产地：QIAGEN
货号：12362
名称：EndoFree Plasmid Maxi Kit
Procedure
Bacterial cells are lysed under alkaline conditions and the crude lysates are cleared using the QIAfilter Maxi Cartridge. At this stage, the Endotoxin Removal Buffer is added to the filtered lysate, which is incubated on ice. The cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and ultrapure plasmid DNA is eluted in high-salt buffer (see flowchart "Comparison of QIAGEN Plasmid Kit Procedures"). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.EndoFree Plasmid Maxi Kit
Principle
The level of endotoxin contamination in purified plasmid DNA depends on the purification method used (see table "Endotoxin levels in plasmid preparations"). Silica-slurry–purified DNA exhibits extremely high endotoxin levels. QIAGEN, QIAfilter, and HiSpeed Plasmid Kits and 2x CsCl ultracentrifugation yield very pure DNA with relatively low levels of endotoxin. EndoFree Plasmid Kits include an integrated endotoxin-removal step to yield plasmid DNA containing <0.1 EU/µg plasmid DNA.EndoFree Plasmid Maxi Kit
QIAfilter Cartridges, provided in QIAfilter, HiSpeed, and EndoFree Plasmid Kits, are special filter units designed to replace centrifugation following alkaline lysis of bacterial cells. QIAfilter Cartridges completely remove SDS precipitates and clear bacterial lysates in a fraction of the time needed for centrifugation, reducing plasmid-purification time by up to 1 hour. QIAfilter Midi and Maxi Cartridges have a syringe-format (see figure "QIAfilter Cartridge") and lysates are cleared in a matter of seconds by pushing the liquid through the filter.
The unique anion-exchange resin in QIAGEN-tips is developed exclusively for the purification of nucleic acids. Its exceptional separation properties result in DNA purity equivalent or superior to that obtained by two successive rounds of CsCl gradient centrifugation. Pre-packed QIAGEN-tips operate by gravity flow and never run dry, minimizing the hands-on time required for plasmid preparation. The entire QIAGEN plasmid purification system avoids the use of toxic substances such as phenol, chloroform, ethidium bromide, and CsCl, minimizing hazard both to the user and the environment.
Applications
EndoFree Plasmid Kits yield endotoxin-free DNA which is ideal for high reproducibility and efficiency in plasmid-mediated gene silencing, transfection (see figures "Plasmid Purification Method vs. Transfection Efficiency" and "Plasmid Purity vs. Transfection Efficiency" and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). QIAGEN ultrapure endotoxin-free DNA is also suitable for gene therapy research and genetic vaccination (see "Ultrapure 100 Column") and other sensitive applications.
EndoFree Plasmid Maxi Kit
纯化得到多至 500 ug无内毒素高转染级纯质粒或科斯质粒DNA
纯化得到的DNA 内毒素低于0.1 EU/µg
包括LyseBlue 试剂优化操作，确保产量
获得高产量的高拷贝质粒DNA
使用LyseBlue，获得最佳的裂解和最大的DNA产量
EndoFree Plasmid Maxi Kit 将高效去除内毒素整合入质粒纯化，无需额外的抽提或亲和层析去除脂多糖。通过QIAfilter Mega-Giga预制试剂条过滤清楚细菌裂解液，在重力流QIAGEN-tip 500 阴离子交换柱上纯化质粒,EndoFree Plasmid Maxi Kit 规格
背景
内毒素，也称作脂多糖或LPS，使革兰氏阴性菌的细胞膜组分，如 E. coli （见图“E.coli 细胞膜图示”）。在抽提质粒过程中的裂解阶段会释放内毒素，它会降低敏感细胞系的转染效率（见图“质粒纯化方法 vs. 转染效率”和 “质粒纯化 vs. 转染效率”及表格“质粒制备的内毒素水平”和“用EndoFree 抽提的 DNA 确保原代细胞高效转染”）。此外，内毒素可以影响转染过程中质粒DNA的吸收， 这是由于它会与DNA竞争转染试剂。内毒素也会激活诸如巨噬细胞和B细胞等免疫细胞中的非特异性免疫反应，导致错误干扰转染结果。这些反应包括合成蛋白和脂质体，如 IL-1 和前列腺素。综上所述，内毒素是转染实验构建中一种不可控制的变量，可影响转染结果的成功率和可重复性，致使转染实验难以比较和说明。在基因治疗研究中，内毒素会引起内毒素休克综合症、促动补体分支，从而造成干扰。
原理
抽提所得质粒DNA的内毒素水平取决于纯化方法（见表格“质粒的内毒素水平”）。泥浆式硅胶法制备的DNA的内毒素水平非常高。QIAGEN、QIAfilter 和HiSpeed Plasmid Kits 以及 2x CsCl超离心法可制备高纯度的DNA，内毒素水平非常低。EndoFree Plasmid Kits 包含整合的内毒素去除步骤，可制备内毒素 <0.1 EU/µg的质粒DNA。
QIAfilter、HiSpeed 和 EndoFree Plasmid Kits中提供的QIAfilter Cartridges是一种独特设计的过滤器，可代替细菌细胞碱裂解后的离心步骤。QIAfilter Cartridges 可完全去除SDS沉淀，清除细菌裂解液，相比离心可节约很多时间。 QIAfilter Midi 和 Maxi Cartridges 是注射器规格（见图“QIAfilter Midi Cartridge”）通过推动可使液体通过过滤器。
IAGEN纯化柱使用独特的阴离子交换树脂，专用于纯化核酸。创新的分离技术确保DNA纯度等同于甚至高于两次 CsCl梯度离心法制备的DNA。 预装的纯化柱以重力流的方式制备，不会流干，将质粒制备的手动时间降至最少。QIAGEN整个质粒制备系统避免使用有毒物质，诸如苯酚、氯仿、 ethidium bromide和CsCl，将对环境和用户的危害降至最低。
过程
在碱性条件下裂解细菌细胞，应用 QIAfilter Mega-Giga Cartridge过滤裂解液（见图“QIAfilter Mega-Giga Cartridge”）。在滤液中加入内毒素去除液，置于冰上孵育。然后将滤液装到阴离子交换柱上，在合适的盐浓度和pH值条件下，质粒DNA会选择性地结合到阴离子交换柱上。RNA、蛋白、代谢物和其他小分子组分通过中等盐浓度去除，应用高盐浓度缓冲夜可洗脱超纯的质粒 DNA（见操作流程）。应用异丙醇沉淀浓缩DNA，离心后可获得DNA。
EndoFree Plasmid Maxi Kit
