高特异性扩增,用于多种PCR分析

  • 高PCR特异性,无需优化
  • 操作简单,室温下进行反应体系的构建
  • 即用型预混液,减少移液步骤
HotStarTaq Master Mix包含HotStarTaq DNA Polymerase、可将优化需求最小化的独特的QIAGEN PCR Buffer、以及dNTPs。预混液中提供各种所需组分,减少移液步骤,降低污染风险,同时提高通量和可重复性。
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HotStarTaq Master Mix Kit (250 U)
3 x 0.85 ml HotStarTaq Master Mix (contains 250 units HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP)and 2 x 1.7 ml RNase-Free Water
203443
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HotStarTaq Master Mix Kit (1000 U)
12 x 0.85 ml HotStarTaq Master Mix (contains 1000 units HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP) and 8 x 1.7 ml RNase-Free Water
203445
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Hot StarTaq Master Mix Kit (2500 U)
1 x 25 ml HotStarTaq Master Mix (contains 2500 units HotStarTaq DNA Polymerase PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP) and 1 x 50 ml RNase-Free Water
203446
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The HotStarTaq Master Mix Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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HotStarTaq procedure.|Superior performance.|Higher specificity with different primer–template systems.|Effect of hot start on RT-PCR performance.|Highly sensitive single-cell PCR.|Tolerance to variable temperatures and magnesium concentrations.|Specific amplification in multiplex PCR.|Increased specificity of primer annealing.|
The HotStarTaq procedure is fast and easy for maximum convenience.|A 497 bp fragment was amplified from 50 copies of an HIV-pol-gene construct which had been added to 1 µg human genomic DNA. Different hot-start enzymes were employed: HotStarTaq DNA Polymerase from QIAGEN (HotStarTaq); hot-start enzyme from Supplier AII (Hot-start enzyme); Taq-antibody mixture from Supplier L (Antibody-mediated); enzyme without hot start from Supplier R (No hot start). Specific PCR product is indicated by the arrow. Equal volumes of the reaction were analyzed on a 2% agarose gel. M: markers.|Three different primer–template systems were amplified under the same conditions with either Taq DNA polymerase from Supplier R (R) or with HotStarTaq DNA Polymerase (H). System 1: A 1.1 kb fragment of a D-IgI homolog was amplified from human genomic DNA. System 2: A 296 bp fragment from the chromosomal region correlated with X-linked juvenile retinoschisis was amplified from human genomic DNA. System 3: A 214 bp fragment of the β-actin gene was amplified from cDNA synthesized from total RNA. M: markers.|A 1.1 kb fragment of the human interleukin 1 receptor (type II) gene was amplified from cDNA. Amplification reactions were prepared in triplicate using Taq DNA polymerase and buffer from Supplier L (No hot start); antibody-mediated hot start using enzyme and buffer from Supplier L (Antibody-mediated); and HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq). M: markers.|A 500 bp fragment of the murine p53 gene was amplified from single cells isolated by flow cytometry and directly sorted into individual PCR tubes. Reactions were prepared in parallel using HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq), a hot-start enzyme and buffer from Supplier AII (Hot-start enzyme), or antibody-mediated hot start and buffer from Supplier L (Antibody-mediated). M: markers.|[A] PCR amplification at the indicated annealing temperatures using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human cystic fibrosis gene was amplified. M: markers. [B] Tolerance to Variable Magnesium Concentration. PCR amplification at the indicated Mg2+ concentrations using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human prion protein gene was amplified. M: markers.|Fragments from the murine p53 gene were amplified from genomic DNA in multiplex PCR. Parallel reactions were prepared using standard reaction conditions and an enzyme from Supplier R (No hot start) or using HotStarTaq Master Mix Kit from QIAGEN (HotStarTaq Master Mix). M: markers.|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.|
Performance

每一批HotStarTaq Master Mix Kit都经过全方位的质量控制测试,包括:严格的PCR特异性和可重复性分析,即使低拷贝的靶分子也可扩增。通过检测,HotStarTaq DNA Polymerase优于其它供应商提供的试剂盒,确保高度特异性和在热启动PCR中的卓越表现(参见"Higher specificity with different primer–template systems"、"Superior performance"和表格)。该试剂盒提供的新型PCR缓冲液使得在多种PCR条件下都维持高特异性,无需优化(参见"Tolerance to variable magnesium concentration")。

高度特异性配合简单的操作使HotStarTaq Master Mix Kit适用于复杂的基因组或cDNA模板(参见"Effect of hot start on RT-PCR performance")、多重引物对(参见"Specific amplification in multiplex PCR")、从珍贵样本或低拷贝靶分子中抽提的模板(参见"Highly sensitive single-cell PCR")。该试剂盒还适用于大量样本的扩增,诸如基因筛查等项目。

热启动模式比较
HotStarTaq DNA Polymerase Supplier AII提供的热启动酶 抗体介导 手动 蜡屏障
特异性扩增 ++ + + +/– +/–
PCR优化需求 ++ +/– +/–
易用性 ++ ++ +
HotStarTaq DNA Polymerase特性

浓度:5 units/µl
重组酶:
底物类似物:dNTP、ddNTP、dUTP、biotin-11-dUTP、DIG-11-dUTP、fluorescent-dNTP/ddNTP
延伸速度:72°C条件下2–4 kb/min
酶半衰期:97°C条件下10 min,94°C条件下60 min
扩增效率:≥105
5'–>3'外切酶活性:
额外添加A:
3'–>5' 外切酶活性:
核酸酶污染:
蛋白酶污染:
RNases污染:
自启活性:否  

Principle

HotStarTaq Master Mix是一种即用型预混液,包含HotStarTaq DNA Polymerase、QIAGEN PCR Buffer和dNTPs。HotStarTaq DNA Polymerase是一种经修饰的Taq DNA Polymerase,确保热启动PCR的高特异性。

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase以非活性形式提供,常温下没有聚合酶活性。避免PCR构建和循环初始阶段低温条件下非特异性引物的延伸和引物二聚体的形成(参见"Superior performance in hot-start PCR"和"Higher specificity with different primer–template systems")。95°C条件下孵育15分钟即可激活HotStarTaq DNA Polymerase,这个步骤可整合入已有的热循环程序中。

QIAGEN PCR Buffer

QIAGEN PCR Buffer通过提高每个PCR扩增退火过程特异性引物的结合比例,确保PCR每个循环特异性扩增(参见"Increased specificity of primer annealing")。独特的KCl和(NH4)2SO4平衡组合,使得该缓冲液与传统的PCR缓冲液相比在很宽的温度和Mg2+浓度范围内都有严格的引物退火条件和高度的特异性,无需进行耗时的优化(参见"Tolerance to variable magnesium concentration")。

Procedure
HotStarTaq Master Mix Kit以方便的预混液形式提供,易于使用。95°C条件下孵育15分钟即可激活HotStarTaq DNA Polymerase,可以整合入已有的热循环程序。在室温下应用该预混液可快速、简单地构建反应体系,只需将25 µl HotStarTaq Master Mix移入每个PCR反应管,并加入25 µl溶于该试剂盒提供的不含RNase的纯水中的引物和模板DNA(参见"HotStarTaq procedure")。移液步骤最少化,降低操作误差和污染风险,确保提高通量和可重复性。该试剂盒提供高效、经优化的实验方案,用于快速、方便的PCR反应体系构建。
Applications

HotStarTaq Master Mix Kit适合多种应用,包括各种富有挑战性的研究,如各种扩增应用:

  • 复杂的基因模板
  • 复杂的cDNA模板(如RT-PCR)
  • 低拷贝的靶分子(如单细胞PCR)
  • 多重引物对反应
Feature
Specifications
Applications PCR, RT-PCR, Complex genomic templates, very low-copy targets
Enzyme activity 5'-> 3' exonuclease activity
Mastermix Yes
Reaction type PCR amplification
Real-time or endpoint Endpoint
Sample/target type Genomic DNA and cDNA
Single or multiplex Single
With/without hotstart With hotstart

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Kit Handbooks
1
HotStarTaq DNA Polymerase; HotStarTaq Master Mix Kit - For highly specific hot-start PCR without optimization  
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User-Developed Protocols
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As starting material, 5 g soil was mixed with different amounts of Bacillus subtilis cells. Sensitivity was 5 x 103 cells/5g soil.
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References
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