QIAquick PCR Purification Kit

纯化多至10 μg PCR产物,100 bp–10 kb

S_1084_5_GEN_V2

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QIAquick PCR Purification Kit (50)

Cat. No. / ID:  28104

For purification of 50 PCR reactions: 50 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml)
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KitColumn
QIAquick PCR Purification Kit
QIAquick PCR & Gel Cleanup Kit
QIAquick Spin Columns
Preparations
50
250
QIAquick PCR Purification Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 即用型DNA回收率高达95%
  • 3个简单步骤即可回收多达10 kb DNA片段
  • 含有凝胶上样染料,方便样本分析

Product Details

QIAquick PCR Purification Kit包含离心柱、缓冲液和收集管,利用硅胶模技术纯化100 bp以上的PCR产物。 通过简单快速的结合、洗涤、洗脱步骤,使用30–50 μl洗脱体积即可纯化多达10 kb的DNA片段。内置的pH指示剂可方便测定DNA与离心柱结合的最佳pH值,整个过程可在QIAcube全自动核酸纯化仪上自动化运行。

Performance

QIAquick PCR Purification操作流程去除引物、核苷酸、酶、矿物油、盐和DNA样本中的其他杂质(参见"Complete primer removal after PCR")。通过使用微型离心机或者真空底座,可以纯化得到100 bp–10 kb的DNA片段。

Principle

QIAquick Kits配备有硅胶膜,在高盐度缓冲液中结合DNA,应用低盐度缓冲液或水洗脱DNA。纯化过程去除引物、核苷酸、酶、矿物油、琼脂糖、溴化乙锭和DNA样本中的其他杂质(参见" Complete primer removal after PCR")。硅胶膜技术避免了松散树脂常见的问题和不便。特制的结合缓冲液可促进吸附特定片段范围内的DNA分子。 

凝胶上样染料

为更快、更方便地处理和分析样本,已提供凝胶上样染料。GelPilot Loading Dye含有3种示踪染料(二甲苯蓝、溴酚蓝和orange G),便于优化跑胶时间,避免小片段DNA跑得过远(参见" GelPilot Loading Dye")。

See figures

Procedure

QIAquick体系采用了简单的结合、洗涤、洗脱的流程(参见" QIAquick and MinElute procedure")。结合缓冲溶液可直接加入到PCR样本或者其他酶试剂中,然后将混合液上样到QIAquick离心柱。结合缓冲液含有pH值指示剂,可方便地确定DNA结合的最佳pH值(参见 "pH Indicator Dye")。核酸在高盐度条件下结合到硅胶膜上,洗去杂质,使用小体积的低盐缓冲液或水洗脱纯DNA,得到的DNA可直接用于后续应用。 

处理过程

QIAquick 离心柱提供两个方便的操作方法。离心柱可放在传统的台式微型离心机上或通过适配器连接到任何真空底座上,如QIAvac 24 Plus和QIAvac Luer Adapters。QIAquick PCR Purification Kit以及其他的QIAGEN离心柱试剂盒可在QIAcube全自动核酸纯化仪器上全自动化运行,确保高产量和标准化的结果(参见"Spin column handling options  A,  B,  C,  D, and  E")。

See figures

Applications

QIAquick系统纯化的DNA片段可直接用于各种应用,包括测序、微阵列分析、连接和转化、限制性酶切、标记、显微注射、PCR和体外转录。

Supporting data and figures

Resources

用户开发的实验方案 (1)
The protocol has been used successfully for Cy3-, Cy5-, and biotin-labeling of cDNA from <50 ng of total RNA or poly A+ mRNA.
试剂盒操作手册 (2)
安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
Certificates of Analysis (1)

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 Buffer PB of the QIAquick PCR Purification Kit and Buffer QG of the QIAquick Gel Extraction Kit interchangeable?

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
Are QIAprep and QIAquick Spin columns interchangeable?
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
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
Do CoralLoad dyes supplied in various QIAGEN PCR Kits interfere with downstream applications?

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
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 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
Do you have a protocol for purification of DNA fragments from Cy3/Cy5 dye-label reactions?
Yes, please follow the Supplementary Protocol 'Purification of DNA fragments from dye-labeled reactions using the QIAquick PCR Purification Kit' (QQ06). 
FAQ ID -947
What is the composition of Buffer PB?
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
Will the QIAquick PCR Purification Kit remove sufficient SYBR Green from real-time PCR reactions to allow sequencing?

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
Do I have to remove the oil from my PCR reaction before using the QIAquick or MinElute PCR Purification Kit?
No - mineral oil will not affect the clean-up procedure with the QIAquick or MinElute PCR Purification Kit.
FAQ ID -575
Can I use the QIAquick PCR Purification Kit for restriction enzyme cleanup?

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
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