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Oligo pools are array-synthesized, user-defined mixtures of single-strandedoligonucleotides that can be used as a source of synthetic DNA for librarycloning. While currently offering the most affordable source of synthetic DNA,oligo pools also come with limitations such as a maximum synthesis length(approximately 350 bases), a higher error rate compared to alternativesynthesis methods, and the presence of truncated molecules in the pool due toincomplete synthesis. Here, we provide users with a comprehensive protocol thatdetails how oligo pools can be used in combination with Golden Gate cloning tocreate user-defined protein mutant libraries, as well as single guide RNAlibraries for CRISPR applications. Our methods are optimized to work within theYeast Toolkit Golden Gate scheme, but are in principle compatible with anyother Golden Gate-based modular cloning toolkit and extendable to otherrestriction enzyme-based cloning methods beyond Golden Gate. Our methods yieldhigh-quality, affordable, in-house variant libraries.

Combining oligo pools and Golden Gate cloning to create protein variant libraries or guide RNA libraries for CRISPR applications