Abstract
A method for the intermediate recovery of plasmid DNA (pDNA) from alkaline lysates is described that comprises differential isopropanol precipitation steps. In a first low-cut precipitation, a smaller amount of isopropanol (20% v/v) is used so that only high molecular weight RNA precipitates. After solid liquid separation, a high-cut precipitation is performed by bringing isopropanol concentration to 70% v/v to precipitate pDNA. Tests made with lysates show that the differential precipitation increases purity threefold compared to the conventional one-step precipitation at 70% v/v without affecting pDNA recovery (>80%).
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Acknowledgments
Funding received by iBB-Institute for Bioengineering and Biosciences from FCT-Portuguese Foundation for Science and Technology, Portugal (grant UID/BIO/04565/2019), the research project PureMab (PTDC/QEQ-PRS/0286/2014), and from Programa Operacional Regional de Lisboa 2020, Portugal (Project N. 007317) is acknowledged. A. Rita Silva-Santos acknowledges FCT for the PhD grant PD/BD/135138/2017 (BIOTECnico program).
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Wagner, A., Silva-Santos, A.R., Rosa, S.S., Gierak, S., Azevedo, A.M., Prazeres, D.M.F. (2021). Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation. In: Sousa, Â. (eds) DNA Vaccines. Methods in Molecular Biology, vol 2197. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0872-2_8
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DOI: https://doi.org/10.1007/978-1-0716-0872-2_8
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