Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation.

Autor: Wagner A; Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal., Silva-Santos AR; Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal., Rosa SS; Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal., Gierak S; Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.; Institut National des Sciences Appliquées de Rouen, Saint-Étienne-du-Rouvray, France., Azevedo AM; Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal., Prazeres DMF; Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. miguelprazeres@tecnico.ulisboa.pt.
Jazyk: angličtina
Zdroj: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2021; Vol. 2197, pp. 151-165.
DOI: 10.1007/978-1-0716-0872-2_8
Abstrakt: 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%).
Databáze: MEDLINE
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