Plasmid Production Protocol

About 2 kg of frozen cell paste of E. coli cells containing a 4.5 kb reporter plasmid, pCAT was thawed and resuspended in 15 liters of 50mM Tris/HCl buffer pH 8.0 containing 10mM EDTA. The cells in suspension were lysed by adding an equal volume of 0.2N NaOH in 1% SDS and gently mixed. After five minutes, two volumes of an unbuffered solution of 7M ammonium acetate in 1M potassium acetate at 4oC was added to each bottle and again gently mixed until a uniform white gelatinous precipitate formed. The lysate was then held overnight at 4oC.

The lysate was then pumped to a filter train consisting of the Pall Preflow^TM capsule (0.45µm) and the Pall Supor® EKV, which is described in Figure 1. Following filtration, the lysate was pooled in a hold tank and diluted with 1.5 volumes of 18 MΩ water. It was mixed thoroughly until the conductivity readings were between 80-85 mS/cm to prevent shear to chromosomal DNA.

The Q anion-exchange capsule (260mL) was equilibrated with 0.5M NaCl in 10mM Tris/HCl buffer pH 8.0 and 0.1mM EDTA at pH 8.0 at 4.1L min-1(~16MV min-1). The diluted lysate (71 liters) was loaded onto the capsule at 4.1L min-1(~16MV min-1), whereby effluent from the capsule was continuously monitored at 260nm and 280nm.

The bound plasmid was eluted from the membrane with 10mM Tris/HCl buffer pH 8.0 containing 1.2M NaCl and 0.1mM EDTA, and the entire peak was collected.

(From: Pora, H. 2005. Efficient Plasmid Purification Strategies Simplify Scale Up of Gene Therapy and DNA Vaccines. Genetic Engineering News, in Press as at April 2005).