Making miniprep solutions for spin columns - Now that we can reuse the columns... (May/04/2007 )

Isolating plasmid DNA from bacteria is a fundamental step in molecular biology. It is often accomplished by an alkaline lysis of bacteria and the subsequent adsorption of nucleic acids to silica oxide in the presence of chaotropic substances. Here we show that the addition of such chaotropic reagents is not required for the efficient DNA isolation with silica oxide. This surprising finding allowed us to purify plasmid DNA with significantly less lipopolysaccharides (LPS), which is otherwise a common bacterial contaminant of silica oxide-isolated DNA and inhibits subsequent applications. In addition, we have implemented a precipitation step that altogether leads to a reduction of the LPS content by a factor of 900 relative to published methods. Our novel protocol facilitates an inexpensive high-throughput analysis of pure plasmids in a 96-well format without the addition of hazardous reagents.

We stepwise reduced the concentration of guanidine hydrochloride, but even when we excluded this chemical altogether, we did not observe a significant drop in the yield of plasmid DNA (Table 1). We also found that chaotropic substances are not required for DNA binding to other silica-based DNA adsorption matrixes such as commercially available spin columns (data not shown).
The DNA binding is dependent on the presence of silica oxide and potassium acetate in the reaction (data not shown). Possibly, the high potassium acetate concentration used to neutralize the alkaline lysis of bacteria is sufficient to facilitate the DNA binding to silica.