QIAquick Nucleotide Removal Kit

Para la limpieza de hasta 10 µg de oligonucleótidos (17–40 mers) y ADN (entre 40 bp y 10 kb) a partir de reacciones enzimáticas

✓ Procesamiento automático sin interrupción de pedidos en línea

✓ Servicio técnico y para productos experto y profesional

✓ Realización y repetición de pedidos rápidas y fiables

QIAquick Nucleotide Removal Kit (250)

Cat. No. / ID: 28306

250 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml)

KitColumn

QIAquick Nucleotide Removal Kit

QIAquick Spin Columns

Inquire

El QIAquick Nucleotide Removal Kit está concebido para su uso en aplicaciones de biología molecular. Este producto no está concebido para el diagnóstico, la prevención ni el tratamiento de enfermedades.

✓ Procesamiento automático sin interrupción de pedidos en línea

✓ Servicio técnico y para productos experto y profesional

✓ Realización y repetición de pedidos rápidas y fiables

Características

Hasta 95 % de recuperación de ADN listo para usarse
Procedimiento rápido y práctico
Limpieza de ADN de hasta 10 kb en tres sencillos pasos
Colorante de carga de gel para el análisis práctico de muestras

Detalles del producto

El QIAquick Nucleotide Removal Kit incluye columnas de centrifugación, tampones y tubos de recogida para la purificación basada en membrana de gel de sílice de nucleótidos y ADN. Se eliminan los nucleótidos no incorporados, las sales y otros contaminantes y se purifican los oligonucleótidos (>17 nt) y los fragmentos de ADN que oscilan entre 40 bp y 10 kb mediante un procedimiento sencillo y rápido de unión, lavado y elución y un volumen de elución de 30–200 µl. El procedimiento se automatiza por completo en el instrumento QIAcube Connect.

Rendimiento

El procedimiento de QIAquick Nucleotide Removal elimina nucleótidos, enzimas, sales y otras impurezas de las muestras de ADN (consulte la figura « Eliminación completa de nucleótidos de los oligonucleótidos marcados»). Con el uso de una microcentrifugadora, se purifica ADN de 17 mer–10 kb. En volúmenes de muestra inferiores a 50 µl, también se puede utilizar el kit DyeEx Spin.

Ver figuras

Eliminación completa de nucleótidos de los oligonucleótidos marcados.

Principio

Los kits QIAquick contienen un ensamblaje de membrana de sílice para la unión del ADN en tampón de alta salinidad y elución con tampón de baja salinidad o agua. El procedimiento de purificación elimina cebadores, nucleótidos, enzimas, aceites minerales, sales, agarosa, bromuro de etidio y otras impurezas de las muestras de ADN (consulte la figura « Eliminación completa de nucleótidos de los oligonucleótidos marcados»). La tecnología de membrana de sílice elimina los problemas e inconvenientes relacionados con las resinas sueltas y los residuos pastosos. Los tampones de unión especializados están optimizados para aplicaciones específicas y promover la adsorción selectiva de moléculas de ADN dentro de rangos de tamaño específicos.

Colorante de carga de gel

Para que el procesamiento y el análisis de las muestras sean más rápidos y prácticos, se proporciona el colorante de carga de gel. El GelPilot Loading Dye contiene tres colorantes de seguimiento (xileno cianol, azul de bromofenol y naranja G) para facilitar la optimización del tiempo de la serie del gel de agarosa y evitar que los fragmentos más pequeños de ADN migren demasiado lejos (consulte la figura « GelPilot Loading Dye»).

Ver figuras

Eliminación completa de nucleótidos de los oligonucleótidos marcados.

GelPilot Loading Dye.

Procedimiento

El sistema QIAquick emplea un procedimiento sencillo de unión, lavado y elución (consulte el diagrama de flujo « Procedimiento del QIAquick Nucleotide Removal»). El tampón de unión se añade directamente a la muestra y la mezcla se aplica a la QIAquick Spin Column. Los ácidos nucleicos se adsorben a la membrana de sílice en las condiciones de alta salinidad generadas por el tampón. Se lavan las impurezas y el ADN puro se eluye con un volumen bajo del tampón de baja salinidad suministrado o de agua, listo para usarse en todas las aplicaciones posteriores.

Manejo

Las QIAquick Spin Columns se han diseñado con dos prácticas opciones de manejo. Las columnas de centrifugación se adaptan a una microcentrifugadora de mesa convencional. El QIAquick Nucleotide Removal Kit, junto con otros kits basados en columna de centrifugación de QIAGEN, se puede automatizar por completo en el instrumento QIAcube Connect, lo que permite incrementar la productividad y la normalización de los resultados (consulte las figuras «Opciones de manejo de la columna de centrifugación A» y « QIAcube Connect»).

Ver figuras

Procedimiento de QIAquick Nucleotide Removal.

Opciones de manejo de la columna de centrifugación — A.

QIAcube Connect.

Aplicaciones

Los fragmentos de ADN purificados con el sistema QIAquick están listos para su uso directo en diversas aplicaciones, incluyendo la secuenciación, el análisis de micromatrices, la ligación y transformación, la digestión de restricción, el marcado y la microinyección.

Datos y cifras de respaldo

Procedimiento de QIAquick Nucleotide Removal.

Specifications

Features	Specifications
Binding capacity	10 µg
Technology	Tecnología de sílice
Recovery: oligonucleotides dsDNA	Recuperación: oligonucleótidos, ADNbc
Format	Tubo
Elution volume	30-50 μl
Processing	Manual
Sample type: applications	ADN, oligonucleótidos: Reacciones de PCR
Removal <10mers 17–40mers dye terminator proteins	Eliminación, <10 mers
Fragment size	40 bp–10 kb

Recursos

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

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

Yes - all QIAquick Spin Kits contain identical columns, but different binding buffers optimized for each specific application.

FAQ ID -577

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

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

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 h to precipitate the DNA
Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 15–20 min
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

Yes, please follow the Supplementary Protocol 'Cleanup of nonradioactive samples using the QIAquick Nucleotide Removal Kit' (QQ04).

FAQ ID -945

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

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

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

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

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

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