( ...)Efficient Recovery of Ultrapure Plasmid DNA

Nicole Passaretti, Vincent Prezioso, PhD, and Susanne Wagner
Nicole Passaretti, Vincent Prezioso, PhDBrinkmann Instruments, Inc., Westbury, NY
Susanne WagnerEppendorfNethelerHinz GmbH, Hamburg, Germany Plasmids are naturally occurring, extrachromosomal DNA in a bacterial cell, which can replicate independently but (usually) do not integrate into the host chromosome. Those used for molecular cloning have been artificially created from various natural plasmids. Plasmids have become the cornerstone of modern molecular biology research, making nucleic acid purification a standard application in most laboratories. It is with these standard methods that users are looking for high-quality and high quantity results obtainable with an easy-to-handle, reproducible method. Perfectprep Plasmid Kits easily meet these essential demands. These ready-to-use kits were designed to suit the throughput and DNA quantities required in the lab: from single purification in a spin column (ranging in size from Mini to XL) to the simultaneous isolation of 96 plasmid samples.

Handling

The general process of plasmid recovery and purification is optimized by the new purification kits. An improved alkaline lysis step is used for plasmid isolation, which enables efficient separation of the plasmid DNA from chromosomal DNA.

Fig. 1: Restriction digestion of 40 individual plasmids with EcoRI (Roche Molecular Biochemicals). Vector pVL1392; length: 9.6 kb; bacteria strain: XL-1 Blue; medium 2x YT. The plasmid DNA was separated using the Perfectprep Plasmid Mini Kit. Each digestion was carried out using 1 g DNA. 100 l was separated using 1% TAE agarose gel and stained with ethidium bromide. Gibco 1 kb ladder as standard; length: 9.6 kb. At the same time, RNA is degraded effectively by RNase A, which is included in the kits. Also, an enhanced DNA binding matrix1 was specially developed for these kits. In the presence of high concentrations of chaotropic salts, plasmid DNA selectively binds to the surface of the spin-column matrix, whereas polysaccharides and proteins show little or no affinity to the matrix at these salt concentrations. After only two washing steps, the pure plasmid DNA can thus be eluted from the silica matrix under low-salt conditions.2 With the Miniprep kit, the need for additional DNA purification via alcohol precipitation is eliminated, and the DNA is ready to be used for restriction digests, PCR,* or automated sequencing.

Advantages

In tandem with the combination of DNA binding matrix and chaotropic salts, the optimized lysis step maximizes the reproducibility of the results (Fig. 1). The DNA yields are as high as, and in fact usually markedly better than, those obtained with other kits on the market (Fig. 2). More than 95% of the extracted plasmid DNA is in the supercoiled form. Since the purity of the plasmid DNA is similar to DNA purified on a CsCI density gradient, it can be used directly for downstream applications, such as restriction digestion, cloning, transformation, DNA sequencing, or PCR. Even for the transfection of different cell lines (including CHO, COS1, COS7, NIH 3T3, HeLa, and HEK 293), the results obtained are excellent, and they are at least as good as those produced using endotoxin-free kits (Fig. 3).

Quality controls

To ensure that they always meet the requirements for reproducible quality and quantity, Eppendorf plasmid kits are subject to stringent quality-control procedures. Control plasmids (high-/low-copy) are isolated in accordance with protocol guidelines, and their quality and quantity is then determined using a variety of methods, including spectrophotometric assays,3 agarose gel electrophoresis, restriction digestion, and automatic fluorescence sequencing. Transfection of culture cells via calcium phosphate provides an additional, and important, proof of quality.

Fig. 2: The concentration of plasmid DNA obtained with the plasmid purification kit from Eppendorf and that obtained with a commonly available kit (mini- and maxi-prep scale). In each case, the concentration was determined spectrophotometrically. Midi-Prep 1 (25 ml preparation): Vector pEGFP-N1; length: 4.7 kb; bacteria strain: HB-101; Midi-Prep 2 (75 ml preparation): Vector pCH110; length: 7.128 kb; bacteria strain: JM-109; Maxi-Prep 1 (100 ml preparation): Vector pCH110; bacteria strain: JM-109; Maxi-Prep 2 (100 ml preparation): Vector pGEMgapdh; length: 2.7 kb; bacteria strain: HB-101. Each experiment was carried out twice. Fig. 3: Comparison between the transfection efficiency of the Maxi plasmid purification kit from Eppendorf (ENH) and that of a commonly available kit (A) and a kit that can be used to extract endotoxin-free DNA (B). Positive transfections were detected via FACS analysis. 100% indicates the total amount of living cells after transfection has taken place. Vector pEGFP; length: 4.7 kb; bacteria strain: JM-109. Each experiment was carried out twice. A: Transfection of COS 7 cells with lipofectamine reagent (Life Technologies). B: Transfection of COS 1 cells with a calcium phosphate transfection kit (Eppendorf 5-Prime). References

1. Marko MA, Chipperfield R, Birnboim HC. Anal Biochem. 1982;121:382-387.
   
2. Manchester KL. Biotechniques. 1995;19:208-210.
3. Wilfinger WW, Mackey K, Chomczynski P. Biotechniques. 1997;22:474-481.
   
   

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