Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

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Abstract

The primary purification of a 6.1kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA.

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Subject classification (UKÄ) – MANDATORY

  • Biological Sciences
  • Biochemistry and Molecular Biology

Keywords

  • Escherichia coli, Aqueous two-phase systems, Thermoseparation, DNA
Original languageEnglish
Pages (from-to)95-104
JournalJournal of Chromatography A
Volume1024
Issue number1-2
Publication statusPublished - 2004
Publication categoryResearch
Peer-reviewedYes