Nicole Passaretti, Vincent Prezioso, PhD, and Susanne Wagner
Nicole Passaretti, Vincent Prezioso, PhD—Brinkmann Instruments, Inc.,
Westbury, NY
Susanne Wagner—Eppendorf–Netheler–Hinz GmbH, Hamburg, Germany
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| 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.
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| 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.
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