Plasmid DNA small-scale preparation based on nuclease inactivation by diethylpyrocarbonate

Abstract

▼For several years now, we have been using a modification of the rapid boiling method (Ref. 1) as a small-scale plasmid preparation procedure. Our method requires a short exposure of bacterial cells to lysozyme to weaken their cell walls, irreversible inactivation of nucleases by diethylpyrocarbonate (Fluka), heating at 70◦C to partially denature genomic bacterial DNA (and proteins) and elimination by sedimentation of the insoluble cloud that is formed upon cooling. Plasmid DNA remains in solution and is recovered by ethanol precipitation. The yield of plasmid DNA is within the range of that obtained using alternative methods (Ref. 1, 2): 10−30 μg of plasmid DNA [pBluescript SK+ plasmid (Stratagene), 25 ± 3 μg DNA; pUC, 30 ± 5 μg DNA; and pUR, 10 ± 1 μg DNA] is obtained from 1.5 ml of Escherichia coli overnight culture in Luria Broth (LB) medium. An important and distinct feature of our method is the use of diethylpyrocarbonate (DEPC) to inactivate nucleases. A titration curve for DEPC against the amount of plasmid DNA recovered is shown (Fig. 1). The yield of plasmid DNA increases with DEPC concentration up to 0.1−0.2% but there is no further increase in plasmid DNA recovery with higher DEPC concentrations. We have also determined that incubation for 5 min at 70◦C in presence of DEPC is enough to recover acceptable amounts of plasmid DNA, regardless of the type of plasmid (data not shown). Most of the plasmid DNA prepared by the DEPC method is in the supercoiled form (>90%) rather than nicked cir-