QIAquick PCR Purification Kit

Para la purificación de hasta 10 µg de productos de PCR, entre 100 bp y 10 kb

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QIAquick PCR Purification Kit (50)

Cat. No. / ID: 28104

Para la purificación de 50 reacciones de PCR: 50 QIAquick Spin Columns, tampones, Collection Tubes (2 ml)

KitColumn

QIAquick PCR Purification Kit

QIAquick PCR & Gel Cleanup Kit

QIAquick Spin Columns

Preparations

250

1000

Inquire

El QIAquick PCR Purification 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.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

Hasta 95 % de recuperación de ADN listo para usarse
Limpieza de ADN de hasta 10 kb en tres sencillos pasos
Colorante de carga de gel para el análisis práctico de muestras
Kit combinado para la extracción en gel y la limpieza de PCR
Las columnas de centrifugación están disponibles por separado

Product Details

El QIAquick PCR Purification Kit incluye columnas de centrifugación, tampones y tubos de recogida para la purificación basada en membrana de gel de sílice de productos de PCR >100 bp. Se purifica el ADN de hasta 10 kb mediante un procedimiento sencillo y rápido de unión, lavado y elución y un volumen de elución de 30–50 μl. Un indicador de pH opcional permite determinar con facilidad el pH óptimo para la unión de ADN a la columna de centrifugación. El procedimiento se puede automatizar por completo en el instrumento QIAcube Connect.

Para obtener resultados óptimos, se recomienda utilizar este producto junto con QIAvac 24 Plus.

Los protocolos estándar de QIAquick PCR Purification también pueden llevarse a cabo mediante el uso del sistema TRACKMAN Connected, combinado con las pipetas PIPETMAN M Connected, ambos de Gilson. El sistema TRACKMAN Connected orienta a los investigadores a lo largo de los protocolos de QIAquick PCR Purification al tiempo que se ajustan automáticamente los parámetros de la pipeta PIPETMAN M Connected con Bluetooth. Cada paso de la ejecución del protocolo se registra para acelerar la elaboración de informes mediante la generación de un informe completo de la serie. Descargar más información.

Performance

El procedimiento de QIAquick PCR Purification elimina cebadores, nucleótidos, enzimas, aceites minerales, sales y otras impurezas de las muestras de ADN (consulte la figura «»). Con el uso de una microcentrifugadora o un colector de vacío, se purifica el ADN que oscila entre 100 bp y 10 kb.

See figures

Eliminación completa de cebadores tras la PCR.

Principle

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 cebadores tras la PCR»). 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 »).

See figures

Eliminación completa de cebadores tras la PCR.

GelPilot Loading Dye.

Procedure

El sistema QIAquick emplea un procedimiento sencillo de unión, lavado y elución (consulte el diagrama de flujo « Procedimiento de QIAquick y MinElute»). El tampón de unión se añade directamente a la muestra de PCR u otras reacciones enzimáticas y la mezcla se aplica a la QIAquick Spin Column. El tampón de unión contiene un indicador de pH, lo que permite determinar con facilidad el pH óptimo para la unión del ADN (consulte la figura «Colorante indicador de pH»). 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 o a cualquier colector de vacío con conectores Luer, como QIAvac 24 Plus con QIAvac Luer Adapters. El QIAquick PCR Purification Kit, junto con otros kits basados en columnas de centrifugación de QIAGEN, se puede automatizar por completo en el instrumento QIAcube, lo que permite incrementar la productividad y la normalización de los resultados (consulte las figuras «Opciones de manejo de la columna de centrifugación A, B, C y D» y « QIAcube Connect»).

See figures

Procedimiento de QIAquick y MinElute.

Colorante indicador de pH.

Opciones de manejo de la columna de centrifugación — A.

Opciones de manejo de la columna de centrifugación — B.

Opciones de manejo de la columna de centrifugación — C.

Opciones de manejo de la columna de centrifugación — D.

QIAcube Connect.

Applications

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, la microinyección, la PCR y la transcripción in vitro.

Supporting data and figures

Procedimiento de QIAquick y MinElute.

Los sistemas QIAquick y MinElute emplean un sencillo procedimiento de unión, lavado y elución con columnas de centrifugación o un colector de vacío.

Specifications

Features	Specifications
Binding capacity	10 µg
Technology	Tecnología de sílice
Sample type: applications	ADNmc o ADNbc a partir de la PCR y otras reacciones enzimáticas
Fragments removed	<40 mers
Elution volume	>30 µl
Fragment size	100 bp–10 kb
Processing	Manual
Format	Tubo
Type(s) of DNA recovered	ADNmc y ADNbc

Resources

The protocol has been used successfully for Cy3-, Cy5-, and biotin-labeling of cDNA from <50 ng of total RNA or poly A⁺ mRNA.

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

Buffer PB of the QIAquick PCR Purification Kit cannot be used to extract DNA from agarose gels. However, Buffer QG of the QIAquick Gel Extraction Kit can be used to remove salt and proteins from enzymatic reactions by adding 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction and proceeding with step 6 of the Gel Extraction Spin Protocol in the QIAquick Spin Handbook. See the QIAquick Spin Handbook for a list of reactions which can be cleaned up with the various QIAquick kits.

FAQ ID -786

No. While columns from the QIAprep Spin Miniprep Kit and the QIAquick PCR Purification- and Gel Extraction Kits are based on silica-gel-membrane technology, each is designed to work optimally within its own kit format. In addition, the binding capacity and DNA recovery size cut-offs of the QIAprep and QIAquick Spin columns are different. QIAprep Spin columns bind up to 20 ug of plasmid DNA up to 50 kb in length, while the QIAquick Spin columns bind up to 10 ug of DNA with a maximum fragment size of 10 kb.

FAQ ID -311

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

CoralLoad dyes supplied in PCR Kits such as, e.g., Taq, HotStarTaq, and TopTaq DNA Polymerase and TopTaq Master Mix do not interfere with most downstream enzymatic applications.

However, for reproducible results, purification of PCR products using the QIAquick or MinElute PCR Purification Kits prior to enzymatic manipulation is recommended.

FAQ ID -1745

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 'Purification of DNA fragments from dye-labeled reactions using the QIAquick PCR Purification Kit' (QQ06).

FAQ ID -947

Buffer PB contains a high concentration of guanidine hydrochloride and isopropanol. The exact composition of Buffer PB is confidential. However, this buffer can be purchased separately: Buffer PB.

FAQ ID -2791

Based on our experience, the QIAquick PCR Purification Kit removes SYBR Green dye efficiently from PCR reactions.

We strongly assume that the QIAquick Gel Extraction Kit will be equally efficient in removing this dye, however, we recommend a 5 min incubation with wash Buffer PE on the QIAquick spin column at step 10 of the QIAquick Gel Extraction Kit Protocol. Efficiency of SYBR Green dye removal has to be validated by the enduser.

FAQ ID -637

No - mineral oil will not affect the clean-up procedure with the QIAquick or MinElute PCR Purification Kit.

FAQ ID -575

Yes. The QIAquick PCR Purification Kit has been used to clean up fragments between 100 bp and 10 kb from a wide range of enzymatic reactions, removing salts, buffers, enzymes, nucleotides, and primers smaller than 40 nucleotides. Reactions that can be cleaned up with the QIAquick PCR Purification Kit include restriction digests, random priming, ligase, kinase, phosphatase, nuclease, nick translation, and cDNA synthesis reactions.
For data and additional information, please see QIAGEN News article Issue No. 5, 1998 "Fast and efficient enzyme removal with QIAquick Spin kits."

FAQ ID -130

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