Simplify plasmid purification with QIAGEN anion-exchange resin
QIAGEN-tips contain a unique, patented anion-exchange resin which eliminates the need for expensive equipment and reagents such as ultracentrifuges, HPLC/FPLC‚ or CsCl. Toxic and mutagenic substances such as phenol, chloroform, and ethidium bromide are also not required. Plasmid purification on QIAGEN resin is based on the interaction between negatively charged phosphates of the DNA backbone and positively charged DEAE groups on the surface of the resin (see figure Chemical structure of positively charged DEAE groups of QIAGEN resin).
Chemical structure of positively charged DEAE groups 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.
The salt concentration and pH conditions of the buffers used determine whether DNA is bound or eluted from the column. The key advantage of QIAGEN anion-exchange resin arises from its exceptionally high charge density. The resin consists of defined silica beads with a particle size of 100 µm, a large pore size, and a hydrophilic surface coating. The large surface area allows dense coupling of the DEAE groups.
Plasmid DNA remains tightly bound to the DEAE groups over a wide range of salt concentrations (see figure Separation of nucleic acids at neutral pH on QIAGEN anion-exchange resin).
Separation of nucleic acids at neutral pH on QIAGEN anion-exchange resin
Impurities such as RNA, protein, carbohydrates, and small metabolites are washed from QIAGEN resin with medium-salt buffers, while plasmid DNA remains bound until eluted with a high-salt buffer. The separation range of QIAGEN resin is extremely broad, extending from 0.1 M to 1.6 M salt (see figure Separation of nucleic acids at neutral pH on QIAGEN anion-exchange resin), and DNA can be efficiently separated from RNA and other impurities.
In contrast, conventional anion-exchangers, based on cellulose, dextran, or agarose, have separation ranges only up to 0.4 M salt, so that binding and elution of all substances is limited to a narrow range of salt concentrations. This means that the elution peaks of proteins, RNA, and DNA overlap extensively with one another, and a satisfactory separation cannot be achieved. Thus, the separation and purification qualities of QIAGEN resin, as well as its ease of use surpass those of conventional anion-exchange resins.
Purity and biological activity
Nucleic acids prepared on QIAGEN resin are of equivalent or superior purity to nucleic acids prepared by two rounds of purification on CsCl gradients. DNA prepared using QIAGEN-tips has been tested with restriction endonucleases, polymerases (including Taq DNA polymerase), DNA ligases, phosphatases, and kinases. Subsequent procedures such as transfection, transformation, sequencing, cloning, and in vitro transcription and translation proceed with optimal efficiency.
Capacity and recovery
The names of the different QIAGEN-tips indicate the binding capacities (in µg) of the columns for double-stranded plasmid DNA, as determined with purified pUC18 DNA. QIAGEN-tip 100, for example, has a binding capacity of 100 µg of plasmid DNA. QIAGEN resin has different binding capacities for different classes of nucleic acids. The capacity of QIAGEN resin for RNA, for example, is twice that for plasmid DNA. Conversely, large nucleic acids, such as lambda, cosmids, and genomic DNA, are bound at a slightly lower capacity than plasmid DNA. This relationship between the binding capacity of the QIAGEN resin and the size of the nucleic acids being prepared must be taken into account when calculating expected yields.
QIAGEN resin is stable for up to six hours after equilibration. Beyond this time, the separation characteristics of the resin will begin to change, and it will no longer be effective. QIAGEN-tips may be reused within six hours for the same sample by re-equilibrating the resin with Buffer QBT after the first elution. QIAGEN resin will not function in the presence of anionic detergents such as SDS, or at a pH less than 4.0.
The binding, washing, and elution conditions for QIAGEN resin are strongly influenced by pH. This figure (Elution points of different nucleic acids from QIAGEN Resin as a function of pH and NaCl concentration) shows the influence of pH on the salt concentration required for elution of various types of nucleic acids. Deviations from the appropriate pH values of the buffers at a given salt concentration may result in losses of the desired nucleic acid. Buffers, such as MOPS, sodium phosphate, Tris•Cl and sodium acetate can be used at the indicated pH. MOPS (3-[N-morpholino]propanesulfonic acid, pKa 7.2) is frequently the buffer of choice in QIAGEN protocols, since it has a higher buffering capacity at pH 7.0 than sodium phosphate, Tris•Cl or sodium acetate buffers. SDS and other anionic detergents interfere with the binding of nucleic acids to QIAGEN resin by competing for binding to the anion-exchange groups. If SDS is used during sample preparation, it must be removed through steps such as potassium acetate precipitation or alcohol precipitation prior to column application. SDS removal steps are incorporated into the QIAGEN protocols.
Elution points of different nucleic acids from QIAGEN Resin as a function of pH and NaCl concentration.