Neudecker F, Grimm S
Max-Planck-Institute for Biochemistry, Martinsried, Germany.
Biotechniques. 2000 Jan;28(1):107-9.
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.
从细菌中分离质粒DNA是分子生物学中的一个基本步骤。通常通过对细菌进行碱裂解，然后在离液剂存在的情况下使核酸吸附到氧化硅上来完成。在这里我们表明，使用氧化硅高效分离DNA时不需要添加此类离液剂。这一惊人发现使我们能够纯化出脂多糖（LPS）含量显著更低的质粒DNA，否则LPS是氧化硅分离DNA时常见的细菌污染物，会抑制后续应用。此外，我们实施了一个沉淀步骤，总体上使LPS含量相对于已发表方法降低了900倍。我们的新方案有助于以96孔板形式对纯质粒进行廉价的高通量分析，且无需添加危险试剂。
