A portion of the harvested plasmid was transformed directly into Thermo Fisher One-Shot Mach1 T1 Chemically Competent cells according to manufacturer’s instructions. 36–48 colonies, each bearing a single library member, were picked for rolling circle amplification and subject to Sanger sequencing with primers:
Clones of interest were then subcloned into pET29 expression vectors.
Alternatively, at the end of the last sort of a given round, the BD FACS Aria Cell Sorter was switched to plate mode, gates adjusted to only collect the top 0.1–0.3% of cells, and single cells collected in each well of a 96-well plate. After growing to saturation, these clones were subject to flow cytometry assays. Top performers were sequenced and then subcloned into pET29 expression vectors.
Clones of interest were then subcloned into pET29 expression vectors.
Alternatively, at the end of the last sort of a given round, the BD FACS Aria Cell Sorter was switched to plate mode, gates adjusted to only collect the top 0.1–0.3% of cells, and single cells collected in each well of a 96-well plate. After growing to saturation, these clones were subject to flow cytometry assays. Top performers were sequenced and then subcloned into pET29 expression vectors.