QIAquick Nucleotide Removal Kit

For up to 10 μg oligonucleotide (17–40mers) and DNA (40 bp to 10 kb) cleanup from enzymatic reactions

Features

  • Up to 95% recovery of ready-to-use DNA
  • Fast and convenient procedure
  • Cleanup of DNA up to 10 kb in three easy steps
  • Gel loading dye for convenient sample analysis
QIAquick Nucleotide Removal Kit (250)

Cat. No. / ID: 28306

250 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml)
₩720,000.00
KitColumn
QIAquick Nucleotide Removal Kit
QIAquick Spin Columns
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250
50
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The QIAquick Nucleotide Removal Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The QIAquick Nucleotide Removal Kit provides spin columns, buffers, and collection tubes for silica-membrane-based purification of oligonucleotides and DNA. Unincorporated nucleotides, salts, and other contaminants are removed and oligonucleotides (>17 nt) and DNA fragments ranging from 40 bp to 10 kb are purified using a simple and fast bind-wash-elute procedure and an elution volume of 30–200 μl. The procedure is fully automated on the QIAcube.

Performance

The QIAquick Nucleotide Removal procedure removes nucleotides, enzymes, salts, and other impurities from DNA samples (see figure " Complete removal of nucleotides from labeled oligos". Using a microcentrifuge, 17mer – 10 kb DNA is purified. For sample volumes smaller than 50 µl the DyeEx Spin kit can also be used.
See figures

Principle

QIAquick Kits contain a silica membrane assembly for binding of DNA in high-salt buffer and elution with low-salt buffer or water. The purification procedure removes primers, nucleotides, enzymes, mineral oil, salts, agarose, ethidium bromide, and other impurities from DNA samples (see figure " Complete removal of nucleotides from labeled oligos"). Silica-membrane technology eliminates the problems and inconvenience associated with loose resins and slurries. Specialized binding buffers are optimized for specific applications and promote selective adsorption of DNA molecules within particular size ranges.

Gel loading dye

To enable faster and more convenient sample processing and analysis, gel loading dye is provided. GelPilot Loading Dye contains three tracking dyes (xylene cyanol, bromophenol blue, and orange G) to facilitate the optimization of agarose gel run time and prevent smaller DNA fragments from migrating too far (see figure " GelPilot Loading Dye").

See figures

Procedure

The QIAquick system uses a simple bind–wash–elute procedure (see flowchart " QIAquick Nucleotide Removal procedure"). Binding buffer is added directly to the sample, and the mixture is applied to the QIAquick spin column. Nucleic acids adsorb to the silica membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in all subsequent applications.

Handling

QIAquick spin columns are designed to provide two convenient handling options. The spin columns fit into a conventional table-top microcentrifuge. The QIAquick Nucleotide Removal Kit, in addition to other QIAGEN spin-column-based kits, can be fully automated on the QIAcube, enabling increased productivity and standardization of results (see figures "Spin column handling options  A, and  B").

See figures

Applications

DNA fragments purified with the QIAquick system are ready for direct use in all applications, including sequencing, microarray analysis, ligation and transformation, restriction digestion, labeling, and microinjection.

Supporting data and figures

Specifications

FeaturesSpecifications
Binding capacity10 µg
TechnologySilica technology
Recovery: oligonucleotides dsDNARecovery: oligonucleotides, dsDNA
FormatTube
Elution volume30–50 µl
ProcessingManual
Sample type: applicationsDNA, oligonucleotides: PCR reactions
Removal <10mers 17–40mers dye terminator proteinsRemoval <10mers
Fragment size40 bp – 10 kb

Resources

Quick-Start Protocols (1)
Supplementary Protocols (1)
QIAquick® Spin Columns can now be used on any vacuum manifold with luer connectors, for example, the QIAvac 6S or QIAvac 24 with QIAvac Luer Adapters. This protocol is designed for removal of primers <10 bases, enzymes, salts, and unincorporated nucleotides from biotin-, or DIG-labeled DNA fragments and oligonucleotides >17 nucleotides.

Publications

Effects of the chemotherapeutic agent doxorubicin on the protein C anticoagulant pathway.
Woodley-Cook J; Shin LY; Swystun L; Caruso S; Beaudin S; Liaw PC;
Mol Cancer Ther; 2006; 5 (12):3303-11 2006 Dec PMID:17172434
Differential effects of NF-kappaB on apoptosis induced by DNA-damaging agents: the type of DNA damage determines the final outcome.
Strozyk E; Pöppelmann B; Schwarz T; Kulms D;
Oncogene; 2006; 25 (47):6239-51 2006 May 15 PMID:16702954
Human enhancer of rudimentary is a molecular partner of PDIP46/SKAR, a protein interacting with DNA polymerase delta and S6K1 and regulating cell growth.
Smyk A; Szuminska M; Uniewicz KA; Graves LM; Kozlowski P;
FEBS J; 2006; 273 (20):4728-41 2006 Sep 19 PMID:16984396
Dual effects of plant steroidal alkaloids on Saccharomyces cerevisiae.
Simons V; Morrissey JP; Latijnhouwers M; Csukai M; Cleaver A; Yarrow C; Osbourn A;
Antimicrob Agents Chemother; 2006; 50 (8):2732-40 2006 Aug PMID:16870766
Autoregulation of the MisR/MisS two-component signal transduction system in Neisseria meningitidis.
Tzeng YL; Zhou X; Bao S; Zhao S; Noble C; Stephens DS;
J Bacteriol; 2006; 188 (14):5055-65 2006 Jul PMID:16816178

FAQ

I bound an 11 kb DNA fragment to a QIAquick column; is it completely lost?

Larger DNA fragments bind more tightly to the QIAquick columns. It is difficult to predict whether a DNA fragment larger than 10 kb can be efficiently recovered, because this depends on base composition as well as fragment size. If the fragment is only a few kb larger than the 10 kb limit, it can be helpful to heat the elution buffer EB to 60°C and let it incubate on the column for a few minutes before centrifuging. However, please note that it will become less likely to recover your sample the larger the fragment size is. As we cannot guarantee recovery of fragments larger than the maximum cutoff size, we do not recommend to purify such fragments using QIAquick Kits.

The QIAEX II Kit can be used to extract DNA fragments up to 50 kb from agarose or polyacrylamide gels.

 

FAQ ID -756
Are the columns of the QIAquick PCR Purification-, Gel Extraction-, and Nucleotide Removal Kit interchangeable?
Yes - all QIAquick Spin Kits contain identical columns, but different binding buffers optimized for each specific application.
FAQ ID -577
How can I improve recoveries when using the QIAquick Kits?

Buffer PE did not contain ethanol

Ethanol must be added to Buffer PE (concentrate) before use. Repeat procedure with correctly prepared Buffer PE.

Inappropriate elution buffer

DNA will only be eluted efficiently in the presence of low-salt buffer (e.g., Buffer EB: 10 mM Tris·Cl, pH 8.5) or water. Elution efficiency is strongly dependent on the salt concentration and pH of the elution buffer. Contrary to adsorption, elution is most efficient under basic conditions and low salt concentrations. DNA is eluted with 50 or 30 µl of the provided Buffer EB (10 mM Tris·Cl, pH 8.5), or water. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water to elute, make sure that the pH is within this range. In addition, DNA must be stored at –20°C when eluted with water since DNA may degrade in the absence of a buffering agent. Elution with TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0) is possible, but not recommended because EDTA may inhibit subsequent enzymatic reactions.

Elution buffer incorrectly dispensed

Add elution buffer to the center of the QIAquick membrane to ensure that the buffer completely covers the membrane. This is particularly important when using small elution volumes (30 µl).

FAQ ID -180
Can QIAquick Kits be used to clean up RNA samples?
Although it is possible to use the QIAquick Nucleotide Removal Kit and QIAquick PCR Purification Kit for RNA purification, the conditions are not optimized for RNA, nor are RNase-free conditions guaranteed. The RNeasy MinElute Cleanup Kit is recommended for clean-up, concentration and desalting of RNAs above 200 bases in length.
FAQ ID -490
How do I perform a DNA precipitation to concentrate my sample?
  • Add 1/10 volume of 3 M Na-Acetate pH 5.2, and 2 to 2.5 volumes of ice-cold 100% ethanol to the DNA sample
  • Mix, and store at -20°C for at least 1 hour to precipitate the DNA
  • Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 15-20 minutes
  • Pour off the ethanol and wash the pellet twice with room-temperature 70% ethanol
  • Allow the DNA pellet to air-dry
  • resuspend the DNA in a suitable volume of sterile TE buffer or distilled water
FAQ ID -305
Do you have a protocol for cleanup of non-radioactive DNA samples?
Yes, please follow the Supplementary Protocol 'Cleanup of nonradioactive samples using the QIAquick Nucleotide Removal Kit' (QQ04).
FAQ ID -945
Why does my DNA sample float out of the slot when loading it onto an agarose gel?

DNA fragments purified with the QIAGEN DNA Cleanup Systems, i.e., the QIAquick PCR Purification Kit, the MinElute Reaction Cleanup Kit, the QIAEX II Gel Extraction Kit etc. may float out of the loading wells of agarose gels due to residual ethanol carried over from the wash step with Buffer PE (despite the addtition of glycerol-containing loading buffer).

Use either of the following options to remove residual ethanol from the eluate:

  • re-purify the sample using a QIAquick-, or MinElute column, or QIAEX II resin
  • incubate the eluate at 56°C for 10 min to evaporate the ethanol
  • dry down the sample in a vacuum centrifuge, and resuspend the pellet in a small volume of sterile water
FAQ ID -205
Is it possible to clean up a methylation reaction containing bisulfite with QIAquick Cleanup Kits?
Yes, bisulfite containing methylation reactions can be cleaned up with our silica-based cleanup products, such as QIAquick and QIAEX II. Please see Goyon et al. (1994),  'Perpetuation of cytosine methylation in Ascobolus immersus implies a novel type of maintenance methylase', published in J Mol Biol. 1994 Jul 1;240(1):42-51, for a reference.
FAQ ID -519
Do you have information about the cleanup of single-stranded DNA (ssDNA) with QIAquick columns?

As a rule of thumb, single-stranded DNA binds to silica with approximately half the affinity of a double-stranded DNA fragment of the same length under the buffer conditions used in the QIAquick and MinElute Kits. Even though no systematic experimental data exists, we expect that recovery of ssDNA fragments of approximately 200 nucleotides and below will not be very efficient after cleanup using the QIAquick PCR Purification Kit or MinElute PCR Purification Kit. By comparison, it should be possible to purify fragments longer than 140 nucleotides using the QIAquick Gel Extraction Kit.

Note that recovery of single strand DNA is influenced to some degree also by factors such as base composition and secondary structure. It has to be determined empirically by the researcher if cleanup of single-stranded DNA with QIAquick columns yields satisfactory results.

FAQ ID -759
What is the composition of Buffer EB?

The composition of Buffer EB is:

  • 10 mM Tris-Cl, pH 8.5

Buffer EB is the elution buffer used in the QIAquick PCR, Gel Extraction, Nucleotide Removal Kits, and MinElute Kits for DNA cleanup, and the QIAprep Miniprep Kits for small-scale plasmid purification. The purified DNA can also be eluted in TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

FAQ ID -199
What is the small band below my fragment of interest on an agarose gel after DNA cleanup using QIAquick?

Occasionally, DNA fragments eluted from the silica matrix of QIAquick, MinElute or QIAEX II Kits will contain denatured single-stranded DNA (ssDNA), appearing as a smaller band on an analytical gel. Under certain conditions, chaotropic agents (present in all silica-based DNA purification methods) can denature DNA fragments. This is a rare event that may be influenced by sequence characteristics such as the presence of inverted repeats or A–T-rich stretches.

Because salt and buffering agents promote renaturation of DNA strands, the following tips are recommended:

  • use the eluted DNA to prepare your downstream enzymatic reaction, but omit the enzyme. Incubate the reaction mix at 95°C for 2 minutes to reanneal the ssDNA, and allow the tube to cool slowly to room temperature before adding the enzyme and proceeding
  • alternatively, the DNA can be eluted from the silica-gel membrane or resin in 10 mM Tris buffer containing 10 mM NaCl. However, the salt concentration of the eluate must then be taken into consideration in downstream applications.
FAQ ID -148
Can I buy QIAquick and MinElute columns separately?

The QIAquick Spin Columns (100) (cat. no. 28115) in the QIAquick PCR Purification, Gel Extraction, Nucleotide Removal and PCR & Gel Cleanup kits are also sold separately from the kits.

The MinElute columns in the MinElute PCR Purification, Gel Extraction and Reaction Cleanup kits are not sold separately.

We always provide extra buffers in our kits so you can scale up reactions, add extra washes or allow for spillage.

FAQ ID -2460