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Title: On the application of 'seeding' techniques in the primary separation of plasmid DNA from neutralised E-coli lysates
Authors: Theodosiou, Eirini
Thomas, Owen R.T.
Keywords: Alkaline lysis
Gene therapy
Genetic vaccination
Magnetic particle seeds
Low-density gas-filled microspheres
Issue Date: 2008
Publisher: John Wiley and Sons Ltd (© Society of Chemical Industry)
Citation: THEODOSIOU, E. and THOMAS, O.R.T., 2008. On the application of 'seeding' techniques in the primary separation of plasmid DNA from neutralised E-coli lysates. Journal of Chemical Technology and Biotechnology, 83 (2), pp.192-200.
Abstract: 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 ten-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.
Description: This is a preprint of an article published in the Journal of Chemical Technology and Biotechnology at http://onlinelibrary.wiley.com/doi/10.1002/jctb.1834/abstract.
Version: Accepted for publication
DOI: 10.1002/jctb.1834
URI: https://dspace.lboro.ac.uk/2134/12016
Publisher Link: http://dx.doi.org/10.1002/jctb.1834
ISSN: 0268-2575
Appears in Collections:Published Articles (Chemical Engineering)

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