High Throughput Methods for Gene Cloning and Expression

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 ligat...

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Bibliographic Details
Published in:Protein expression and purification 2002-06, Vol.25 (1), p.1-7
Main Authors: Dieckman, Lynda, Gu, Minyi, Stols, Lucy, Donnelly, Mark I., Collart, Frank R.
Format: Article
Language:English
Subjects:
Automation
Cloning, Molecular - methods
DNA Primers - pharmacology
Escherichia coli - metabolism
high throughput
ligation-independent cloning
Mutagenesis, Site-Directed
Plasmids - metabolism
Polymerase Chain Reaction
protein expression
structural genomics
Time Factors
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Summary: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.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.2001.1602