HotStarTaq Plus Master Mix Kit
For fast and highly specific amplification in all applications
- Fast 5-minute enzyme activation time
- Fewer pipetting steps reduces the risk of contamination
- High PCR specificity without the need for optimization
- Optional ready-to-load buffer additive for easier handling
HotStarTaq Plus Master Mix contains HotStarTaq Plus DNA Polymerase, the unique QIAGEN PCR Buffer that minimizes the requirement for optimization, and dNTPs. The HotStarTaq Plus Master Mix Kit provides the same unrivalled highly specific and sensitive PCR as the HotStarTaq Master Mix Kit, but combined with a fast 5-minute enzyme activation time. In addition, CoralLoad Concentrate, containing two gel-tracking dyes, is also provided for improved pipetting visualization and immediate gel-loading of PCR products.
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HotStarTaq Plus Master Mix Kit (250)
For 250 x 20 μl reactions: 3 x 0.85 ml HotStarTaq Plus Master Mix (contains 250 units of HotStarTaq Plus DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 μM of each dNT), 1 x 0.55 ml CoralLoad Concentrate, 2 x 1.9 ml RNase-Free Water
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203643
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HotStarTaq Plus Master Mix Kit (1000)
For 1000 x 20 μl reactions: 12 x 0.85 ml HotStarTaq Plus Master Mix (contains 1000 units of HotStarTaq Plus DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 μM of each dNTP), 4 x 0.55 ml CoralLoad Concentrate, 8 x 1.9 ml RNase-Free Water
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203645
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HotStarTaq Plus Master Mix Kit (2500)
For 2500 x 20 μl reactions: 25 ml HotStarTaq Plus Master Mix (contains 2500 units of HotStarTaq Plus DNA Polymerase total, PCR Buffer with 3 mM MgCl2, and 400 μM of each dNTP), 5.5 ml CoralLoad Concentrate, 2 x 20 ml RNase-Free Water
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203646
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HotStarTaq Plus Master Mix 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。
|HotStarTaq Plus实验流程。|高特异性。|对不同的引物-模板体系均有较高的特异性。|RT-PCR中高效的热启动。|多重PCR反应中的特异性扩增。|高度灵敏的单细胞PCR。|退火时引物结合的特异性增强。|
[A] CoralLoad染料包含两种凝胶示踪染料,能够便利的进行PCR反应体系构建时的移液操作、PCR产物凝胶上样,[B] 以及清楚的看到DNA的迁移。|HotStarTaq Plus实验流程快速、简单,十分便利。|使用QIAGEN的HotStarTaq Plus DNA Polymerase、HotStarTaq DNA Polymerase和Taq DNA Polymerase,以及指定供应商的三种热启动PCR酶进行PCR反应。使用50 ng人基因组DNA,遵循供应商的建议进行并行反应。采用35个PCR循环扩增1.5 kb的人CFTR基因片段。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。注意:图示数据来自HotStarTaq Master Mix Kit的实验,使用HotStarTaq Plus Master Mix Kit进行实验获得了相同的灵敏度和特异性。|使用HotStarTaq和HotStarTaq Plus DNA Polymerase对500 bp的鼠p53基因片段进行单细胞PCR,重复两次。两种酶具有同样高的特异性和灵敏度。M:分子量标准。注:显示的是HotStarTaq Plus DNA Polymerase的数据;使用HotStarTaq Plus Master Mix Kit可获得相同的灵敏度和特异性。|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 Plus DNA Polymerase 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 Plus Master Mix Kit outperforms kits from other suppliers and ensures high specificity and superior performance in hot-start PCR (see figures "Highest specificity" and "Higher specificity with different primer–template systems", and table). The innovative PCR buffer provided with the kit ensures specificity over a wide range of PCR conditions, minimizing the need for optimization. CoralLoad Concentrate, also included with the kit, ensures greater convenience by improving pipetting visibility and allowing direct loading of PCR products onto a gel. CoralLoad Concentrate can be added to the PCR without affecting amplification sensitivity or specificity. PCR fragments amplified in the presence of CoralLoad Concentrate have been successfully tested for cloning and restriction digestion without prior purification.
The combination of high specificity and easy handling makes the HotStarTaq Plus 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"). The HotStarTaq Plus Master Mix Kit is also suitable for projects such as genetic screening, in which large numbers of samples are amplified.
| Specific amplification |
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| Minimal PCR optimization |
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| Easy to use |
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| Speed of activation |
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HotStarTaq Plus 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 Plus Master Mix is a ready-to-use mixture of HotStarTaq Plus DNA Polymerase, QIAGEN PCR Buffer, MgCl2, and dNTPs. HotStarTaq Plus DNA Polymerase provides the unrivaled performance of HotStarTaq DNA Polymerase with a shortened activation time of just 5 minutes.
HotStarTaq Plus DNA Polymerase, a modified form of QIAGEN Taq DNA Polymerase, is supplied in an inactive state that 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 "Highest specificity" and "High specificity with different primer–template systems"). HotStarTaq Plus DNA Polymerase is activated by a short 5-minute incubation at 95°C, which can be easily 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.
CoralLoad Concentrate
The HotStarTaq Plus Master Mix Kit contains CoralLoad Concentrate (see figure "CoralLoad Concentrate"), which contains a gel-loading reagent and 2 gel-tracking dyes. CoralLoad Concentrate 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.
操作流程
HotStarTaq Plus Master Mix Kit is supplied in a convenient master mix format for maximum ease of use. HotStarTaq Plus DNA Polymerase is activated by a 5-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 10 µl HotStarTaq Plus Master Mix into each PCR tube and add 10 µl of primers and template DNA diluted in the RNase-free water provided with the kit (see figure " HotStarTaq Plus 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. CoralLoad Concentrate, also provided with the kit, ensures improved pipetting visibility and enables direct loading of PCR products onto a gel for enhanced convenience.
应用
The HotStarTaq Plus 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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图片
[A] CoralLoad染料包含两种凝胶示踪染料,能够便利的进行PCR反应体系构建时的移液操作、PCR产物凝胶上样,[B] 以及清楚的看到DNA的迁移。
HotStarTaq Plus实验流程。
HotStarTaq Plus实验流程快速、简单,十分便利。
高特异性。
使用QIAGEN的HotStarTaq Plus DNA Polymerase、HotStarTaq DNA Polymerase和Taq DNA Polymerase,以及指定供应商的三种热启动PCR酶进行PCR反应。使用50 ng人基因组DNA,遵循供应商的建议进行并行反应。采用35个PCR循环扩增1.5 kb的人CFTR基因片段。M:分子量标准。
对不同的引物-模板体系均有较高的特异性。
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。
RT-PCR中高效的热启动。
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。
多重PCR反应中的特异性扩增。
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。注意:图示数据来自HotStarTaq Master Mix Kit的实验,使用HotStarTaq Plus Master Mix Kit进行实验获得了相同的灵敏度和特异性。
高度灵敏的单细胞PCR。
使用HotStarTaq和HotStarTaq Plus DNA Polymerase对500 bp的鼠p53基因片段进行单细胞PCR,重复两次。两种酶具有同样高的特异性和灵敏度。M:分子量标准。注:显示的是HotStarTaq Plus DNA Polymerase的数据;使用HotStarTaq Plus Master Mix Kit可获得相同的灵敏度和特异性。
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。
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