QIAGEN-tip質(zhì)粒提取試劑盒

Note: For other miniprep spin and vacuum formats, See Plasmid Miniprep Kit Selection guide.
* Actual yields depend on plasmid copy number, size of insert, host strain, culture medium, and culture volume.
? Culture volumes depend on plasmid copy number, size of insert, host strain, and culture medium.

Principle

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 (see Figure "Ultrapure Plasmid DNA Yielded by QIAGEN Plasmid kits"). 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.

Ultrapure Plasmid DNA Yielded by QIAGEN Plasmid kits

Electron micrograph of pCMVLuc DNA prepared using a QIAGEN-tip 2500. (Data kindly provided by E. Spiess, German Cancer Research Center, Heidelberg, Germany.)

Procedure

With QIAGEN Plasmid Kits, bacterial lysates are cleared by centrifugation. 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). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.

Applications

Plasmid DNA purified with QIAGEN Plasmid Kits is ideal for use in applications such as plasmid-mediated gene silencing, transfection (see figure "Transfection Efficiency vs Plasmid Purification Method "), cloning, manual or automated sequencing, including capillary sequencing, and in vitro transcription.

Transfection Efficiency vs Plasmid Purification Method

Different pRSVcat DNA preparations using the methods indicated were introduced into the indicated cell lines by liposome-mediated transfection, and the efficiencies determined by measuring CAT expressed after 40 h. Each bar represents the mean of 4 independent transfections (2 transfections with each of 2 independent plasmid preparations).