Read phase-out notice below For highly reliable end-point PCR with unrivaled ease of use
- Ready-to-use master mix with minimal pipetting steps
- Fridge storage eliminates thawing time
- Reliable high-yield PCR performance
- Convenient room-temperature handling
- Optimized protocol suitable for all PCR assays
The TopTaq Master Mix Kit offers maximum convenience by combining all the benefits of specific TopTaq DNA Polymerase with the advantage of a ready-to-use master mix. It contains TopTaq DNA Polymerase, the unique TopTaq PCR Buffer that minimizes the requirement for optimization, and dNTPs. The unique TopTaq Stabilizer included in the enzyme storage buffer allows storage of TopTaq Master Mix at 4°C and reaction setup at room temperature, without time-consuming thawing of reagents. Providing all components in a ready-to-use master mix reduces pipetting steps, which lowers the risk of contamination. High yields of PCR product are achieved, even after storing the TopTaq Master Mix for extended periods of time at 25°C or 4°C.
IMPORTANT NOTE: This product is phasing-out until October 15, 2020, and afterward, available only until stocks last. Visit the product page of the successor kit to view improved features or to request a trial kit.
For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.
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TopTaq Master Mix Kit (250)
For 200 x 50 µl reactions: 2x TopTaq Master Mix containing 250 units of TopTaq DNA Polymerase in total, 10x CoralLoad Concentrate, and RNase-Free Water
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200403
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The TopTaq Master Mix Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
PCR反应的产量高,不受储存条件影响。|便利的CoraLoad染料。|TopTaq实验流程。|无需优化,获得高产量PCR产物。|退火时引物结合的特异性增强。|4°C时保持稳定。|
使用TopTaq Master Mix进行PCR,预混液可在–20°C、4°C或25°C条件下保存4个月。使用标准优化流程从人基因组DNA中扩增两个不同的基因(prp, 750 bp和HugL, 1200 bp。结果表明可获得高产量特异性PCR产物。[A]QIAxcel System获得的凝胶图片。Markers: GelPilot 1 kb Plus Marker和QX Alignment Marker 15 bp/10 kb。[B] QIAxcel System一次运行可对至多96个样本进行完全自动化的灵敏、高分辨率的毛细管电泳。|[A] CoralLoad染料包含两种凝胶示踪染料,能够便利的进行凝胶上样,[B] 以及清楚的看到DNA的迁移。|由于酶储存缓冲液中含有的TopTaq Stabilizer具有独特性质,TopTaq DNA Polymerase是第一个可以在4°C下保存的PCR试剂盒。TopTaq Stabilizer结合到TopTaq DNA Polymerase上,阻止聚合酶的失活。在低温下,模板DNA和引物也不能结合到聚合酶上。在变性步骤初期,TopTaq Stabilizer从聚合酶上自由分离,不影响聚合酶活性。这增加了快速构建反应体系的便利性,无需使用冰,节省了融化试剂所需的时间。|[A]按照标准的已优化的实验方案,在相同的循环条件下,使用TopTaq DNA Polymerase扩增哺乳动物基因组DNA的14个PCR产物,片段大小在100 bp至2 kb之间。[B]使用其他供应商的DNA聚合酶扩增同样的4个靶序列(11–14泳道),循环条件相同。对于不同的片段大小,TopTaq DNA Polymerase (QIAGEN) 的特异性PCR产物均有更高产量。|QIAGEN PCR缓冲液中的铵态氮和钾离子能够在退火时促进引物的特异性结合。K+结合到DNA主链上的磷酸基团(P–)上,稳定结合到模板上的引物。在热循环中,NH4+以铵离子和氨的形式存在,可以与碱基(B)之间的氢键发生相互作用,使错配碱基间的不稳定的氢键容易断开。两种阳离子的共同作用,在广泛的温度范围内保持特异性结合比例高。|[A] TopTaq DNA Polymerase在聚合酶的表面有小分子量的TopTaq Stabilizer,因此可在4°C或室温下保持稳定。[B]更高温度下,TopTaq Stabilizer与聚合酶分离开,不影响酶活性。|
性能
TopTaq Master Mix Kit is used for standard and specialized end-point PCR applications without the need for optimization. High yields of PCR product are achieved, even after storing TopTaq Master Mix for 4 months at 25°C or 4°C, demonstrating the fact that TopTaq Master Mix offers a very robust and reliable amplification system (see figure Reliable high-yield PCR). TopTaq Stabilizer binds to TopTaq DNA Polymerase, preventing polymerase inactivation during long-term storage. With its balanced potassium and ammonium ions, the unique PCR buffer included in the master mix promotes specific primer–template annealing and simultaneously reduces nonspecific annealing. TopTaq DNA Polymerase outperformed DNA polymerases tested from other suppliers and ensures high yields of specific products, even when using low template amounts, and independent of fragment size (see figure High PCR product yields without optimization).
原理
TopTaq Master Mix Kit has been developed by QIAGEN to provide highly reliable end-point PCR with unrivaled ease of use. Until the development of the TopTaq PCR system, all PCR enzymes required storage at –20°C. Due to the unique proprietary TopTaq Stabilizer contained in the enzyme storage buffer, TopTaq Master Mix Kit is the first PCR kit that can be stored at 4°C. This ready-to-use master mix format ensures greater convenience. High yields of specific products are achieved due to TopTaq DNA Polymerase, even when amplifying fragments of different sizes (see figure "High PCR product yields without optimization").
TopTaq PCR Buffer
The innovative TopTaq PCR Buffer facilitates the amplification of specific PCR products. During the annealing step of every PCR cycle, the buffer greatly increases the ratio of specific primer binding over nonspecific primer binding. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer-annealing conditions over a wider range of temperatures and Mg2+ concentrations than conventional PCR buffers (see figure "Increased specificity of primer annealing"). The need to optimize PCR by varying the annealing temperature or the Mg2+ concentration is dramatically reduced, or often not required.
TopTaq Stabilizer
TopTaq Stabilizer binds to TopTaq DNA Polymerase, preventing polymerase inactivation during long-term storage (see figure "Stabilization at 4°C"). Template DNA and primers are also prevented from binding to the polymerase at low temperatures. During the initial denaturation step, TopTaq Stabilizer freely dissociates from the polymerase without compromising polymerase activity.
CoralLoad Concentrate
For enhanced convenience, TopTaq Master Mix Kit includes CoralLoad Concentrate. CoralLoad Concentrate contains a gel-loading reagent and two gel-tracking dyes. This unique reagent improves pipetting visibility and facilitates estimation of DNA migration distance and optimization of agarose gel run time. When using CoralLoad Concentrate, PCR products can be directly loaded onto an agarose gel without prior addition of loading buffer (see figure "Convenient CoralLoad Concentrate").
操作流程
The TopTaq Master Mix Kit offers all of the benefits of specific TopTaq DNA Polymerase combined with the advantage of a ready-to-use master mix. With the TopTaq Master Mix Kit, separate pipetting of individual components is not required and the risk of contamination and pipetting variability is minimized, making PCR setup quick and easy. During the initial denaturation step, the TopTaq Stabilizer freely dissociates from the polymerase without compromising polymerase activity. This added convenience allows immediate reaction setup without the use of ice — saving the time usually required for reagent thawing (see flowchart " TopTaq procedure").
Reliable high-yield PCR without the need for optimization
The optimized protocol suitable for all PCR assays eliminates the need for lengthy optimization of experimental parameters for individual primer–template systems, resulting in substantial time and cost savings. High yields of specific PCR product are achieved, even after storing the TopTaq Master Mix for 4 months at 25°C or 4°C (see figure "Reliable high-yield PCR").
应用
The TopTaq Master Mix Kit is highly suited to all end-point PCR applications.
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Feature
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Specifications
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Applications
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PCR, RT-PCR, DNA Fingerprinting
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dNTP's included
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Yes (in Master Mix)
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Enzyme activity
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5' -> 3' exonuclease activity
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Mastermix
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Yes
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Reaction type
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PCR amplification
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Real-time or endpoint
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Endpoint
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Sample/target type
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Genomic DNA and cDNA
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Single or multiplex
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Single
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Storage conditions
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Fridge storage and room temperature setup
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With/without hotstart
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Without Hotstart
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For standard and specialized end-point PCR applications without the need for optimization
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Show details
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Download Safety Data Sheets for QIAGEN product components.
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View
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Images
Reliable high-yield PCR, independent of storage conditions.
PCR was performed using TopTaq Master Mix stored at –20°C, 4°C, and 25°C for 4 months. Two different genes from human genomic DNA (prp, 750 bp and HugL, 1200 bp) were amplified according to the standard optimized protocol (duplicates shown). The results show high yields of specific PCR product. [A] Gel image produced by the QIAxcel System. Markers: GelPilot 1 kb Plus Marker and QX Alignment Marker 15 bp/10 kb. [B] The QIAxcel System fully automates sensitive, high-resolution capillary electrophoresis of up to 96 samples per run.
Convenient CoralLoad Concentrate.
[A] CoralLoad Concentrate contains two gel-tracking dyes, enabling immediate gel loading of PCR products for [B] easy visualization of DNA migration.
TopTaq procedure.
Due to the unique proprietary TopTaq Stabilizer contained in the enzyme storage buffer, TopTaq DNA Polymerase is the first PCR kit that can be stored at 4°C. TopTaq Stabilizer binds to TopTaq DNA Polymerase, preventing polymerase inactivation. Template DNA and primers are also prevented from binding to the polymerase at low temperatures. During the initial denaturation step, the TopTaq Stabilizer freely dissociates from the polymerase without compromising polymerase activity. This added convenience allows immediate reaction setup without the use of ice — saving the time usually required for reagent thawing.
High yields of PCR product without optimization
[A] TopTaq DNA Polymerase was used to amplify 14 different PCR products from mammalian genomic DNA ranging in size from 100 bp to 2 kb according to the standard preoptimized protocol and using identical cycling conditions. [B] The same four targets (shown in lanes 11–14) were amplified under identical cycling conditions using DNA polymerases from the indicated suppliers. TopTaq DNA Polymerase (QIAGEN) resulted in high yields of specific PCR products, regardless of fragment size.
Increased specificity of primer annealing.
Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P–) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.
Stabilization at 4°C.
[A] TopTaq DNA Polymerase is stable at 4°C or room temperature due to the presence of low-molecular-weight molecules (TopTaq Stabilizer) on the surface of the polymerase. [B] At higher temperatures, the TopTaq Stabilizer completely dissociates from the polymerase without compromising polymerase activity.
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