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Strategies for High-Throughput Gene Cloning and Expression

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Genetic Engineering

Part of the book series: Genetic Engineering: Principles and Methods ((GEPM,volume 27))

Summary

High-throughput approaches for gene cloning and expression require the development of new, nonstandard tools for use by molecular biologists and biochemists. We have developed and implemented a series of methods that enable the production of expression constructs in 96-well plate format. A screening process is described that facilitates the identification of bacterial clones expressing soluble protein. Application of the solubility screen then provides a plate map that identifies the location of wells containing clones producing soluble proteins. A series of semi-automated methods can then be applied for validation of solubility and production of freezer stocks for the protein production group. This process provides an 80% success rate for the identification of clones producing soluble protein and results in a significant decrease in the level of effort required for the labor-intensive components of validation and preparation of freezer stocks. This process is customized for large-scale structural genomics programs that rely on the production of large amounts of soluble proteins for crystallization trials.

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Author information

Authors and Affiliations

  1. Biosciences Division, Argonne National Laboratory, Argonne, Illinois, 60439

    L. J. Dieckman, W. C. Hanly & F. R. Collart

Authors
  1. L. J. Dieckman
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  2. W. C. Hanly
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  3. F. R. Collart
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Editor information

Editors and Affiliations

  1. Brookhaven National Laboratory, Upton, New York

    Jane K. Setlow

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© 2006 Springer Science+Business Media, Inc.

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Dieckman, L.J., Hanly, W.C., Collart, F.R. (2006). Strategies for High-Throughput Gene Cloning and Expression. In: Setlow, J.K. (eds) Genetic Engineering. Genetic Engineering: Principles and Methods, vol 27. Springer, Boston, MA. https://doi.org/10.1007/0-387-25856-6_10

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