biostudies-literatureMedina-Cucurella AVDefense Advanced Research Projects AgencyNIAID NIH HHSNational Institutes of HealthDepartment of DefenseNIGMS NIH HHS41-45https://www.ebi.ac.uk/biostudies/studies/S-EPMC7212264biostudies-literatureUnknown32(1)User-defined mutagenic libraries are fundamental for applied protein engineering workflows. Here we show that unamplified oligo pools can be used to prepare site saturation mutagenesis libraries from plasmid DNA with near-complete coverage of desired mutations and few off-target mutations. We find that oligo pools yield higher quality libraries when compared to individually synthesized degenerate oligos. We also show that multiple libraries can be multiplexed into a single oligo pool, making preparation of multiple libraries less expensive and more convenient. We provide software for automatic oligo pool design that can generate mutagenic oligos for saturating or focused libraries.Protein engineering, design & selection : PEDSUser-defined single pot mutagenesis using unamplified oligo pools.PMC7212264T32 GM110523R01 AI141452T32-GM110523R01AI141452Borelli ANKirby MBWhitehead TAMedina-Cucurella AVSteiner PJBeltran JCutler SRFaber MSfalseUser-defined single pot mutagenesis using unamplified oligo pools.User-defined mutagenic libraries are fundamental for applied protein engineering workflows. Here we show that unamplified oligo pools can be used to prepare site saturation mutagenesis libraries from plasmid DNA with near-complete coverage of desired mutations and few off-target mutations. We find that oligo pools yield higher quality libraries when compared to individually synthesized degenerate oligos. We also show that multiple libraries can be multiplexed into a single oligo pool, making preparation of multiple libraries less expensive and more convenient. We provide software for automatic oligo pool design that can generate mutagenic oligos for saturating or focused libraries.2019-01-01T00:00:00Z2019 Sep2025-04-21T20:12:41.364Z2025-04-05T17:53:56.949ZS-EPMC72122643129752310.1093/protein/gzz013