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

d DNA was tested in automated fluorescent sequencing. Plasmid DNA purified by a StrataPrep standard midiprep, a StrataPrep endotoxin-free midiprep, twice-banded CsCl density gradient centrifugation, and anion exchange methods were sequenced at Sequetech Corporation. High-signal, low-background sequence data was obtained with all four plasmid preparations. Figure 4 shows representative sequence data for StrataPrep endotoxin-free midiprep purified DNA. Sequence read length for the four templates was approximately 680 bases. There were a number of sequence ambiguities generated from each template, starting arbitrarily from nucleotide 20: one for a StrataPrep midiprep standard, one for a StrataPrep midiprep endotoxin-free, zero for CsCl density gradient centrifugation, and four for anion exchange.

Figure 4

Increased Transfection Efficiency

Endotoxins are toxic to mammalian cell cultures and can greatly affect transfection efficiencies. The StrataPrep midiprep endotoxin-free extraction protocol reduces endotoxin levels to between 0.1 to 0.2 EU/g plasmid DNA. LipoTAXI transfection reagent3,4 and calcium phosphate5 reagent methods were used to transfect StrataPrep endotoxin-free purified DNA into Cos-7, DU 145, and JAR cell lines and resulted in higher transfection efficiencies, compared to anion exchange enndotoxin-free purified plasmid DNA (Figure 5).

Figure 5

Supplemental Components

Endonuclease A removal buffer is available separately when purifying plasmid DNA from End A+ bacterial strains such as JM101. A simple wash step remov
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