138. Purification of Plasmid DNA on an Anion Exchange Column without the Use of RNase To Process Scale

• Published in: Molecular therapy Vol. 9; no. S1; pp. S53 - S54
• Main Authors: Pitarresi, Tina M, Herzer, Sibylle U, Kennedy, Bob, Moore, Peter
• Format: Journal Article
• Language: English
• Published: Milwaukee Elsevier Inc 01-05-2004; Elsevier Limited
• Subjects: Adenoviruses; Biodistribution; Gene expression; Gene therapy; Genomes; Lymphatic system; Plasmids; Spleen; Transgenic animals; Vectors (Biology)
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2004.06.493

We report here the use of a simple, straightforward RNase free method for the purification of plasmid DNA on ANX Sepharose 4 FF high sub exploiting the use of compacting agents and salts to improve selectivity, capacity and recovery. We have used generic procedures with standard protocols for alkaline lysis with commonly used buffers. The method was applicable to high and low copy number plasmids generated in different bacterial strains. A combination of isopropanol for compaction of supercoiled plasmid during adsorption and removal of residual RNA with ammonium acetate during a wash step allowed omission of RNA pretreatment. Material generated by this one column procedure was sufficiently pure to allow transfection of cell lines.