Functional enrichment by direct plasmid recovery after Fluorescence Activated Cell Sorting
Frei, Christopher S.
Cirino, Patrick C.
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Iterative screening of expressed protein libraries using fluorescence-activated cell sorting (FACS) typically involves culturing the pooled clones after each sort. In these experiments, if cell viability is compromised by the sort conditions and/or expression of the target protein(s), rescue PCR provides an alternative to culturing but requires re-cloning and can introduce amplification bias. We haveoptimized a simple protocol using commercially available reagents to directly recover plasmid DNA from sorted cells for subsequenttransformation. We tested our protocol with 2 different screening systems in which <10% of sorted cells survive culturing and demonstrate that >60% of the sorted cell population was recovered.