HotStarTaq Plus Master Mix Kit

Fast & specific amplification을 위한 premixed solution

이 제품은 2023년 11월 30일에 단종됩니다.

지금 바로 후속 AllTaq PCR 키트로 바꿔 보세요. 전환에 대해 자세히 알아보고 상세한 FAQ를 확인해 보세요.

HotStarTaq Plus Master Mix Kit (1000)

Cat. No. / ID: 203645

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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HotStarTaq Plus Master Mix Kit은/는 분자생물학 분야에 사용하기 위한 것입니다. 이 제품은 질병의 진단, 예방, 또는 치료용이 아닙니다.

이 제품은 2023년 11월 30일에 단종됩니다.

지금 바로 후속 AllTaq PCR 키트로 바꿔 보세요. 전환에 대해 자세히 알아보고 상세한 FAQ를 확인해 보세요.

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특징

빠른 5분 enzyme activiation 시간
Master mix 형 — 편리함, pipetting steps 최소, contamination 위험 최소
Non-specific amplification을 생성되지 않고 보다 더 민감한 PCR
Optional ready-to-load buffer additive 사용으로 쉬운 조작과 로딩

제품 세부 정보

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.

성능

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.

Comparison of hot-start methods
	HotStarTaq Plus DNA Polymerase	HotStarTaq DNA Polymerase	Hot-start enzyme from Supplier A_II	Supplier R	Supplier I (antibody-mediated)	Manual	Wax barrier
Specific amplification	++	++	+	++	+	+/–	+/–
Minimal PCR optimization	++	++	+/–	+/–	+/–	–	–
Easy to use	+++	++	++	+	+	–	–
Speed of activation	++	+	++	++	++	–	–

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

원리

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.

그림 참조

Increased specificity of primer annealing.

CoralLoad Concentrate.

절차

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.

응용 분야

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

지원되는 데이터 및 수치

CoralLoad Concentrate.

Effect of hot start on RT-PCR performance.

Higher specificity with different primer–template systems.

Specific amplification in multiplex PCR.

Specifications

Features	Specifications
Applications	PCR, RT-PCR, Complex genomic templates, very low-copy targets
Enzyme activity	5' -> 3' exonuclease activity
Sample/target type	Genomic DNA and cDNA
Single or multiplex	Single
Real-time or endpoint	Endpoint
Mastermix	Yes
With/without hotstart	With hotstart
Reaction type	PCR amplification

리소스

Second edition — innovative tools

Addressing critical factors and new solutions

For highly specific hot-start PCR without optimization

FAQ

HotStart PCR is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. Lack of sensitivity or specificity is most often caused by the amplification of nonspecific priming events, such as primer dimers, that usually occur at the lower temperatures when reactions are set up. Although thermostable DNA-dependent DNA polymerases have optimal activity at higher temperatures, they do also have some activity at lower temperatures when they may amplify these nonspecific priming events. HotStart enzymes are inactive at room temperature, and require heating at nucleic acid melting temperatures in order to be activated. In this way, nonspecific priming events are melted before the enzyme can amplify them. During PCR cycles, the temperature never drops low enough during annealing of gene-specific primers for nonspecific priming events to occur, resulting in amplification exclusively of the target of interest. When using a HotStart DNA polymerase, it is critical that the initial denaturation step in the experiment be of sufficient duration to fully activate the enzyme.

FAQ ID -2676

Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides' (PCR03).

FAQ ID -961

Not necessarily. In a lot of cases, the uniquely formulated PCR Buffer provided in the HotStarTag Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and QIAGEN Multiplex PCR Kits provides optimal amplification of specific PCR products. The usefulness of Q-Solution needs to be determined empirically for each primer/template setup, by running parallel PCR reactions with and without Q-Solution under the same cycling conditions.

Q-Solution changes the melting behavior of DNA and will often improve a suboptimal PCR caused by templates that have a high degree of secondary structure or high GC-contents. For more details on the effects of Q-Solution on PCR amplification, please see the Q-Solution sections of the HotStarTaq Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase, and the QIAGEN Multiplex PCR Handbooks.

FAQ ID -380

CoralLoad gel tracking dye contained in Taq, HotStarTaq, TopTaq DNA Polymerase and TopTaq Master Mix Kits separates into 2 fragment-size dependent colors (orange and red) when loaded onto an agarose gel. Sigma Red buffer only has one color which is harder to visualize.

FAQ ID -1644