Contaminant eluted from solid-phase plasmid affinity-purification protocolcolumns is not found using liquid-phase methods and can be prevented

The preparation of high quality plasmid DNA is a necessary requirement for most molecular biology applications. We compared four different large plasm id preparation protocols, which were based on either a liquid-phase approac h (Triton lysis) or purification of alkaline lysis bacterial extracts follo wed by supercoiled plasmid purification on affinity columns. Two host Esche richia coli strains, JM 109 and INV alpha LF', were used to grow the test p lasmids for comparison of product plasmid DNA produced from the four differ ent plasmid isolation methods. While the DNA grown in E. coli strain JM109, prepared by liquid-phase Triton lysis was appropriately restricted by 12 r estriction enzymes, this was not the case for any of the JM109-grown DNA pu rified by any of the affinity column solid-phase approaches. In contrast to this, when the plasmid DNA was grown in E. coli strain INV alpha F', most restriction enzymes cut DNA appropriately, irregardless of the plasmid prep aration protocol used. It seems that an impurity commonly eluted with the D NA from all three of the solid-phase DNA columns had an equal effect on the above enzymes using the common host strain JM109, but not strain INV alpha F'. (C) 1999 Elsevier Science B.V. All rights reserved.