HotStar HiFidelity Polymerase Kit

Read phase-out notice below
For highly sensitive and reliable high-fidelity hot-start PCR
  • High sensitivity and specificity
  • Unique UA/TA cloning feature
  • Fidelity 10-fold higher than Taq DNA Polymerase
  • Room-temperature setup and fast enzyme activation

This ready-to-use, optimized kit includes everything required for high-fidelity PCR — enzyme, buffers, and dNTPs. HotStar HiFidelity DNA Polymerase is a hot-start proofreading enzyme uniquely modified to produce A overhangs, enabling direct and streamlined UA/TA cloning. The buffer contains Factor SB to prevent degradation of primers and template during PCR setup, providing highly sensitive and reliable high-fidelity PCR. In addition, Q-Solution enables efficient amplification of "difficult" (e.g., GC rich) templates.

 

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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产品 货号 目录价:
HotStar HiFidelity Polymerase Kit (100)
100 units HotStar HiFidelity DNA Polymerase, 5x HotStar HiFidelity PCR Buffer (incl. dNTPs), 5x Q-Solution, 25 mM MgSO4, RNase-Free Water
202602
S Fr. 254.00
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HotStar HiFidelity Polymerase Kit (1000)
1000 units HotStar HiFidelity DNA Polymerase, 5x HotStar HiFidelity PCR Buffer (incl. dNTPs), 5x Q-Solution, 25 mM MgSO4, RNase-Free Water
202605
S Fr. 1'756.00
加入购物车

HotStar HiFidelity Polymerase 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。


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高效的TA/UA克隆。|高度灵敏、可靠的PCR。|扩增困难模板。|退火时引物结合的特异性增强。|
使用HotStar HiFidelity Polymerase Kit和标准的Taq DNA Polymerase,从100 ng的人基因组DNA中扩增955 bp的片段。使用市售的TA或UA克隆试剂盒克隆PCR产物(各4 µl)。利用蓝白斑筛选确定克隆效率。|使用HotStar HiFidelity DNA Polymerase (QIAGEN) 和指定供应商的4种高保真PCR酶进行PCR反应。使用10 ng和1 ng人基因组DNA,遵循供应商的建议进行并行反应。对Supplier I的两种不同的高保真酶进行检测。采用40个PCR循环扩增2.3 kb的人IL9R基因片段。M:分子量标准。|使用QIAGEN PCR Buffer和Taq DNA Polymerase,不加入 (–) 或者加入 (+) 1x Q-Solution,对两种不同的引物-模板系统进行扩增,重复两次。Q-Solution可实现难以处理的模板的特异性扩增。[A] 人血管紧张素受体II基因;[B] 小鼠蛋白激酶C基因;M:分子量标准。|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。|
性能

HotStar HiFidelity Polymerase Kit includes a hot-start proofreading enzyme uniquely modified to prevent degradation of primers and template during PCR setup, providing reliable high-fidelity PCR without the need for optimization. The HotStar HiFidelity DNA Polymerase Kit outperformed kits tested from other suppliers and ensures highly specific and sensitive amplification (see figure "Highly sensitive and reliable PCR"). HotStar HiFidelity DNA Polymerase fidelity is over 10-times higher than Taq DNA polymerase. Proprietary Factor SB contained in the novel HotStar HiFidelity PCR Buffer improves the sensitivity and reliability of PCR, especially when low amounts of starting template are used. Suboptimal PCR can be improved with Q-Solution, also provided with the kit (see figure "Amplification of difficult templates").

HotStar HiFidelity DNA Polymerase specifications

Concentration: 2.5 units/μl 
5'–>3' exonuclease activity: No 
3'–>5' exonuclease activity: Yes
Extra A addition (terminal transferase activity): Yes
Half-life: >4 hours at 95°C
Nuclease contamination: No
Protease contamination: No
RNase contamination: No

 

原理

High-fidelity enzymes generally utilize a 3'→5' exonuclease activity to remove incorrectly incorporated bases. However, this activity can also degrade primers during setup and the start of PCR, which can result in nonspecific priming, smearing, or even failure to amplify any product at all — especially when using low amounts of template.

HotStar HiFidelity DNA Polymerase

Uniquely isolated from a novel Pyrococcus spp. source, HotStar HighFidelity DNA Polymerase is chemically modified to inactivate both the polymerase and the exonuclease activity. A simple 5-minute hot start activates both enzymatic components, allowing PCR to start with high proofreading ability. This unique modification ensures that primers and template remain intact during setup, preventing mispriming. The reliable performance of HotStar HiFidelity DNA Polymerase ensures highly specific and sensitive amplification.

HotStar HiFidelity PCR Buffer

Novel HotStar HiFidelity PCR Buffer contains the proprietary PCR additive, Factor SB, as well as optimized concentrations of dNTPs and MgSO4. Factor SB is a unique PCR additive that improves PCR sensitivity and reliability (see figure "Highly sensitive and reliable PCR"). Innovative Factor SB ensures that the template DNA, which is often degraded by the 3'→5' exonuclease activity of high-fidelity enzymes, remains intact.

The preoptimized formulation provides reliable amplification of specific PCR products with a very low error rate. The buffer accomplishes this by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle (see figure "Increased specificity of primer annealing"). Owing to a uniquely balanced combination of KCl and (NH4)2SO4, HotStar HiFidelity PCR 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.

HotStar HiFidelity PCR Buffer also contains specifically optimized concentrations of dNTPs for convenient PCR setup and reliable results. The buffer also maximizes the fidelity of HotStar HiFidelity DNA Polymerase, which ensures a very low error rate. The HotStar HiFidelity Polymerase Kit is highly suitable for any high-fidelity PCR application due to the increased sensitivity, specificity, and minimal optimization requirements afforded by the HotStar HiFidelity PCR Buffer.  

Q-Solution

The HotStar HiFidelity Polymerase Kit includes Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or GC-rich templates (see figure "Amplification of difficult 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. Adding Q-Solution to the PCR does not compromise PCR fidelity.

Complete kit format

The HotStar HiFidelity Polymerase Kit includes everything required for PCR. No additional reagents or buffers need to be purchased separately.

操作流程

The HotStar HiFidelity procedure is straightforward — simply follow the step-by-step, optimized protocol included with the kit. HotStar HiFidelity DNA Polymerase has been chemically modified to temporarily inactivate not only the polymerase activity, but also the 3'–>5' exonuclease activity of the enzyme. This prevents excessive degradation of primers and template during PCR setup and the initial PCR cycles. Both polymerase and proofreading activities are easily restored by a 5-minute, 95°C incubation step, which can be readily incorporated into the PCR cycling program. This modification allows convenient room-temperature reaction setup without compromising PCR specificity or product yield.

TA/UA cloning

In contrast to all other high-fidelity DNA polymerases, PCR products generated using HotStar HiFidelity DNA Polymerase can be used directly in TA- or UA-cloning procedures (see figure "Efficient TA/UA cloning").

Product length and sensitivity

Due to the high sensitivity of the HotStar HiFidelity Polymerase Kit, it is possible to amplify fragments with very low amounts of starting material (e.g., as little as 1 ng human genomic DNA). Product lengths of up to 5 kb and 10 kb have been achieved using genomic DNA and lambda DNA respectively, thus indicating the suitability of the HotStar HiFidelity Polymerase Kit for any application, including the amplification of long DNA fragments for cloning.

应用

HotStar HiFidelity Polymerase Kit is highly suitable for all hot-start PCR applications that require high sensitivity combined with a low error rate, such as cloning and site-directed mutagenesis.

特点
参数
应用 Cloning, mutation analysis, TA- or UA-cloning procedures
酶活 3' -> 5' exonuclease activity, A-addition
预混液 No
反应类型 PCR amplification, proofreading
real-time或终点法PCR Endpoint
样本/目标类型 Genomic DNA and cDNA
单一或多重 Single
有/无热启动酶 With hotstart

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产品介绍与指南
2
试剂盒操作手册
1
For highly sensitive and reliable high-fidelity hot-start PCR
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图片
Efficient TA/UA cloning
高效的TA/UA克隆。
使用HotStar HiFidelity Polymerase Kit和标准的Taq DNA Polymerase,从100 ng的人基因组DNA中扩增955 bp的片段。使用市售的TA或UA克隆试剂盒克隆PCR产物(各4 µl)。利用蓝白斑筛选确定克隆效率。
Highly sensitive and reliable PCR
高度灵敏、可靠的PCR。
使用HotStar HiFidelity DNA Polymerase (QIAGEN) 和指定供应商的4种高保真PCR酶进行PCR反应。使用10 ng和1 ng人基因组DNA,遵循供应商的建议进行并行反应。对Supplier I的两种不同的高保真酶进行检测。采用40个PCR循环扩增2.3 kb的人IL9R基因片段。M:分子量标准。
Amplification of Difficult Templates with Q-Solution
扩增困难模板。
使用QIAGEN PCR Buffer和Taq DNA Polymerase,不加入 (–) 或者加入 (+) 1x Q-Solution,对两种不同的引物-模板系统进行扩增,重复两次。Q-Solution可实现难以处理的模板的特异性扩增。[A] 人血管紧张素受体II基因;[B] 小鼠蛋白激酶C基因;M:分子量标准。
NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing
退火时引物结合的特异性增强。
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。