 2 are shown with a dashed line. The process began with design of primers for each ORF in the genome (Step 1A). The primers were used to amplify ORFs from genomic DNA (Step 1B). Subsequent amplification with universal primers (Step 1C) generated ORF sequences flanked by complete recombinational cloning sites for capture by BP (Step 3). For amplicons captured by In-Fusion in Phase 2, universal primed PCR was not necessary (Step 2B) as the capture reaction completes creation of the recombinational cloning sites. Successful PCR was monitored by agarose gel electrophoresis (Steps 2A and B). In Phase 1 all products were purified from preparative gels (Step 2B) and cloned into a recombinational cloning vector via the BP clonase reaction (Steps 3), whereas in Phase 2, the capture method depended on ORF size as indicated, with only diagnostic gels needed for short amplicons (Step 2A) and preparative gels needed when In-Fusion capture was performed. Competent bacteria were transformed with the reaction mix to yield colonies which were isolated robotically, cultured in liquid media and stored as 15% glycerol stocks (Step 4).

The label NTFT02#### indicates the unique identifier for each ORF of Francisella tularensis. The expected molecular weights (based on predicted protein coding sequences of the ORFs) are shown below each lane. Black arrows (left side) indicate protein bands observed at approximately the expected molecular weight. MW, molecular weight; NS, no sample.