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Functional enrichment by direct plasmid recovery after fluorescence activated cell sorting.

ABSTRACT: 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 have optimized a simple protocol using commercially available reagents to directly recover plasmid DNA from sorted cells for subsequent transformation. 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.

SUBMITTER: Ramesh B

PROVIDER: S-EPMC5173293 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Functional enrichment by direct plasmid recovery after fluorescence activated cell sorting.

Ramesh Balakrishnan B Frei Christopher S CS Cirino Patrick C PC Varadarajan Navin N

BioTechniques 20150901 3

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 have optimized a simple protocol using commercially available reagents to directly recover plasmid DN ...[more]

PMID: 26345509

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