title: Methods and results for semi-automated cloning using integrated robotics.
abstract: The Joint Center for Structural Genomics (JCSG) has emphasized automation and parallel processing approaches. Here, we describe automated methods used across the cloning process with results from JCSG projects. The protocols for PCR, restriction digests and ligations, as well as for gel electrophoresis and microtiter plate assays have all been automated. The system has the capacity to routinely process 384 clones a week. This throughput can adequately supply our expression and purification pipeline with expression-ready clones, including novel targets and truncations. The utility of our system is demonstrated by our results from three diverse projects. In summary, 94% of the PCR amplicons generated to date have been successfully cloned and verified by sequencing (83% of the total attempted targets). Our results demonstrate the capabilities of this robotic platform to provide an avenue to high-throughput cloning which requires little manpower and is rapid and cost-effective while providing insights for method optimization.
authors.affiliation: Joint Center for Structural Genomics, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
authors.forename: Heath E
authors.initials: HE
authors.lastname: Klock
authors.forename: Aprilfawn
authors.initials: A
authors.lastname: White
authors.forename: Eric
authors.initials: E
authors.lastname: Koesema
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics / Q000379:methods*; D009841:Oligonucleotides; D012371:Robotics / Q000295:instrumentation* / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D009841:Oligonucleotides