Plasmid DNA Preparation

As mentioned earlier, plasmid DNA is present as small supercoiled circular double-stranded DNA in bacteria. In this conformation, plasmid DNA is more resistant to alkaline denaturation than is host genomic DNA. Hence disruption of cells bearing plasmid DNA followed by the addition of alkali and subsequent neutralization and centrifugation leads to the precipitation of denatured genomic DNA and proteins, whereas plasmid DNA remains in the solution [7], Plasmid DNA can be recovered from the supernatant by ethanol precipitation and may be further purified [3,4], 

In order to grow bacteria for plasmid DNA isolation, typically, a single bacterial colony is inoculated into 5 mL of LB medium. If the plasmid codes for antibiotic resistance, that antibiotic is added to the LB medium. The bacterial culture is grown to saturation overnight at 37°C on a shaker. The cells are centrifuged in 1.5-mL Eppendorf tubes in microcentrifuge for 2 minutes at maximum speed. After removal of the supernatant, the pellet is resuspended in 100 pL of GTE solution (50 mM glucose/25 mMTris-Cl, pH 

0/10 mM EDTA). After the addition of 200 pL of NaOH/SDS solution (0.2 M NaOH/1% SDS), the sample is mixed by inversion several times. After 5 minutes, 150 pL of 3 M potassium acetate solution (pH 4.8) is added, mixed by inversion several times, and incubated in ice for 5 minutes. After centrifugation for 5 minutes in microcentrifuge at 14,000 x g for 5 minutes, the clear supernatant is extracted with phenol, phenol-chloroform, and chloroform. The plasmid DNA is then precipitated after the addition of 2 volumes of ethanol. After incubation at room temperature for 5 minutes, the DNA is centrifuged in microcentrifuge by centrifugation for 10 minutes at room temperature. The pellet is then washed with 70% ethanol and dried under vacuum. 

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The plasmid DNA prepared as described above still has some broken RNA fragments and polysaccharides and may contain some bacterial DNA. Various approaches exist [3,4] for the purification of plasmid DNA. 

Davis, H. L., Schleef, M Moritz, P Mancini, M., Schorr, J., and Whalen, R. G. (1996) Comparison of plasmid DNA preparation methods for direct gene transfer and genetic immunization. Biotechnology 21(1), 92-99. 

Cotten, M., Baker, A., Saltik, M Wagner, E., and Buschle, M. (1994) Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus. Gene Ther. 1,239-246. 

Five colonies for each UGTIAI variant are picked at random and plasmid DNA prepared. The insert DNA is then sequenced on both strands. DNA verified as correct is then subcloned into pcDNA3.1(-) as described previously. 

Generally, the volume of DNA should not exceed 10% of the volume of competent cells added. If it does, then lOX LiAc/TE should be added so that the transformation reaction is maintained in a IX LiAc/TE buffer. We have found that for most routine experiments, crude miniprep-DNA prepared with standard alkaline lysis protocols works well in this transformation procedure However, if the highest efficiencies are required, one should use more purified plasmid DNAs preparations (e g, cesium banded or Qiagen columns)... 

Positive colonies picked into 15-mL polypropylene tubes containing 3-4 mL LBamp can be grown up and plasmid DNA prepared using a variety of mmi-preparation protocols This DNA will be used m subsequent experiments as it represents a single, homogeneous, DNA fragment... 

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

Prazeres, D.M.F., Schluep, T., and Cooney, C., Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography, ]. Chromatogr. A, 806, 31, 1998. 

Southern hybridization experiments, employing the cloned PHA synthase structural gene of Acinetobacter sp. and sucrose gradient fractions of DNA preparations separated in plasmid and chromosomal DNA fractions gave two hybridization signals and revealed some but not yet conclusive evidence for... 

Blanche et al. [45] showed that the P-CAC technology is very promising for the purification of Plasmid DNA at preparative scale especially when resins with low binding capacities for the product of interest are used. The aim of the study was to purify the Plasmid DNA out of a clear lysate of E. coli. The lysate containing RNA, nicked DNA, as well as the Plasmid DNA was loaded onto the annular column filled with Poros 20 R2 beads as the stationary phase. The chromatographic process for the purification is shown in Fig. 7. 

An additional opportunity that arises from applying polymer-based materials is the preparation of water-dispersible composites, which is an essential feature for biomedical purposes, as it is possible to attach bio-active molecules to the poly-mer/CNTs systems and specifically deliver them to cells. In this manner, plasmid DNA, siRNA (Fig. 3.11) and several anticancer agents have been successfully bound and delivered [61]. The stratagem to generate materials with good solubility in aqueous media usually involves the presence of water-soluble polar groups (e.g. phosphates, protonated amines etc.) embedded in the polymer chain. 

Entrapment of plasmid DNA and/or protein into liposomes entails the preparation of a lipid film from which multilamellar vesicles and, eventually, small unilamellar vesicles (SUVs) are produced. SUVs are then mixed with the plasmid DNA and/or protein destined for entrapment and dehydrated. The dry cake is subsequently broken up and rehydrated to generate multilamellar dehydration-rehydration vesicles (DRV) containing the plasmid DNA and/or protein. On centrifugation, liposome-entrapped vaccines are separated from nonentrapped materials. When required, the DRV are reduced in size by microfluidization in the presence or absence of nonentrapped materials or by employing an alternative method (7) of DRV production, which utilizes sucrose (see below). 

Quantitative entrapment of vaccines into small (up to about 200 nm diameter) liposomes in the absence of microfluidization (which can damage DNA and other labile materials when extensive) can be carried out by a novel one-step method (7) as follows SUVs (e.g., cationic) prepared as in section Preparation of Small Unilamellar Vesicles are mixed with sucrose to give a range of sucrose-to-lipid weight/weight ratio of 1.0 to 5.0 and the appropriate amount of plasmid DNA (e.g., 10-500 pg) and/or protein (e.g., up to 1 mg). The mixture is then rapidly frozen and subjected to dehydration by freeze-drying, followed by rehydration as in section Preparation of Vaccine-Containing Dehydration-Rehydration Vesicles. ... 

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