Plasmid-DNA purification

Fig. 7. Unwrapped P-CAC cylinder showing the configuration for the Plasmid DNA purification...

Wizard SV 96 Plasmid DNA Purification System (Promega Corp., Madison, Wl, USA). 

For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to plasmid preparation using the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of resultant plasmid solution is applied to agarose gel electrophoresis to examine the yields of plasmids. 

Three to several colonies for each sample are inoculated into the same 600 pL of LB medium containing 50 pg/mL of kanamycin and cultured at 37°C overnight. For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to the plasmid preparation with the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of the resultant plasmid solution is directly applied to agarose gel electrophoresis for estimation of the size of plasmid in a form of covalently closed circular. Similarly, another 1 pL of the plasmid solution is treated with S fi/Pmel and analyzed by agarose gel electrophoresis for an insert size check. This plasmid solution set is the final product and is reserved in a freezer. 

Two microliters of pFlK-ORF plasmid solutions prepared with the Wizard SV 96 Plasmid DNA Purification System are treated with 5 U of S ifi and 5 U of Pmel in a 10 pL reaction volume containing lx Flexi Digest Buffer at 37°C for 60 min, and then the enzymes are inactivated by incubating at 65°C for 20 min. 

Clones with ampicillin resistance but without kanamycin resistance are examined with the CloneChecker System. By this antibiotic selection, the undesired vector heterodimer is removed. If both of the clones are false, additional clones are analyzed using the same protocol. When appropriate clones are obtained for almost all of the genes, they are inoculated from the ampicillin plates to prepare plasmids in a 96-well format by using the Wizard SV 96 Plasmid DNA Purification System see Note 6). 

The plasmid preparation using the Wizard SV 96 Plasmid DNA Purification System follows the protocol from Promega, except that TE buffer instead of H O is used for the final plasmid elution buffer. This alteration is applied to all the plasmid preparation processes in this chapter. 

Urthaler J, Schleg R, Podgomik A, Strancar A, Jungbauer A, and Necina R. Application of monoliths for plasmid DNA purification. Development and transfer to production. J. Chromatogr. A 2005 1065 93-106. 

SMB and column chromatography for plasmid DNA purification and Troger s base enantiomer separation Maximization of productivity and minimization of solvent consumption. NSGA Optimization results show that SMB is better tiian column chromatography for the two applications studied. Paredes and Mazzotti (2007)... 

Paredes, 1. and Mazzotti, M. (2007). Optimization of simulated moving bed and column chromatography for a plasmid DNA purification step and for a chiral separation, J. Chromatogr. A., 1142, pp. 56-68. 

Plasmid is routinely recovered from bacteria by an alkaline lysis procedure, which lyses the bacterial cell while maintaining bacterial DNA attachment to the cell wall. This procedure enables subsequent precipitation of bacterial DNA and cellular debris, leaving a crude preparation enriched in plasmid. We routinely use a plasmid DNA purification kit provided by Qiagen that utilizes the alkaline lysis method for harvesting, and anion exchange column chromatography for rapid purification. We refer the reader to the detailed instructions provided in the kit by the manufacturer, which we have not found necessary to modify for purification of laboratory-use plasmid DNA. 

Figure 8.3 shows the process flow sheet for large-scale purification of plasmid DNA. Table 8.14 presents a comparison of laboratory methods and laige-scale pharmaceutical processes for plasmid DNA purification. 

Table 8.15. The purification of clinical-grade plasmid DNA Purification strategy [Refs in 415].

M. Muller, Considerations for the scale-up of plasmid DNA purification in Nucleic Acid Isolation Methods Eds. B. Bowlen, P. Dtirre), American Scientific Publishers, New York, 2003. 

Shao D, Xia A, Hu J, et al. (2008) Monodispersed magnetite/silica composite microspheres preparation and application for plasmid DNA purification. Colloid Surf A Physicochem Eng Asp 322 61-65... 

Harvest cells and extract DNA using a plasmid DNA purification kit (see Note 9). 

The DNA plasmid is transformed into E. coli strain DH5a using the well-established heat-shock method (16). Then, plasmid DNA is purified using QIAprep kit for plasmid DNA purification (QIAGEN) following manufecturer s instructions. 

Introduction Advances in Plasmid DNA Purification Advances in Electrophoretic DNA Separations Advances in Affinity-Based DNA and RNA Recovery Conclusions References... 

Horn, N.A. et al.. Cancer gene therapy using plasmid DNA Purification of DNA for human clinical trials. Human Genetic Therapy, 1995 6(5) 565-573. 

Pereira, L.R., D.M.F. Prazeres, and M. Mateus, Hydrophobic interaction membrane chromatography for plasmid DNA purification Design and optimization. Journal of Separation Science, 2010 33(9) 1175-1184. 

Barbosa, H.S.C. et al.. Dual affinity method for plasmid DNA purification in aqueous two-phase systems. Journal of Chromatography, A, 2010 1217(9) 1429-1436. 

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