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Aurum Plasmid 96 Kit Tech Note, Rev A

Neil Duldulao, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA


Introduction
E. coli bacteria have long been used as hosts for plasmid propagation. Several methods have been used for preparing the plasmid, including alkaline lysis of the bacteria and alcohol precipitation of the DNA (Birnboim and Doly 1979). Once DNA had been observed to bind to glass (silicon dioxide) in the presence of chaotropic agents such as sodium iodide and sodium perchlorate, plasmid purification using glass powders and diatomaceous earth began (Vogelstein and Gillespie 1979, Yang et al. 1979, Marko et al. 1982). Typically, plasmid purification kits use alkaline lysis followed by guanidinium salts to bind the plasmid DNA to glass membranes. However, genomics laboratories with automated fluorescence-based sequencing systems have created a demand for high-throughput plasmid purification in a 96-well format. Accordingly, high-throughput plasmid purification kits must take into account the following criteria: sample purity, mass yield, concentration, extent of handling, and cost per sample. The plasmid samples must also give useful sequencing data from an automated fluorescent DNA sequencer. A total of six 96-well plasmid purification kits from various manufacturers were assessed using these criteria (Table 1).


Methods
Bacterial Propagation
E. coli strain DH5α (Invitrogen) containing plasmid pCMV (Stratagene) was streaked out on an LB-ampicillin (100 g/ml) agar plate and incubated at 37C overnight. The following day, 20 ml LB-ampicillin medium in a 50 ml centrifuge tube was inoculated with one bacterial colony from the
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