Increased yield of plasmid DNA during removal of CsCl by ethanol precipitation.

Our laboratory is interested in intramuscular injection of plasmid DNA as a method of eliciting immune responses. This requires large-scale preparation of plasmid DNA because 4–8 mg of plasmid DNA can be required for a single experiment. Several techniques are available for large-scale preparation of plasmid DNA, including ion-exchange resin and CsCl gradient centrifugation (1,2). We have been purifying plasmid DNA using CsCl gradient centrifugation and have found that an additional centrifugation step, during removal of CsCl, increases the DNA yield at least twofold. Following the protocol established by Maniatis et al. (2), we purified the plasmid DNA on CsCl gradients, extracted the ethidium bromide (EtdBr) with isoamyl alcohol and removed the CsCl by adding 3 vol of H2O (to dilute the CsCl) and 2 vol of 100% ethanol (to precipitate the DNA) as described. While this will precipitate some of the plasmid DNA, we have found that a significant proportion of it is retained in the supernatant. We added 2 vol of 70% ethanol to the decanted supernatant to determine whether we could recover any of the residual plasmid DNA. By spectrophotometric analysis, we observed that we had isolated an additional 17 mg of plasmid DNA with a high level of purity (Table 1). To determine if the material was similar to the plasmid DNA isolated in the first precipitation, we subjected both plasmid DNAs to restriction enzyme digestion and analysis by gel electrophoresis. As shown in Figure 1, cutting the vector pcDNA-IL-12p40 with BglII and XhoI (Promega, Madison, WI, USA) generated an expected band of approximately 1.9 kb from both the first and second precipitations of plasmid DNA (lanes 2 and 4, respectively). In addition, uncut plasmid DNA from both precipitations generated similar bands after gel electrophoresis (lanes 1 and 3, respectively). We have also performed this procedure with another vector (pcDNA3; Invitrogen, San Diego, CA, USA) and obtained similar results; 5 mg were recovered in the first precipitation and 11 mg were recovered in the second precipitation. We next determined whether