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Efficient Recovery of Ultrapure Plasmid DNA

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 a
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