title: High throughput methods for gene cloning and expression.
abstract: We outline a high throughput process for the production of bacterial expression clones using automated liquid handlers. The protocol consists of a series of interlinked methods representing liquid manipulations or incubations on various stations of the automation system. The methods employ the ligation-independent cloning approach that enables the simultaneous production of plasmids for different expression systems. The current cloning protocol spans 3 days with a linear throughput of 400 targets per production run. This automated approach enables the production of large numbers of bacterial expression clones and ultimately purified proteins. Although they were developed for structural genomics, these molecular protocols can also be applied in high throughput strategies such as those used for site-specific mutagenesis or protein interaction studies.
authors.affiliation: Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
authors.forename: Lynda
authors.initials: L
authors.lastname: Dieckman
authors.forename: Minyi
authors.initials: M
authors.lastname: Gu
authors.forename: Lucy
authors.initials: L
authors.lastname: Stols
authors.forename: Mark I
authors.initials: MI
authors.lastname: Donnelly
authors.forename: Frank R
authors.initials: FR
authors.lastname: Collart
mesh_terms: D001331:Automation; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013997:Time Factors
chemical_list: D017931:DNA Primers