Introduction
Plasmid Purification Technologies
QIAGEN anion-exchange, Plasmid Plus and silica membrane technologies
We have a wide range of specialized nucleic acid purification products for you to explore. These products are based on anion-exchange technology, and chaotropic and non-chaotropic silica-based (membranes and beads).
Use the table/video/slide show below to compare and find one to match your needs.
QIAGEN anion-exchange resin – solid-phase anion-exchange separations
The binding principle of QIAGEN resin. Chemical structure of positively charged DEAE groups of QIAGEN resin and negatively charged groups of the DNA backbone which interact with the resin.
Applications
QIAGEN anion-exchange resin gives you high yields of high-quality DNA highly suitable for sensitive downstream biological applications, such as transfection, microinjection, in vitro transcription, sequencing, gene therapy/gene editing research, and production of RNA therapeutics and vaccines.
Advantages
QIAGEN resin works effectively over a wide range of pH conditions (pH 6-9) and/or salt concentrations (0.1-1.6 M). This property optimizes the separation of nucleic acids – highly negatively charged, linear polyanions – from other substances and provides the highest possible nucleic acid quality.
In contrast, conventional anion exchangers (based on cellulose, dextran or agarose) were developed to purify proteins – generally globular and irregular in their physiochemical properties – using varying salt concentrations only. Furthermore, salt concentrations that can be used for separation with these resins range only between 0.1 M and 0.6 M due to their lower charge densities. Under these conditions, the elution range of proteins, RNA and DNA overlap extensively, making separation difficult. Purification technologies such as gel filtration cannot discriminate between molecules of similar molecular weight, while products based solely on glass powder or silica gel cannot provide the degree of purity obtained with QIAGEN resin.
Separation of nucleic acids at neutral pH on QIAGEN anion-exchange resin
HiSpeed Tips, provided in our HiSpeed Plasmid Kits, contain resin with a higher capacity, allowing you to get higher yields of high-copy plasmid DNA than from classic tips. HiSpeed Tips are designed to allow a higher flow rate, saving you time during the DNA binding, washing and elution steps.
QIAGEN non-chaotropic silica membrane technologies
Principle
We have developed a wide range of chaotropic and non-chaotropic, silica-based technologies that selectively bind DNA and separate nucleic acids within specific size parameters. Various optimized binding buffers are used to obtain maximum discrimination between nucleic acids during the adsorption and washing steps. Silica membranes are particularly well-suited for use in spin columns or multi-well units designed for high-throughput procedures.
Procedure
Purification using our silica membrane technologies is based on a simple bind-wash-elute procedure. For chaotropic binding, nucleic acids are adsorbed to the silica membrane in the presence of chaotropic salts, which remove water from hydrated molecules in solution. Polysaccharides and proteins do not adsorb and are removed. For non-chaotropic binding, a non-chaotropic binding buffer is added to the lysate, ensuring highly selective binding conditions similar to anion-exchange chromatography.
After a wash step, pure nucleic acids are eluted in small volumes under low- or no-salt conditions, ready for immediate use without further concentration.
Applications
Our silica membrane technologies yield high-purity nucleic acids suitable for most molecular biology and clinical research applications, such as:
- transfection of robust cells
- restriction digestion
- ligation
- labeling
- amplification
- sequencing
Advantages
Purification of nucleic acids with silica membranes is fast, convenient and economical. There are no time-consuming phenol-chloroform extractions, or alcohol or PEG precipitations, and you can avoid the handling inconveniences of loose silica resins or slurries and the problem of silica carryover, which can interfere with downstream applications. The quality of silica membranes used in our products ensures consistent yields of high-purity nucleic acids.
Non-chaotropic silica membrane technology
Principle
If you’re looking for extremely fast and easy large-scale preparation of transfection-grade plasmid DNA with the same performance and quality as anion-exchange technology, try the QIAGEN Plasmid Plus, QIAGEN Plasmid Plus 96 or QIAprep 96 Plus Kits. You can carry out the procedure in 20 (Midi and Maxi), 40 (Mega) or 50 minutes (Giga) using a vacuum and centrifuge. Optimized high-yield protocols and extra buffer volumes are provided with the kit, giving you yields from 250 μg (Midi) to 10 mg (Giga)
The design and binding chemistry of the QIAGEN Plasmid Plus spin columns offer a simple bind-wash-elute procedure based on a proprietary non-chaotropic chemistry. The resulting highly concentrated DNA is ready for immediate use in subsequent applications. For preparing transfection-grade plasmid DNA in a 96-well format, you can use the QIAGEN Plasmid Plus 96, QIAprep 96 Plus Kits and BioRobot Kits. Samples can be conveniently processed using the QIAvac 96 and/or centrifuge. Due to the proprietary binding chemistry, you can get up to 50 μg of transfection-grade plasmid DNA per well from up to 5 mL of an E. coli culture. The innovative binding buffer included in the kits ensures very specific binding conditions, providing DNA quality comparable to anion-exchange preps.
Applications
QIAGEN Plasmid Plus, QIAGEN Plasmid Plus 96, QIAprep 96 Plus Kits provide transfection-grade plasmid DNA, highly suited for all applications such as:
- Transfection into most cell lines (including sensitive cell lines such as Huh-7)
- Enzymatic modification
- Restriction digestion
- Cloning
- In vitro transcription
- In vitro translation
- Preparation of short hairpin vectors (sh-vectors)
- Sequencing
Advantages
QIAGEN Plasmid Plus, QIAGEN Plasmid Plus 96, QIAprep 96 Plus and BioRobot Kits provide transfection-grade plasmid with very low endotoxin levels (see figures Low endotoxin levels, highly efficient transfection into a sensitive cell line and Successful transfection into sensitive cell lines), high yields, fast procedures, as well as convenient and flexible processing options.
Successful transfection into sensitive cell lines: Plasmid pCMVβ DNA was prepared using the indicated preparation method with standard and high-yield (HY) protocols for QIAGEN Plasmid Plus Kits or the recommended protocol from the supplier indicated. Huh-7 cells were transfected using 200 ng plasmid DNA and 0.5 µL Attractene Transfection Reagent or 300 ng plasmid DNA and 0.75 µL Attractene Transfection Reagent.
Products using QIAGEN non-chaotropic silica membrane technology
Chaotropic silica membrane technology
Principle
The QIAprep 2.0 Spin Columns in our QIAprep Spin Miniprep Kits and our eco-friendlier alternative, the QIAwave Plasmid Miniprep Kit, as well as the QIAprep 96 plates in our QIAprep Turbo Kits contain a silica membrane. This membrane binds up to 20 µg DNA in the presence of a high chaotropic salt concentration and allows elution in a small volume of low salt buffer. QIAprep membrane technology eliminates time-consuming phenol-chloroform extraction and alcohol precipitation, as well as the problems and inconveniences associated with loose resins and slurries. High-purity plasmid DNA eluted from QIAprep membrane technology is immediately ready to use – there is no need to precipitate, concentrate or desalt.
Procedure
Plasmid purification using QIAprep Kits follows a simple bind-wash-elute procedure. First, bacterial cultures are lysed, and the lysates are cleared by centrifugation or filtration. The cleared lysates are then applied to the QIAprep 2.0 spin columns or a QIAprep 96 plate where plasmid DNA adsorbs to the silica membrane. Impurities are washed away, and pure DNA is eluted in a small volume of elution buffer or water.
In addition to plasmid purification from Escherichia coli, QIAprep Kits can be used to purify plasmid DNA from Saccharomyces cerevisiae, Bacillus subtilis and Agrobacterium tumefaciens. Contact QIAGEN Technical Services or your local distributor for protocols for these applications.
You can also automate the QIAprep Spin Miniprep Kit and the QIAwave Plasmid Miniprep Kit protocols on the QIAcube Connect.
Applications
The QIAprep Miniprep Kits provide reproducible yields of high-purity DNA suitable for use in most applications, including:
- PCR
- Restriction digestion
- Ligation and transformation
- Sequencing
- Screening