Combining oligo pools and Golden Gate cloning to create protein variant libraries or guide RNA libraries for CRISPR applications
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arXiv
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| Autori principali: | , , , |
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| Natura: | Preprint |
| Pubblicazione: |
2024
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| _version_ | 1866914647328686080 |
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| author | Valero, Alicia Maciá Prins, Rianne C. de Vroet, Thijs Billerbeck, Sonja |
| author_facet | Valero, Alicia Maciá Prins, Rianne C. de Vroet, Thijs Billerbeck, Sonja |
| contents | Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. 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 alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries. |
| format | Preprint |
| id |
arxiv_https___arxiv_org_abs_2401_11746 |
| institution | arXiv |
| publishDate | 2024 |
| record_format | arxiv |
| spellingShingle | Combining oligo pools and Golden Gate cloning to create protein variant libraries or guide RNA libraries for CRISPR applications Valero, Alicia Maciá Prins, Rianne C. de Vroet, Thijs Billerbeck, Sonja Quantitative Methods Biomolecules Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. 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 alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries. |
| title | Combining oligo pools and Golden Gate cloning to create protein variant libraries or guide RNA libraries for CRISPR applications |
| topic | Quantitative Methods Biomolecules |
| url | https://arxiv.org/abs/2401.11746 |