Summary
A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone. Smaller numbers of cloning reactions can be undertaken by appropriate scaling of the indicated reaction volumes.
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Acknowledgments
Protein Structure Initiative Grant 1U54 GM074901 (J.L. Mar-kley, PI; G.N. Phillips, Jr. and B.G. Fox, Co-Investigators) and a Sponsored Research Agreement from Promega Corporation (B.G. Fox, PI) generously supported this research. The authors enthusiastically acknowledge the efforts all other coworkers of the University of Wisconsin Center for Eukaryotic Structural Genomics for their work in establishing our complete pipeline effort and thank Dr. Mike Slater (Promega) for many useful scientific discussions.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Blommel, P.G., Martin, P.A., Seder, K.D., Wrobel, R.L., Fox, B.G. (2009). Flexi Vector Cloning. 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_4
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DOI: https://doi.org/10.1007/978-1-59745-196-3_4
Publisher Name: Humana Press
Print ISBN: 978-1-58829-879-9
Online ISBN: 978-1-59745-196-3
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