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

Abstract

AbstractBACKGROUND: 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. Copyright © 2007 Society of Chemical Industry