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- W4382182712 abstract "Construction of user-defined long circular single stranded DNA (cssDNA) and linear single stranded DNA (lssDNA) is important for various biotechnological applications. Many current methods for synthesis of these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology for generating user-defined cssDNA employing Golden Gate assembly, a nickase, and exonuclease degradation. Our technique is demonstrated for three plasmids with insert sizes ranging from 2.1 to 3.4 kb, requires no specialized equipment, and can be accomplished in 5 h with a yield of 33%–43% of the theoretical. To produce lssDNA, we evaluated different CRISPR-Cas9 cleavage conditions and reported a 52 ± 8% cleavage efficiency of cssDNA. Thus, our current method does not compete with existing protocols for lssDNA generation. Nevertheless, our protocol can make long, user-defined cssDNA readily available to biotechnology researchers." @default.
- W4382182712 created "2023-06-28" @default.
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- W4382182712 date "2023-06-27" @default.
- W4382182712 modified "2023-10-17" @default.
- W4382182712 title "A method for generating user‐defined circular single‐stranded DNA from plasmid DNA using Golden Gate intramolecular ligation" @default.
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- W4382182712 doi "https://doi.org/10.1002/bit.28471" @default.
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