Summary
We describe a plate-based cloning and expression strategy for efficient high-throughput generation of validated expression clones in Escherichia coli. The process incorporates 48- or 96-well plates at all stages including the cloning and colony selection phases which are often performed manually. A 48-grid agar growth plate has been integrated into the colony selection component to improve throughput at the cloning stage. The combinations of 48- and 96-well plate formats are compatible with automated liquid handlers and multichannel pipettes. This revised cloning and expression pipeline increases throughput significantly, and also results in a reduction in both time and material requirements. The system has been validated by the production and screening of several thousand clones at the Midwest Center for Structural Genomics.
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Acknowledgments
The authors would like to thank Kendall Nettles (for technical assistance with using 48-grid plates), and Joseph Gregar (for preparation of the 130-mm Borosilicate Glass rod Petri Dish Spreaders). This work was supported by NIH grants GM62414 and GM074942, Andrzej Joachimiak, PI, and by the U.S. Department of Energy, Office of Science, under Contract DE-AC02-06CH11357 to UChicago Argonne LLC.
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Abdullah, J.M., Joachimiak, A., Collart, F.R. (2009). “System 48” High-Throughput Cloning and Protein Expression Analysis. In: Doyle, S.A. (eds) High Throughput Protein Expression and Purification. Methods in Molecular Biology, vol 498. Humana Press. https://doi.org/10.1007/978-1-59745-196-3_8
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DOI: https://doi.org/10.1007/978-1-59745-196-3_8
Publisher Name: Humana Press
Print ISBN: 978-1-58829-879-9
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