QIAGEN Multiplex PCR Kit

用于高特异性和灵敏度的多重PCR，无需优化

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QIAGEN Multiplex PCR Kit (100)

Cat. No. / ID: 206143

For 100 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix (providing a final concentration of 3 mM MgCl₂, 3 x 0.85 ml), 5x Q-Solution (1 x 2.0 ml), RNase-Free Water (2 x 1.7 ml)

Product

QIAGEN Multiplex PCR Kit (100)

QIAGEN Multiplex PCR Kit (1000)

Inquire

QIAGEN Multiplex PCR 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。

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特点

无需优化步骤
高特异性和高灵敏度，热启动
高度适合用于各种类型的多重PCR应用
使用简单，经济实惠

产品详情

QIAGEN Multiplex PCR Kit提供方便的即用型预混液。QIAGEN Multiplex PCR Master Mix包含HotStarTaq DNA Polymerase和含有新型合成因子MP的独特PCR缓冲液。配合优化的盐浓度，MP因子可以促进引物与模板的特异性结合，使反应体系中所有的引物都能有效延伸，无需额外优化。该试剂盒还包含新型的辅助剂Q-Solution，有助于实现“困难”模板（比如，GC含量高的模板）的高效扩增。

绩效

QIAGEN Multiplex PCR Kit可确保高特异性和灵敏度的多重PCR扩增（参见" Successful 16-plex PCR "）。该试剂盒能成功应用于各种多重应用，如转基因生物分型（参见" Genotyping transgenic mice"）和微卫星分析（参见" Successful microsatellite analysis "）。该混合物包含HotStarTaq DNA Polymerase，用于高效的平行扩增多个靶。扩增效率经新型的PCR缓冲液进一步优化，该缓冲液也包含在混合物中。独特的缓冲液确保了在广泛的PCR条件下PCR的特异性，无需优化步骤。试剂盒还包含辅助剂Q-Solution，用于扩增高GC含量的模板，优化PCR反应。

HotStarTaq DNA Polymerase规格

浓度：5单位/µl
重组酶：有
底物类似物：dNTP、ddNTP、dUTP、biotin-11-dUTP、DIG-11-dUTP、荧光-dNTP/ddNTP
延伸率：72°C下2–4 kb/分钟
半衰期：97°C下10分钟；94°C下60分钟
扩增效率：≥10⁵倍
5'–>3'外切酶活性：有
Extra A辅助剂: 有
3'–>5'外切酶活性：无
污染核酸：无
污染RNA酶：无
污染蛋白酶：无
自吸泵活性：无

查看图表

Successful 16-plex PCR.

Genotyping transgenic mice.

Successful microsatellite analysis.

原理

QIAGEN Multiplex PCR Kit是首个专为多重PCR研发的试剂盒，有即用型混合液规格。QIAGEN Multiplex PCR Master Mix包含预优化浓度的HotStarTaq DNA Polymerase和MgCl₂、dNTPs和新型的PCR缓冲液专为多重PCR研发。该试剂盒在首次尝试多重PCR即获得成功。由于试剂盒内独特的预优化的试剂无需优化反应条件（比如，引物浓度，Mg²⁺和Taq DNA聚合酶）和循环参数。

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase对 Taq DNA聚合酶进行改善，在室温下无聚合酶活性。这可以防止非特异性退火引物和在低温条件下的PCR设置和初始的PCR循环中形成引物二聚体。在95°C下，经15分钟诱导可激活HotStarTaq DNA Polymerase，可纳入各种现有的热循环程序中。

多重PCR缓冲液

这特殊的缓冲液包含优化的由K⁺和NH^₄+以及独特的PCR辅助剂、MP因子可增加模板引物的局部浓度。配合K⁺和其他阳离子、MP因子稳定特异性结合的引物，并能够用HotStarTaq DNA Polymerase进行高效的引物延伸（参见" Stable and efficient primer annealing"）。通过在每个PCR循环中高比率的特异性-非特异性引物结合，该新型的缓冲液维持每个PCR循环的特异性扩增。由于KCl和(NH₄)₂SO₄独特的平衡结合，相比传统的PCR缓冲液，该缓冲液能在更广泛的退火温度和Mg²⁺浓度下，提供严格的引物退火条件。通过改变退火温度或Mg²⁺浓度可优化PCR，因此通常将优化过程最小化或无需要优化。

Q-Solution

Q-Solution是一种新型的PCR辅助剂，有助于扩增由DNA融化特性修改的困难模板，同时配合HotStarTaq DNA Polymerase提供。该独特的试剂可优化高度二级结构或富含GC的模板所致的效果欠佳的PCR。不同于其他常用的PCR辅助剂如DMSO，Q-Solution只用于一种工作浓度，并保证无毒和PCR纯度好。

查看图表

Stable and efficient primer annealing.

程序

QIAGEN Multiplex PCR Kit含有即用型、预优化的混合液，非常方便。使用混合液节省时间、简化反应体系构建流程，并通过消除移液失误和污染，增加可靠性，减少移液步骤和繁琐的计算。只需加入引物和模板来制备最终扩增混合液。混合液可在2–8°C储存，可快速设置多重PCR检测。该试剂盒提供的精简的实验方案确保快速简单的PCR设置。可在室温下设置反应，简单方便，易于使用。HotStarTaq DNA Polymerase可在95°C下孵育15分钟激活，因此可整合入各种现有的热循环流程中。

应用

QIAGEN Multiplex PCR Kit高度适用于各种多重应用，包括：

转基因生物的分型和分析
微卫星的扩增和分析
细菌和病毒的分型和检测
用于SNP分析的多个DNA扩增

辅助数据和图表

Successful 16-plex PCR.

Multiplex PCR of 16 targets (99–955 bp) was carried out for 35 cycles using standard conditions for the QIAGEN Multiplex PCR Kit, without further optimization or using a variety of conditions with a hot-start DNA polymerase from Supplier A_II. [A] Comparison using 2.5 units per 50 µl reaction of the hot-start DNA polymerase from Supplier A_II and with the indicated Mg²⁺ concentrations. [B] Comparison using the optimized Mg²⁺ concentration (3.5 mM) for the hot-start DNA polymerase from Supplier A_II (part A) and the indicated amounts of enzyme per 50 µl reaction. Successful results were ensured with the QIAGEN Multiplex PCR Kit. M: markers.

Specifications

Features	Specifications
ApplicationsZH	PCR, RT-PCR, multiplex PCR, typing, detection
Enzyme activity	5' -> 3' exonuclease activity
Reaction type	PCR amplification
With/without hotstart	With hotstart
Single or multiplex	Multiplex
Real-time or endpoint	Endpoint
Mastermix	Yes
Sample/target type	Genomic DNA and cDNA

资源

Second edition — innovative tools

Addressing critical factors and new solutions

For fast and efficient multiplex PCR without optimization

Download Safety Data Sheets for QIAGEN product components.

FAQ

The concentration of the agarose gel for separation of multiplex PCR products should be appropriate for the overall size of products generated and can be adjusted for resolving small size differences between PCR fragments. For optimal results, we recommend the use of 1x TAE buffer for preparation and running of the gel. Use the general guidelines listed in the table below for choosing the percentage of agarose.

Minimum difference in size of PCR products	Maximum size of fragments	Concentration of agarose
>200 bp	2000 bp	1.3%
>100-200 bp	1000 bp	1.4-1.6%
>50-100 bp	750 bp	1.7-2.0%
20-50 bp	500 bp	2.5-3.0%
<20bp*	250 bp	3.0-4.0%

*Efficient separation of PCR products differing in size by about 20 bp is usually possible using standard molecular-biology–grade agarose. For separation of fragments that differ in size by less than 20 bp, we recommend using high-resolution agarose, for example MetaPhor® agarose (FMC Bioproducts). For more information, visit www.cambrex.com.

Please refer to the QIAGEN Multiplex PCR Handbook for additional information, and for details on successful multiplex PCR using the QIAGEN Multiplex PCR Kit.

FAQ ID -800

Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification. PCR sensitivity and efficiency can be reduced by the presence of impurities in nucleic acid preparations or in biological samples. These PCR inhibitors are completely removed when template is prepared using QIAGEN Kits for nucleic acid purification. Please refer to the Brochure "Maximizing PCR and RT-PCR success" for additional information.

The optimal primer–template ratio has to be determined empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. Generally, no more than 1 ug of template DNA should be used per PCR reaction. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed below.

Spectrophotometric conversions for nucleic acid templates

1 A₂₆₀ unit*	Concentration (ug/ml)
Double-stranded DNA	50
Single-stranded DNA	33
Single-stranded RNA	40

*Absorbance at 260 nm = 1

Molar conversions for nucleic acid templates

Nucleic Acid	Size	pmol/ug	Molecules/ug
1 kb DNA	1000 bp	1.52	9.1 x 10¹¹
pUC 19 DNA	2686 bp	0.57	3.4 x 10¹¹
pTZ18R DNA	2870 bp	0.54	3.2 x 10¹¹
pBluescript II DNA	2961 bp	0.52	3.1 x 10¹¹
Lambda DNA	48,502 bp	0.03	1.8 x 10¹⁰
Average mRNA	1930 nt	1.67	1.0 x 10¹²
Genomic DNA
Escherichia coli	4.7 x 10⁶*	3.0 x 10^-4	1.8 x 10⁸**
Drosophila melanogaster	1.4 x 10⁸*	1.1 x 10^-5	6.6 x 10⁵**
Mus musculus (mouse)	2.7 x 10⁹*	5.7 x 10^-7	3.4 x 10⁵**
Homo sapiens (human)	3.3 x 10⁹*	4.7 x 10^-7	2.8 x 10⁵**

* Base pairs per haploid genome

** For single-copy genes

FAQ ID -74

Always start with the cycling conditions specified in the handbook for the QIAGEN Multiplex PCR Kit to guarantee best performance.

FAQ ID -287

No, we do not sell Q-Solution separately. It is available only as a component of the Taq DNA Polymerase, Taq PCR Core, HotStarTaq DNA Polymerase, QIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.

FAQ ID -204

Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides' (PCR03).

FAQ ID -961

Not necessarily. In a lot of cases, the uniquely formulated PCR Buffer provided in the HotStarTag Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and QIAGEN Multiplex PCR Kits provides optimal amplification of specific PCR products. The usefulness of Q-Solution needs to be determined empirically for each primer/template setup, by running parallel PCR reactions with and without Q-Solution under the same cycling conditions.

Q-Solution changes the melting behavior of DNA and will often improve a suboptimal PCR caused by templates that have a high degree of secondary structure or high GC-contents. For more details on the effects of Q-Solution on PCR amplification, please see the Q-Solution sections of the HotStarTag Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and the QIAGEN Multiplex PCR Handbooks.

FAQ ID -380

The exact composition of the QIAGEN Multiplex PCR Buffer supplied in the QIAGEN Multiplex PCR Kit is proprietary. The buffer contains a specially developed balanced combination of salts and additives to ensure comparable efficiencies for annealing and extension of all primers in the reaction, thereby facilitating the amplification of multiple PCR products in a single tube.

The QIAnews article 'Highly efficient multiplex PCR using novel reaction chemistry' provides additional details and describes the advantages of this buffer in contrast to conventional PCR reagents.

FAQ ID -289

Yes, the Multiplex PCR Master Mix will freeze at -20°C. Freeze-thaw cycles up to 10 times will not negatively influence the performance of the kit. The 2x QIAGEN Multiplex PCR Master Mix can also be stored at 2-8°C for up to 6 months.

FAQ ID - 525

Yes. Please see Table 3 in our brochure Maximizing PCR and RT-PCR success. We tested the effects of different inhibitory substances in a number of PCR systems. We also analyzed the effect of including different volumes of reverse transcription (RT) reaction mixtures in PCR. Please see the table below for a list of commonly encountered template impurities and their inhibitory effects on PCR.

Impurities showing inhibitory effects on PCR

Substance	Inhibitory concentration
SDS	>0.005% (w/v)
Phenol	>0.2% (v/v)
Ethanol	>1% (v/v)
Isopropanol	>1% (v/v)
Sodium Acetate	≥5 mM
Sodium Chloride	≥25 nM
EDTA	≥0.5 mM
Hemoglobin	≥1 mg/ml
Heparin	≥0.15 i.U./ml
Urea	>20 mM
RT reaction mixture	≥15%

FAQ ID -818

Yes. Precipitates may form when the buffer freezes. We recommend that you thaw the buffer on ice, then vortex the tube at room temperature until the precipitate has re-dissolved. Do not centrifuge the tube. Do not heat the buffer.

FAQ ID -216

No, the initial activation time of 15 minutes at 95°C is crucial. Enzyme activation will be incomplete when using shorter activation times, resulting in inefficient PCR product amplification.

FAQ ID -565

The DNA yield obtained in a PCR reaction depends on the size of the amplicon, design of the primers, starting amount of template and primers, amplification efficiency, reaction volume, numbers of PCR cycles etc. Therefore it is really difficult to predict what yield to expect. Nevertheless, in our experience, approximately 1 µg is a good guess for most cases.

FAQ ID -750

To determine the optimal annealing temperature for a PCR assay, a Temperature Gradient experiment should be performed. To do this, you will set up several PCR reactions in duplicate for the same primer/template combination, using the same PCR chemistry, and subject each of the reactions to a slightly different annealing temperature within a specified range. If a thermal cycler with a temperature gradient function can be used, you can simply program a temperature range for adjacent wells in the cycling block. If no cycler with a gradient function exists in your lab, you will either have to perform duplicate reactions at different temperatures in different machines (if available), or back to back in the same machine.

FAQ ID -288