Pronobis Mira I, Deuitch Natalie, Peifer Mark
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
Department of Biology, University of North Carolina at Chapel Hill, CB#3280, Chapel Hill, NC, United States of America.
PLoS One. 2016 Aug 3;11(8):e0160509. doi: 10.1371/journal.pone.0160509. eCollection 2016.
Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs.
质粒纯化是分子生物学家的一项基本工具。尽管利用硅胶离心柱的质粒分离试剂盒的开发减少了质粒纯化所需的时间和人力,但要获得大量质粒DNA产量仍需要大量时间和精力。在此,我们介绍Miraprep,这是一种快速方法,可使用商业小量制备试剂盒分离质粒DNA,但其DNA产量与商业大量制备质粒纯化相当。通过将乙醇沉淀与离心柱纯化相结合,我们创建了一种DNA分离方法,可在不到30分钟的时间内获得高浓度的质粒DNA样品。我们表明,Miraprep分离的质粒与通过标准程序分离的质粒一样稳定,可用于包括DNA测序在内的标准分子生物学程序,并且可以有效地转染到哺乳动物细胞中。这种新的质粒DNA分离方法将在不增加成本的情况下显著减少时间和人力。