On the application of ‘seeding’ techniques in the primary separation of plasmid DNA from neutralisedE. coli lysates

Eirini Theodosiou, Owen Thomas

Research output: Contribution to journalArticle

3 Citations (Scopus)


BACKGROUND: Initial extraction of plasmid DNA from Escherichia coli and its separation from host-derived contaminants is a difficult task to perform. Here, we examine the application of particle 'seeding' solid-liquid separation methods for primary recovery of plasmid DNA from neutralised alkaline cell lysates. RESULTS: Planting magnetic particle 'seeds' during cell lysis resulted in enhanced phase separation, facile magnetic separation of the floc, slight improvements in plasmid purity, but diminished plasmid recoveries. When CaCO3-coated low-density microspheres were seeded into flocs, phase separation was impaired, shear-induced floc damage and contamination of the plasmid liquor with genomic DNA and cell debris occurred, but plasmid DNA recovery was improved. Introduction of hydrophobic low-density microspheres into the floc dramatically improved floc stiffness, phase separation and flotation efficiency, and reduced the solids content in the plasmid liquor 10-fold. However, strong reinforcement of the cell debris lattice by these microspheres hindered plasmid release into the liquor beneath. CONCLUSION: By incorporating magnetic or buoyant seeds during cell lysis we have identified new routes for separation of shear-sensitive cell debris solids from crude plasmid-containing liquors. Effective use of seeding approaches for difficult solid-liquid separation tasks will require evaluation of a wide range of seeds of varying architecture, size, shape, density and chemistry. (c) 2007 Society of Chemical Industry.
Original languageEnglish
Pages (from-to)192-200
Number of pages9
JournalJournal of Chemical Technology and Biotechnology
Issue number2
Early online date1 Jan 2008
Publication statusPublished - 1 Feb 2008


  • alkaline lysis
  • gene therapy
  • flotation
  • low-density gas-filled microspheres
  • genetic vaccination
  • magnetic particle seeds
  • fillers


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