Laboratory of Protein Dynamics and Signaling, National Cancer Institute, National Institutes of Health, NCI-Frederick, Frederick, Maryland 21702, USA.
Biotechnol Bioeng. 2011 Aug;108(8):1872-82. doi: 10.1002/bit.23116. Epub 2011 Mar 17.
Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.
离子交换色谱法是质粒 DNA 纯化的标准技术,是一项重要的分子生物学操作。非离子型去污剂 (NIDs) 已被用于质粒 DNA 纯化,但尚不清楚霍夫迈斯特盐序 (HSS) 是否会改变特定 NIDs 的溶解度和相分离特性,从而增强质粒 DNA 的纯化效果。在对 NID 介导的质粒 DNA 分离进行放大后,我们发现 HSS 溶液中的 NIDs 可最大程度减少质粒 DNA 与蛋白质的污染。此外,大规模的 NID/HSS 溶液比 Qiagen 离子交换柱更有效地去除质粒 DNA 中的脂多糖 (LPS) 污染。与碱分离/柱纯化相比,大规模 NID 分离/NID 纯化可提高高质量 DNA 的产量。这项工作描述了 HSS 如何增强 NID 介导的质粒 DNA 纯化,并证明 LPS 从质粒 DNA 中去除不需要 NID 相转变。特定的 NIDs,如 IGEPAL CA-520,可以用于快速、廉价且高效的基于实验室的大规模质粒 DNA 纯化,优于基于 Qiagen 的柱程序。