For highly specific and sensitive multiplex PCR without optimization requirements
- No optimization required
- High specificity and sensitivity with a built-in hot start
- Highly suited for many types of multiplex PCR applications
- Easy to use and cost-effective
The QIAGEN Multiplex PCR Kit is available in a convenient ready-to-use master mix format. The QIAGEN Multiplex PCR Master Mix includes HotStarTaq DNA Polymerase and a unique PCR buffer containing the novel synthetic Factor MP. Together with optimized salt concentrations, this additive stabilizes specifically bound primers and enables efficient extension of all primers in the reaction without the need for optimization. Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates, is also supplied.
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QIAGEN Multiplex PCR Kit (100)
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)
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206143
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QIAGEN Multiplex PCR Kit (1000)
For 1000 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix (providing a final concentration of 3 mM MgCl2, 1 x 25 ml), 5x Q-Solution (1 x 10 ml), RNase-Free Water (1 x 20 ml)
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206145
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QIAGEN Multiplex PCR Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。
成功的16重PCR。|对转基因小鼠进行基因分型。|成功的微卫星分析。|稳定、高效的退火。|
在标准条件下使用QIAGEN Multiplex PCR Kit,且未进一步优化,或者在多种反应条件下使用Supplier AII的热启动DNA聚合酶,对16个靶点进行35个循环的多重PCR (99–955 bp)。[A] 每50 µl反应使用2.5个单位的Supplier AII的热启动DNA聚合酶以及标示Mg2+浓度的比较。[B] 每50 µl反应使用优化的Mg2+浓度 (3.5 mM) 以及标示量的Supplier AII (图A) 热启动DNA聚合酶的比较。使用QIAGEN Multiplex PCR Kit可确保获得成功。M:分子量标准。|使用QIAGEN Multiplex PCR Kit及3种引物对转基因小鼠进行筛选,鉴别野生型 (wt)、杂合突变体 (ht) 和纯合突变体 (hm) 小鼠。[A] 使用靶向重组激活基因2位点的引物组。[B] 使用靶向γ干扰素基因位点的引物组。M:分子量标准。(数据由德国不伦瑞克国家生物技术研究中心的S. zur Lage和S. Weiss提供。)|使用1 ng的K562人基因组DNA和荧光素标记的引物进行微卫星位点D3S1358、TH01、D21S11、D18S51和Penta E分析。在ABI PRISM 377 Sequencer上对反应进行分析。上:QIAGEN Multiplex PCR Kit确保了高敏感度和一致的信号强度。下:使用Supplier AII的热启动DNA聚合酶获得的结果。|Multiplex PCR Buffer确保了稳定且高效的引物退火。[A] NH4+离子可防止非特异性引物与模板的退火结合。[B] Synthetic factor MP是一种新型PCR添加剂,可增加模板周围的局部引物浓度。Synthetic factor MP与K+及其他阳离子相结合,可稳定特异性结合的引物,实现HotStarTaq DNA Polymerase介导的高效引物延伸。|
性能
The QIAGEN Multiplex PCR Kit outperformed kits tested from other suppliers and ensures highly specific and sensitive multiplex PCR amplification (see figure "Successful 16-plex PCR "). The kit can be successfully used for various multiplex applications such as typing of transgenic organisms (see figure "Genotyping transgenic mice") and microsatellite analysis (see figure figure "Successful microsatellite analysis "). The master mix includes HotStarTaq DNA Polymerase for efficient amplification of multiple targets in parallel. Amplfication effiency is further improved by an innovative PCR buffer, also included in the master mix. The unique buffer ensures PCR specificity over a wide range of PCR conditions, minimizing the need for optimization. Suboptimal PCR can be improved with Q-Solution — an additive for the amplification of GC-rich templates — also provided with the kit .
HotStarTaq DNA Polymerase specifications
Concentration: 5 units/µl Recombinant enzyme: Yes Substrate analogs: dNTP, ddNTP, dUTP, biotin-11-dUTP, DIG-11-dUTP, fluorescent-dNTP/ddNTP Extension rate: 2–4 kb/min at 72°C Half-life: 10 min at 97°C ; 60 min at 94°C Amplification efficiency: ≥105 fold 5'–>3' exonuclease activity: Yes Extra A addition: Yes 3'–>5' exonuclease activity: No Contaminating nucleases: No Contaminating RNases: No Contaminating proteases: No Self-priming activity: No
原理
The QIAGEN Multiplex PCR Kit is the first kit specifically developed for multiplex PCR and is provided in an easy-to-use master-mix format. QIAGEN Multiplex PCR Master Mix contains preoptimized concentrations of HotStarTaq DNA Polymerase and MgCl2, plus dNTPs and an innovative PCR buffer specially developed for multiplex PCR. The kit enables success in multiplex PCR at the first attempt. There is no need to optimize reaction conditions (e.g., the concentrations of primers, Mg2+, and Taq DNA polymerase) and cycling parameters due to unique preoptimized reagents included in the kit.
HotStarTaq DNA Polymerase
HotStarTaq DNA Polymerase is a modified form of Taq DNA polymerase and has no polymerase activity at ambient temperatures. This prevents extension of nonspecifically annealed primers and primer dimers formed at low temperatures during PCR setup and the initial PCR cycle. HotStarTaq DNA Polymerase is activated by a 15-minute incubation at 95°C which can be incorporated into any existing thermal-cycler program.
Multiplex PCR Buffer
This special buffer contains an optimized combination of K+ and NH4+, as well as the unique PCR additive, Factor MP, which increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase (see figure "Stable and efficient primer annealing"). The innovative buffer maintains specific amplification in every cycle of PCR by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is therefore often minimal or not required.
Q-Solution
Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA, is also provided with HotStarTaq DNA Polymerase. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or GC-rich templates. Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed.
操作流程
The QIAGEN Multiplex PCR Kit is provided in a ready-to-use, preoptimized master mix for greater convenience. Use of a master mix saves time, simplifies handling for reaction setup, and increases reproducibility by eliminating many possible sources of pipetting errors and contamination — pipetting steps are minimized and tedious calculations are eliminated. Only primers and template need to be added to prepare the final amplification mix. The master mix can be stored at 2–8°C, allowing even faster setup of multiplex PCR assays. The streamlined, step-by-step protocol provided with the kit ensures fast and easy PCR setup. Reactions can be set up at room temperature, ensuring greater convenience and ease of use. HotStarTaq DNA Polymerase is activated by a 15-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs.
应用
The QIAGEN Multiplex PCR Kit is highly suited for various multiplex applications, including:
- Typing and analysis of transgenic organisms
- Amplification and analysis of microsatellites
- Typing and detection of bacteria and viruses
- Amplification of multiple DNA regions for SNP analysis
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特点
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参数
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应用
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PCR, RT-PCR, multiplex PCR, typing, detection
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酶活
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5' -> 3' exonuclease activity
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预混液
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Yes
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反应类型
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PCR amplification
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real-time或终点法PCR
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Endpoint
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样本/目标类型
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Genomic DNA and cDNA
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单一或多重
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Multiplex
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有/无热启动酶
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With hotstart
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Second edition — innovative tools
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For fast and efficient multiplex PCR without optimization
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显示更多
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图片
成功的16重PCR。
在标准条件下使用QIAGEN Multiplex PCR Kit,且未进一步优化,或者在多种反应条件下使用Supplier AII的热启动DNA聚合酶,对16个靶点进行35个循环的多重PCR (99–955 bp)。[A] 每50 µl反应使用2.5个单位的Supplier AII的热启动DNA聚合酶以及标示Mg2+浓度的比较。[B] 每50 µl反应使用优化的Mg2+浓度 (3.5 mM) 以及标示量的Supplier AII (图A) 热启动DNA聚合酶的比较。使用QIAGEN Multiplex PCR Kit可确保获得成功。M:分子量标准。
对转基因小鼠进行基因分型。
使用QIAGEN Multiplex PCR Kit及3种引物对转基因小鼠进行筛选,鉴别野生型 (wt)、杂合突变体 (ht) 和纯合突变体 (hm) 小鼠。[A] 使用靶向重组激活基因2位点的引物组。[B] 使用靶向γ干扰素基因位点的引物组。M:分子量标准。(数据由德国不伦瑞克国家生物技术研究中心的S. zur Lage和S. Weiss提供。)
成功的微卫星分析。
使用1 ng的K562人基因组DNA和荧光素标记的引物进行微卫星位点D3S1358、TH01、D21S11、D18S51和Penta E分析。在ABI PRISM 377 Sequencer上对反应进行分析。上:QIAGEN Multiplex PCR Kit确保了高敏感度和一致的信号强度。下:使用Supplier AII的热启动DNA聚合酶获得的结果。
稳定、高效的退火。
Multiplex PCR Buffer确保了稳定且高效的引物退火。[A] NH4+离子可防止非特异性引物与模板的退火结合。[B] Synthetic factor MP是一种新型PCR添加剂,可增加模板周围的局部引物浓度。Synthetic factor MP与K+及其他阳离子相结合,可稳定特异性结合的引物,实现HotStarTaq DNA Polymerase介导的高效引物延伸。
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