Abstract
A variety of applications require the creation of custom-designed plasmids, including transgenic reporters, heterologous gene fusions, and phenotypic rescue plasmids. These plasmids are created traditionally using restriction digests and in vitro ligation reactions, but these techniques are dependent on available restriction sites and can be laborious given the size and number of fragments to be ligated. The baker’s yeast Saccharomyces cerevisiae provides a powerful platform to create nearly any plasmid through PCR-directed yeast-mediated ligation. This technique can ligate complex plasmids of up to 50 kilobasepairs (kb) in vivo to produce plasmids with precisely defined sequences.
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Acknowledgments
The author would like to thank Dr. Leonid Kruglyak for financial support and laboratory space. Additionally, Amy Caudy, Justin Gerke, and Robyn Tanny for many helpful comments. This protocol was adapted from one used by the laboratory of Dr. Jay Dunlap (Dartmouth Medical School) and communicated to the author by Dr. Allan Froelich. Many others have contributed to protocols and studies using yeast to create custom vectors and some of whom are referenced below. The author would like to apologize for any omissions of published works or protocols relevant to these studies. E.C.A. is supported by a Ruth L. Kirschstein National Research Service Award from the National Institutes of Health.
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Andersen, E.C. (2012). PCR-Directed In Vivo Plasmid Construction Using Homologous Recombination in Baker’s Yeast. In: Orgogozo, V., Rockman, M. (eds) Molecular Methods for Evolutionary Genetics. Methods in Molecular Biology, vol 772. Humana Press. https://doi.org/10.1007/978-1-61779-228-1_24
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DOI: https://doi.org/10.1007/978-1-61779-228-1_24
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