Flowchart of high-throughput recombinational cloning using the Gateway system. The schematic follows a single malaria ORF (CSP, green) as one example for the workflow. (1) The establishment of master clones is generated in a 96-well format including: ORF amplification, PCR purification, BP reaction, E. coli transformation, and recombinant clone screening. On average, we achieved 84% cloning efficiency for the BP reaction. Quality control is monitored and documented using PCR and DNA sequencing followed by archiving the master clones as purified DNA and glycerol stocks. (2) The simultaneous subcloning of ORFs into multiple expression vectors is achieved though the LR reactions. The illustration shows three destination vector examples: DNA vaccine (red), recombinant protein (green), and Y2H (yellow). Following the LR reaction and E. coli transformation (3), recombinant clones are screened by either single-colony (100% efficiency) or bulk-culture (93% efficiency) PCR as represented by the deep-well plate. (4) After screening and DNA purification, expression clones are then processed directly into functional assays.
