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A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

机译:一步纯化质粒DNA和Lambda噬菌体颗粒的小量制备

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摘要

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E.coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.
机译:质粒DNA小量制备是分子生物学中的基本技术。当前的质粒DNA小量制备使用三溶液形式的碱和阴离子去污剂SDS。此外,碱微量制备通常需要额外的基于柱的纯化步骤,并且不能分离其他染色体外元素,例如噬菌体。非离子型去污剂(NIDs)有时已用作多溶液质粒DNA微量制备液的成分,但尚未开发出一步法。在这里,我们建立了一个单管,一溶液的NID质粒DNA miniprep,并且我们证明了该方法还可以分离噬菌体的lambda颗粒。 NID小量制备比碱小量制备更省时,NID质粒DNA在许多下游应用中的性能优于碱DNA。实际上,NID粗裂解物DNA足够纯净,可用于消化和测序反应。显微镜分析表明,NID程序将大肠杆菌细胞破碎成小的原生质体样成分,这至少可以部分解释这种方法的有效性。这项工作表明一步NID小量制备是生成高质量质粒DNA的可靠方法,NID方法还可以分离噬菌体λ颗粒,其性能优于目前的标准碱基小量制备。

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