Titlebook: Directed Evolution; Methods and Protocol Andrew Currin,Neil Swainston Book 2022 Springer Science+Business Media, LLC, part of Springer Natu

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https://doi.org/10.1007/978-3-642-71028-5to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.

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SpeedyGenesXL: an Automated, High-Throughput Platform for the Preparation of Bespoke Ultralarge Varbinatorial-OR-type libraries based on the SpeedyGenes methodology. Together this offers a flexible platform for library synthesis, capable of generating many different bespoke, diverse libraries simultaneously.

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Facile Assembly of Combinatorial Mutagenesis Libraries Using Nicking Mutagenesis, 14 mutated positions, a total of 16,384 library variants, and a protocol for the generation of large, user-defined combinatorial libraries. The reader can use this protocol to create such libraries in 2 days.

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GeneORator: An Efficient Method for the Systematic Mutagenesis of Entire Genes,mental workload. The method describes library synthesis using asymmetric PCR, in which mutagenic primers are designed to create OR-type mutations at multiple sites of variation in a two-step protocol. As an example, we show how this can be utilized for controlled and economical mutagenesis of every amino acid codon in a gene.

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Application of Restriction Free (RF) Cloning in Circular Permutation,to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.