Functional enrichment by direct plasmid recovery after fluorescence activated cell sorting
| Autor: | Balakrishnan Ramesh, Christopher S Frei, Patrick C Cirino, Navin Varadarajan |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 59, Iss 3, Pp 157-161 (2015) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/000114329 |
| Popis: | 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 60% of the sorted cell population was recovered. |
| Databáze: | Directory of Open Access Journals |
| Externí odkaz: |
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