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A Simple and Rapid Technique to Purify High-Quality Midiprep DNA


High-quality plasmid DNA for all demanding molecular biology applications

Scott Basehore Jeff Braman
Stratagene

A new technique is now available to quickly, easily, and reliably isolate high-purity plasmid DNA. The convenient spin-format eliminates the need for resin suspensions, gravity flow columns, and alcohol precipitations, while easily accommodating simultaneous multiple purifications. The purified DNA is ready for all applications, including sensitive automated sequencing and mammalian cell transfection.

StrataPrep midiprep kit purification of plasmid DNA1 is based on alkaline lysis of bacteria and binding of the liberated plasmid DNA to a glass fiber matrix contained in a midispin cup. Washing the spin cup removes contaminants, and the DNA is eluted in a small volume of buffer. The new midispin cup design and specially formulated alkaline lysis reagents allow up to 350 g of plasmid DNA to be recovered from a single device. The resulting DNA can be used for applications such as restriction digestion, cloning, PCR, site-directed mutagenesis, manual and automated fluorescent sequencing, and mammalian transfection.

Endotoxin, prevalent in plasmid preparations, is difficult to separate from plasmid DNA and results in toxicity to cultured mammalian cells.2 The StrataPrep EF midiprep protocol provides an optional extraction of endotoxins, which are reduced to 0.1 to 0.2 EU/g DNA (endotoxin units per microgram of plasmid DNA); hence, high-efficiency transfection results.

Fast and Easy Method

Fig.1

The StrataPrep midiprep procedure includes a unique color-indicating method that facilitates successful DNA purification (Figure 1). A modified alkaline lysis and centrifugation produces a cl
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