Zhu Kaichun, Jin Huali, He Zhonghuai, Zhu Qinghong, Wang Bin
The People's Hospital of Xinjiang Uygur Autonomous Region, 91 Tianchi Road, Urmuqi, Xinjiang 830001, China.
Nat Protoc. 2006;1(6):3088-93. doi: 10.1038/nprot.2006.452.
This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 degrees C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l(-1) culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA.
本方案描述了一种通过连续热裂解提取质粒DNA的简化方法，该方法是对基本煮沸裂解技术的改进，用于简化大量大肠杆菌培养物的处理。使用中空纤维膜组件收获发酵细菌，并在通过设置为70℃的热交换盘管裂解细胞之前用溶菌酶进行预处理，然后进入冰上并列的冷却盘管。随后通过离心将裂解并冷却的细菌与裂解物分离，并从上清液中沉淀质粒DNA以进行进一步纯化。使用蠕动泵和两个恒温加热盘管而不使用离心，使裂解过程变得恒定且可控，提供了一种用于细胞裂解和质粒DNA提取的连续流程方案。2小时内可处理大量细菌培养物（20升），每升培养物可产生约100毫克质粒DNA，这使得该方案成为一种有吸引力的用于一致且大规模制备质粒DNA的方法。
