产地：QIAGEN
货号：12462
名称：Large-Construct Kit
Principle Large-construct DNA purification with this kit follows an optimized gravity-flow procedure which yields DNA of significantly greater purity than that obtained with other commonly used methods. A unique integrated treatment with ATP-Dependent Exonuclease enables efficient removal of genomic DNA (see figure "Efficient Removal of Genomic DNA").
The unique anion-exchange resin in QIAGEN-tips, included in the QIAGEN Large-Construct Kit, 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 (see figure "QIAGEN Plasmid Kit Anion-Exchange 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 The absence of genomic DNA contamination means that DNA quantitation is accurate and reliable, enabling sensitive experiments to be carried out under defined and reproducible conditions. The ultrapure large-construct DNA is suitable for any application, including:
Subcloning and preparation of shotgun libraries
Direct sequencing of large-construct DNA
Transfection
Procedure Following alkaline lysis of up to 500 ml culture, a unique integrated digestion step with ATP Dependent Exonuclease provided with the kit, ensures selective removal of contaminating genomic DNA (see figure "Efficient Removal of Genomic DNA") as well as nicked or damaged construct DNA.
The sample is then loaded onto the anion-exchange tip where construct DNA selectively binds under appropriate low-salt and pH conditions (see flowchart "Comparison of QIAGEN Plasmid Kit Procedures"). RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash. Genomic DNA-free ultrapure DNA is eluted in high-salt buffer. The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.
纯化至多50μg的BAC、PAC、P1或最多可达200ug的粘粒DNA，无基因组DNA污染
高效去除基因组DNA污染
转染级别纯度的高分子量DNA
快速简单的操作
原理 此试剂盒运用重力沉降原理，纯化高分子量DNA；产物DNA的纯度比其他常用方法得到的DNA的纯度高。该试剂盒自带独特的ATP外切酶消化过程，可高效的去除基因组DNA（参见 "Efficient Removal of Genomic DNA"）。
QIAGEN Large-Construct Kit含有的QIAGEN-tips中独特的离子交换树脂专为核酸纯化设计。其出色的核酸分离特性可纯化得到高纯度DNA，相当于经过两次氯化铯梯度离心得到的纯度。该试剂盒中含有的QIAGEN-tips（参见"QIAGEN Plasmid Kit Anion-Exchange Tips"）运用重力沉降原理，减少了质粒制备过程所需的手工操作的时间。QIAGEN质粒纯化系统不使用苯酚、氯仿、溴乙锭和氯化铯等有毒物质，最大程度上减小了对使用者及环境的危害。
流程 使用碱裂解法裂解至多500 ml的培养液后，使用试剂盒中含ATP的外切酶进行消化，确保特异性去除基因组DNA（参见"Efficient Removal of Genomic DNA"）及缺损的高分子量DNA。
之后将样本上样至离子交换柱，在合适的盐浓度和pH值溶液中，高分子量DNA与树脂特异性结合（参见 "Comparison of QIAGEN Plasmid Kit Procedures"）。用中等盐浓度的缓冲液洗涤，去除RNA、蛋白质、代谢物和其他小分子的杂质。用高盐缓冲液洗脱已去除基因组DNA的纯化DNA。加入异丙醇使DNA脱盐，之后离心收集。
