Abstract
We describe a simple and easy protocol to introduce random mutations into plasmid DNA: error-prone rolling circle amplification. A template plasmid is amplified via rolling circle amplification with decreased fidelity in the presence of MnCl2 and is used to transform a host strain resulting in a mutant library with several random point mutations per kilobase through the entire plasmid. The primary advantage of this method is its simplicity. This protocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis.
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Acknowledgments
This study was supported in part by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
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Fujii, R., Kitaoka, M., Hayashi, K. (2014). Error-Prone Rolling Circle Amplification Greatly Simplifies Random Mutagenesis. In: Gillam, E., Copp, J., Ackerley, D. (eds) Directed Evolution Library Creation. Methods in Molecular Biology, vol 1179. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1053-3_2
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DOI: https://doi.org/10.1007/978-1-4939-1053-3_2
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