QIAGEN Purification Technologies

QIAGEN anion-exchange, Plasmid Plus, silica gel membrane, and magnetic particle technologies 

QIAGEN technologies have revolutionized nucleic acid purification by substantially reducing preparation times and eliminating the need for costly equipment, such as ultracentrifuges, and toxic chemicals, such as phenol. QIAGEN offers a wide range of specialized nucleic acid purification products based on four highly developed purification technologies: solid-phase, anion-exchange technology; QIAGEN Plasmid Plus technology; silica gel membrane technology; and magnetic particle technology.

  QIAGEN
anion-exchange
resin 
QIAGEN
Plasmid
Plus technology
Silica gel
membrane
technology 
Magnetic
particle
technology
Separation
principle

Solid-phase,
anion-exchange
chromatography
Novel, proprietary
chemistry
(patent pending)
Selective adsorption
to silica gel membranes
under controlled ionic
conditions
Binding to magnetic
silica particles
Procedure
Binding: variable
salt and pH

Elution: variable
salt and pH

Alcohol
precipitation
Binding: variable
salt and pH

Elution: variable
salt and pH

Alcohol precipitation
Binding: high salt
Elution: low salt
Ready-to-use eluate
Binding: high salt
Elution: low salt
Ready-to-use eluate
Formats
Gravity-flow
columns,
8-well strips,
96-well plates
Spin columns
and 96-well plates;
can be automated
on the BioRobot
Universal System
Spin columns,
8-well strips,
96-well plates
Automated 1–6,
1–15, 6–48, or up
to 96 samples
Advantages

• Delivers ultrapure,
transfection grade DNA for
optimal results in sensitive
applications
• Available in versatile
formatsfor all scales of
purification

• Delivers
transfection-grade
plasmid DNAfor
use in all downstream
applications
• High yields
• Fast, inexpensive
• Vaccum, centrifuge,
and automated
processing options

• Delivers high-purity
nucleic acids for
use in most downstream
applications
• Fast, inexpensive
• No silica-slurry
carry over,
no alcohol precipitation

• Delivers high-purity
nucleic acids for
use in most downstream
applications
• Fast, inexpensive
• Available in versatile
formats for all scales
of purification
• Easy automation


Principle

QIAGEN resin is a macroporous silica-based resin with a high density of diethylaminoethyl (DEAE) groups that was developed exclusively for isolation of nucleic acids. Purification on QIAGEN resin is based on the interaction between negatively charged phosphates of the nucleic acid backbone and positively charged DEAE groups on the surface of the resin (see figure Binding principle of QIAGEN resin). The salt concentration and pH conditions of the buffers used in each step control binding, wash stringency, and elution of nucleic acids.

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 yields DNA or RNA of a purity and biological activity equivalent to at least two rounds of purification in CsCl gradients, in a fraction of the time. Purified nucleic acids are of the highest possible quality and are highly suitable for sensitive downstream biological applications, such as transfection, microinjection, sequencing, and gene therapy research.
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. It also allows the separation of different classes of nucleic acids from one another by step elution using simple, pH and salt optimized buffers.

In contrast, conventional anion-exchangers (based on cellulose, dextran, or agarose) were developed for purification of proteins — which are generally globular and irregular in their physico-chemical properties — using varying salt concentration 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 (see figures Separation of nucleic acids at neutral pH on anion-exchange resins and Comparison of structure of QIAGEN and polysaccharide-based anion-exchange resins). Under these conditions, the elution range of proteins, RNA, and DNA overlap extensively, making satisfactory separation impossible. 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
Separation of nucleic acids at neutral pH on anion-exchange resins
Comparison of standard anion-exchange and QIAGEN anion-exchange resin selectivity: A: Plasmid DNA and RNA co-elute using conventional anion-exchange resin, while B: QIAGEN anion-exchange resin allows the elution of plasmid DNA and RNA at distinct salt concentrations.

HiSpeed Midi Tips, provided in the HiSpeed Plasmid Midi Kit, contain a newly developed anion-exchange resin. The resin has a higher capacity, allowing higher yields of high-copy plasmid DNA to be obtained from HiSpeed Midi Tips than from classic midi tips. Furthermore, HiSpeed Tips are designed to permit a higher flow rate, allowing DNA binding, washing, and elution steps to proceed faster.

Products using QIAGEN anion-exchange technology

Productline Applications
QIAGEN Plasmid Kits
Small-to large-scale plasmid purification
QIAwell 96 Ultra Plasmid Kit
Plasmid minipreps in 96-well format
QIAGEN Genomic-Tips
High-molecular-weight genomic DNA isolation
Principle

QIAGEN Plasmid Plus technology delivers the same performance and quality as anion-exchange technology. QIAGEN Plasmid Plus Kits provide a novel patent-pending method for extremely fast and easy large-scale preparation of transfecton-grade plasmid DNA. The procedure can be performed 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, enabling yields from 250 μg (Midi) to 10 mg (Giga). The design and unique binding chemistry of the QIAGEN Plasmid Plus spin columns allow a simple bind-wash-elute procedure based on a novel chemistry. The resulting highly concentrated DNA is ready for immediate use in subsequent applications. For preparation of transfection-grade plasmid DNA in 96-well format, QIAGEN Plasmid Plus 96 Miniprep and BioRobot Kits are available. Samples can be conveniently processed using the QIAvac 96 and/or a centrifuge or automated on the BioRobot Universal System. Due to the proprietary binding chemistry, up to 50 μg of transfection-grade plasmid DNA per well can be obtained from up to 5 ml of an E. coli culture. The innovative binding buffer included in kits ensures very specific binding conditions, providing DNA quality that is comparable to anion-exchange preps.

Applications

QIAGEN Plasmid Plus Kits and QIAGEN Plasmid Plus 96 Miniprep and BioRobot 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 Kits and QIAGEN Plasmid Plus 96 Miniprep and BioRobot Kits provide transfection-grade plasmid DNA 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 are just some of the benefits experienced with this unique technology. 

Low endotoxin levels: Purification per pellet-wet weight (g/L) for midi prep using Buffer ETR is shown. QIAGEN Plasmid Plus technology generally results in low endotoxin levels. Use of Buffer ETR further decreases the low levels of endotoxins obtained using QIAGEN Plasmid Plus technology.
Highly efficient transfection into a sensitive cell line: pCMVß DNA was prepared using the indicated preparation method. Huh-7 cells (4 x 104) were transfected using 200 ng plasmid DNA and 0.75 µl Attractene Transfection Reagent. ß-gal activity and protein content were measured after 48 hours. Plasmid DNA prepared with QIAGEN Plasmid Plus 96 Miniprep and BioRobot Kits resulted in highly efficient transfection into sensitive cell lines.
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 anion-exchange technology
Productline Applications 
QIAGEN Plasmid Plus Kits
Small-to large-scale plasmid purification 
QIAGEN Plasmid Plus 96 Miniprep and BioRobot Kits
Plasmid minipreps in 96-well format 

Principle

QIAGEN has developed a wide range of silica gel membrane products that selectively bind either RNA or DNA and separate nucleic acids within certain size parameters. A variety of modified silica gel surfaces and optimized binding buffers are used to obtain maximum discrimination between nucleic acids during adsorption and washing steps. Silica gel membranes are particularly well-suited for use in spin columns or multiwell units designed for high-throughput procedures.

Procedure

Purification using QIAGEN silica gel membrane technology is based on a simple bind-wash-elute procedure. Nucleic acids are adsorbed to the silica gel membrane in the presence of chaotropic salts, which remove water from hydrated molecules in solution. Polysaccharides and proteins do not adsorb and are removed. After a wash step, pure nucleic acids are eluted under low- or no-salt conditions in small volumes, ready for immediate use without further concentration. 

Applications

QIAGEN silica gel membrane technology yields high-purity nucleic acids suitable for most molecular biology and clinical research applications, such as restriction digestion, ligation, labeling, amplification, and radioactive and fluorescent sequencing.

Advantages

Purification of nucleic acids with silica gel membrane products is fast, convenient, and economical. With QIAGEN silica gel membrane purification, there are no time-consuming phenol-chloroform extractions, or alcohol or PEG precipitations. QIAGEN silica gel membrane technology also avoids 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 gel membranes used in QIAGEN products ensures consistent yields of high-purity nucleic acids.

Products using QIAGEN silica gel membrane technology
Productline
Applications 
QIAprep Miniprep Kits
Plasmid minipreps 
QIAprep Spin M13 Kit  Single-stranded M13 DNA purification 
QIAquick DNA cleanup kits  DNA cleanup and gel extraction 
MinElute PCR Purification Kits  DNA cleanup and gel extraction 
DNeasy Blood & Tissue Kits  DNA isolation from animal tissues and cells
DNeasy Plant Kits  DNA isolation from plants and fungi 
QIAamp Kits  DNA and RNA isolation from various samples 
RNeasy Kits  RNA isolation from various samples 

Principle

The use of magnetic particles allows a rapid purification procedure to be performed, from the initial binding of target molecules (e.g., genomic DNA) to the particles, through to washing of the particles and elution of pure target molecules.

Procedure

Purification using QIAGEN magnetic particle technology is based on a simple bind-wash-elute procedure. Nucleic acids are isolated from lysates through binding to the magnetic particles in the presence of a chaotropic salt, which removes water from hydrated molecules in solution. The particles are separated from the lysates using a magnet. The nucleic acids are then efficiently washed and eluted under low- or no-salt conditions in small volumes of elution buffer.

Advantages

Magnetic particle technology combines the speed and efficiency of silica-based DNA purification with convenient handling and ease of automation. Magnetic particle technology removes the need for centrifugation or vacuum processing, eliminating tedious and time-consuming processing steps.

Products using QIAGEN magnetic particle technology
Productline Applications 
EZ1 Kits  Low-throughput, automated nucleic
acid purification from a wide range
of clinical samples 
MagAttract M48 Kits  Medium-throughput, automated nucleic
acid purification from a wide range
of clinical samples 
MagAttract M96 Kits
High-throughput, automated DNA
purification from clinical samples,
such as blood and tissue 
BioSprint 15 DNA Blood Kit   Low-throughput, automated purification
of DNA from cells, tissue, and blood
or blood-related products 
BioSprint 96 DNA Blood Kit   High-throughput, automated purification
of DNA from cells, tissue, and blood or
blood-related products 
MagAttract 96 DNA Plant Core Kit   High-throughput, manual or automated
purification of total cellular DNA from
plant tissue 
QIAsymphony DNA Kits   Low- to High-throughput, automated
nucleic acid purification from a wide
range of samples 

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