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 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.

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Citation

Copyright 2016 BioTechniques.Recommended citation:Ramesh, Balakrishnan, Christopher S. Frei, Patrick C. Cirino, and Navin Varadarajan. "Functional enrichment by direct plasmid recovery after fluorescence activated cell sorting." BioTechniques 59, no. 3 (2015): 157-161.doi:10.2144/000114329. URL: https://www.future-science.com/doi/pdf/10.2144/000114329. Reproduced in accordance with the original publisher's liceinsing terms and with permission from the authors.