HotStarTaq Master Mix Kit

For highly specific amplification for any PCR application

  • High PCR specificity without the need for optimization
  • Easy reaction setup at room temperature
  • Ready-to-use master mix format reduces pipetting steps
HotStarTaq Master Mix contains HotStarTaq DNA Polymerase, the unique QIAGEN PCR Buffer that minimizes the requirement for optimization, and dNTPs. Providing all components in a master mix reduces pipetting steps and the risk of contamination, while increasing throughput and reproducibility.
Product Cat. no. List price:
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
HK$1,990.00
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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
HK$7,150.00
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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
HK$16,390.00
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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实验流程。|出色的表现。|对不同引物-模板体系均有较高的特异性。|RT-PCR中高效的热启动。|高度灵敏的单细胞PCR。|对不同镁离子浓度的耐受性。|多重PCR中特异性结合。|退火时引物结合的特异性增强。|
HotStarTaq实验流程快速、简单,十分便利。|将50拷贝的HIV-pol基因重组体加入1 µg人基因组DNA中,扩增497 bp的片段。采用不同的热启动酶:QIAGEN的HotStarTaq DNA Polymerase(HotStarTaq);Supplier AII的热启动酶(Hot-start enzyme);Supplier L的Taq抗体混合物(Antibody-mediated);Supplier R的非热启动酶(No hot start)。特异性的PCR产物如箭头所示。取等体积的反应产物在2%的琼脂糖凝胶上分析。M:分子量标准。|在相同的条件下,使用Supplier R的Taq DNA聚合酶 (R) 或HotStarTaq DNA Polymerase (H),对三种不同的引物-模板系统进行扩增。System 1:从人基因组DNA中扩增1.1 kb的D-IgI同系物片段。System 2:从人基因组DNA中扩增与X染色体链锁型青年型视网膜裂损症相关的296 bp的染色体区域片段。System 3:利用总RNA合成的cDNA,扩增214 bp的β-actin基因片段。M:分子量标准。|利用cDNA扩增1.1 kb的人白介素1受体 (II型) 片段。使用Supplier L的Taq DNA聚合酶和缓冲液(No hot start);使用Supplier L的抗体介导的热启动酶和缓冲液(Antibody-mediated);使用QIAGEN的HotStarTaq DNA Polymerase和PCR Buffer (HotStarTaq)制备扩增反应,重复三次。M:分子量标准。|利用流式细胞术分离单细胞,并直接分选至单个PCR管内,扩增500 bp的鼠p53基因片段。使用QIAGEN的HotStarTaq DNA Polymerase和PCR Buffer(HotStarTaq)、Supplier AII的热启动酶和缓冲液(Hot-start enzyme)或Supplier L的抗体介导的热启动酶和缓冲液(Antibody-mediated)制备并行反应。M:分子量标准。|[A] 使用QIAGEN PCR Buffer和Taq DNA Polymerase (QIAGEN)在图示退火温度下进行PCR扩增。同时使用另一供应商(Supplier AII)的PCR缓冲液和Taq DNA聚合酶进行平行反应。扩增单拷贝人类囊性纤维化基因。M:分子量标准。[B] 对不同镁离子浓度的耐受性。使用标示浓度的Mg2+Taq DNA Polymerase及QIAGEN PCR Buffer (QIAGEN) 构建PCR反应。使用另一家供应商 (Supplier AII) 提供的PCR缓冲液和Taq DNA聚合酶,同时进行相同的PCR反应。扩增单拷贝的人朊病毒蛋白基因。M:分子量标准。|利用多重PCR,从基因组DNA中扩增鼠p53基因片段。在标准反应条件下使用Supplier R的酶(No hot start),或者使用QIAGEN的HotStarTaq Master Mix Kit(HotStarTaq Master Mix)制备并行反应。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

Each lot of HotStarTaq Master Mix Kit is subjected to a comprehensive range of quality control tests, including a stringent PCR specificity and reproducibility assay in which low-copy targets are amplified. HotStarTaq Master Mix Kit outperformed kits tested from other suppliers and ensures high specificity and superior performance in hot-start PCR (see figures "Higher specificity with different primer–template systems" and "Superior performance " and table). The innovative PCR buffer provided with the kit ensures specificity over a wide range of PCR conditions, minimizing the need for optimization (see figure "Tolerance to variable temperatures and magnesium concentrations").

The combination of high specificity and easy handling makes the HotStarTaq Master Mix Kit suitable for use with complex genomic or cDNA templates (see figure "Effect of hot start on RT-PCR performance"), multiple primer pairs (see figure "Specific amplification in multiplex PCR"), and templates isolated from difficult sources or very low-copy targets (see figure "Highly sensitive single-cell PCR"). It is also suitable for projects such as genetic screening, in which large numbers of samples are amplified.

Comparison of hot-start methods 
HotStarTaq DNA Polymerase Hot-start enzyme from Supplier AII Antibody-mediated Manual Wax barrier
Specific amplification ++ + + +/– +/–
Minimal PCR optimization ++ +/– +/–
Easy to use ++ ++ +
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

HotStarTaq Master Mix is a ready-to-use mixture of HotStarTaq DNA Polymerase, QIAGEN PCR Buffer, and dNTPs. HotStarTaq DNA Polymerase, a modified form of Taq DNA Polymerase, provides high specificity in hot-start PCR.

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase is supplied in an inactive state 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 (see figures "Superior performance in hot-start PCR" and "Higher specificity with different primer–template systems"). HotStarTaq DNA Polymerase is activated by a 15-minute incubation at 95°C, which can be incorporated into any existing thermal-cycler program.

QIAGEN PCR Buffer

QIAGEN PCR 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 (see figure "Increased specificity of primer annealing"). 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 (see figures "Wide annealing temperature window" and "Tolerance to variable magnesium concentration").

操作流程
HotStarTaq Master Mix Kit is supplied in a convenient master mix format for maximum 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. Room-temperature reaction setup using the master mix is fast and easy — simply pipet 25 µl HotStarTaq Master Mix into each PCR tube and add 25 µl of primers and template DNA diluted in the RNase-free water provided with the kit (see figure "HotStarTaq procedure"). Pipetting steps are minimized, reducing the possibility of errors and contamination, and ensuring increased throughput and reproducibility. The kit includes a streamlined, optimized protocol for fast and easy PCR setup.
应用

HotStarTaq Master Mix Kit is highly suitable for a wide variety of applications, including challenging applications such as amplification of: 

  • Complex genomic templates
  • Complex cDNA templates (e.g., RT-PCR)
  • Very low-copy targets (e.g., single-cell PCR)
  • Reactions with multiple primer pairs

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Kit Handbooks
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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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Safety Data Sheets
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Download Safety Data Sheets for QIAGEN product components.
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Highly specific PCR results with both manual and automated PCR setup
HotStarTaq procedure.

The HotStarTaq procedure is fast and easy for maximum convenience.

Superior Performance in Hot-Start PCR
Superior performance.
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.
Higher Specificity with Different Primer–Template Systems
Higher specificity with different primer–template systems.
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.
Effect of Hot Start on RT-PCR Performance
Effect of hot start on RT-PCR performance.
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.
Single-Cell PCR
Highly sensitive single-cell PCR.
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.
Wide Annealing-Temperature Window
Tolerance to variable temperatures and magnesium concentrations.
[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.
Specific Amplification in Multiplex PCR
Specific amplification in multiplex PCR.
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.
NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing
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.