QIAGEN Plasmid Purification System

The QIAGEN Plasmid Purification System provides the fastest and most economical way to prepare 20 µg to 100 mg ultrapure plasmid or cosmid DNA. Gravity-flow QIAGEN anion-exchange tips are provided in EndoFree Plasmid Kits, HiSpeed Plasmid Kits, QIAfilter Plasmid Kits, and QIAGEN Plasmid Kits see Large-Scale Plasmid Kit Selection Guide), as well as in the QIAGEN Large-Construct Kit for purification of genomic DNA-free BAC, PAC, P1, and cosmid DNA.

Features and benefits

• Ultimate DNA purity — equivalent or superior to that obtained by 2x CsCl-gradient centrifugation
• Ultrafast procedure — for reproducible yields of ultrapure DNA
• Nontoxic reagents — no ethidium bromide, no phenol, no chloroform, no CsCl
• Cost efficiency — for mini*, midi, maxi, mega and giga, and pilot-scale DNA preparations

* For other miniprep spin and vacuum formats, see plasmid miniprep kit selection guide.

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"). For more information on the separation properties of QIAGEN Resin, see pages 321 and 322. 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.

Plasmid purification time is further reduced by using QIAfilter Cartridges for rapid and efficient clearing of bacterial lysates and QIAprecipitator modules for collecting DNA following isopropanol precipitation. Both of these modules replace time-consuming centrifugation steps in the classic anion-exchange procedure.

For the best and highly reproducible results in sensitive applications such as transfections, an endotoxin removal step integrated into both the EndoFree and Ultrapure 100 Column procedures provides the ultimate in DNA purity. For purification of genomic DNA-free BAC, PAC, P1, and cosmid DNA, a unique integrated treatment with ATP-Dependent Exonuclease in the QIAGEN Large-Construct Kit procedure ensures efficient removal of genomic DNA.

Contents of QIAGEN Plasmid Purification Kits
	QIAGEN anion-exchange tips	QIAfilter Cartridges	QIAprecipitator modules	Special features
QIAGEN Plasmid Kits		X	X	–
QIAfilter Plasmid Kits			X	–
HiSpeed Plasmid Kit				–
EndoFree Plasmid Kits			X	Integrated endotoxin removal
QIAGEN Large-Construct Kit		X	X	Integrated exonuclease digestion

Comparison of QIAGEN Plasmid Kit Procedures

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

Bacterial cells are lysed under alkaline conditions and the crude lysates are cleared using either the QIAfilter Cartridge (see figures "QIAfilter Midi Cartridge" and "QIAfilter Mega-Giga Cartridge") or centrifugation (see flowchart "Comparison of QIAGEN Plasmid Kit Procedures"). At this stage, an incubation step with a special Endotoxin Removal Buffer can be performed for effective removal of bacterial lipopolysaccharides (EndoFree Plasmid Kits and Ultrapure 100 Column). 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. The DNA is concentrated and desalted by isopropanol precipitation and collected either using the QIAprecipitator module (HiSpeed Plasmid Kits) or by centrifugation. For recommendations on culture volumes and expected yields with QIAGEN-tips, see QIAGEN Plasmid Kits. For details on the optimized procedure for purification of genomic DNA-free BAC, PAC, P1, and cosmid DNA, see QIAGEN Large-Construct Kit.

Applications

Plasmid and cosmid DNA purified with QIAGEN-tips is ideal for use in all applications including:

• Transfection (see Figure "Transfection Efficiency vs Plasmid Purification Method ")
• Microinjection
• In vitro transcription
• Manual and automated sequencing, including capillary sequencing
• Restriction analysis and enzymatic modification

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).