Description
The QIAGEN Large-Construct Kit provides gravity-flow, anion-exchange columns for purification of large-molecular-weight DNA. A unique integrated ATP-dependent exonuclease digestion step ensures selective removal of contaminating genomic DNA. The purified DNA is equivalent to that obtained by 2 x CsCl gradient centrifugation and is suitable for transfection.
DNA purified using the QIAGEN Large-Construct Kit is free of genomic DNA contamination (see figure "Efficient removal of genomic DNA"). This removal of genomic DNA ensures accurate and reliable DNA quantitation, which enables sensitive experiments to be carried out under defined and reproducible conditions.
DNA purification using the QIAGEN Large-Construct Kit uses 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. 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. Prepacked QIAGEN-tips (see figure "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.
Following alkaline lysis of up to 500 ml culture (see flowchart "QIAGEN Plasmid Kit procedures"), a unique integrated digestion step with ATP-dependent exonuclease provided with the kit, ensures selective removal of contaminating genomic DNA, as well as nicked or damaged construct DNA. The sample 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. Genomic DNA-free, pure plasmid DNA is eluted in high-salt buffer. The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.
