Plasmid Production Protocol
The lysate was then pumped to a filter train consisting of the Pall PreflowTM 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).