HotStarTaq Master Mix Kit

高特异性扩增，用于多种PCR分析

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HotStarTaq Master Mix Kit (250 U)

Cat. No. / ID: 203443

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

JP¥39,000

单位

250 U

1000 U

2500 U

HotStarTaq Master Mix Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

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特点

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

产品详情

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。

绩效

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 A_II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：是
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倍
5'–>3'外切酶活性：是
额外添加A：是
3'–>5' 外切酶活性：否
核酸酶污染：否
蛋白酶污染：否
RNases污染：否
自启活性：否

查看图表

Higher specificity with different primer–template systems.

Superior performance.

Tolerance to variable temperatures and magnesium concentrations.

Effect of hot start on RT-PCR performance.

Specific amplification in multiplex PCR.

Highly sensitive single-cell PCR.

原理

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"）。

查看图表

Effect of hot start on RT-PCR performance.

Higher specificity with different primer–template systems.

Increased specificity of primer annealing.

Tolerance to variable temperatures and magnesium concentrations.

程序

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""）。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。

查看图表

HotStarTaq procedure.

应用

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

辅助数据和图表

HotStarTaq procedure.

The HotStarTaq procedure is fast and easy for maximum convenience.

Specifications

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

资源

As starting material, 5 g soil was mixed with different amounts of Bacillus subtilis cells. Sensitivity was 5 x 10³ cells/5g soil.

HotStarTaq DNA Polymerase; HotStarTaq Master Mix Kit - For highly specific hot-start PCR without optimization

PCR 実験における重要項目と新技術

Addressing critical factors and new solutions

Download Safety Data Sheets for QIAGEN product components.

Publications

Rapid detection of point mutations conferring resistance to fluoroquinolone in gyrA of Helicobacter pylori by allele-specific PCR.

Nishizawa T; Suzuki H; Umezawa A; Muraoka H; Iwasaki E; Masaoka T; Kobayashi I; Hibi T;

J Clin Microbiol; 2006; 45 (2):303-5 2006 Nov 22 PMID:17122023

Quantitative detection of DNA methylation states in minute amounts of DNA from body fluids.

Paliwal A; Vaissière T; Herceg Z;

Methods; 2010; 52 (3):242-7 2010 Apr 1 PMID:20362673

Quantitative PCR-based approach for rapid phage display analysis: a foundation for high throughput vascular proteomic profiling.

Ballard VL; Holm JM; Edelberg JM;

Physiol Genomics; 2006; 26 (3):202-8 2006 May 16 PMID:16705020

Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells.

Wolff NA; Abouhamed M; Verroust PJ; Thévenod F;

J Pharmacol Exp Ther; 2006; 318 (2):782-91 2006 May 11 PMID:16690719

Cytochrome P450 gene induction in rats ex vivo assessed by quantitative real-time reverse transcriptase-polymerase chain reaction (TaqMan).

Baldwin SJ; Bramhall JL; Ashby CA; Yue L; Murdock PR; Hood SR; Ayrton AD; Clarke SE;

Drug Metab Dispos; 2006; 34 (6):1063-9 2006 Mar 10 PMID:16531474

FAQ

Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification. PCR sensitivity and efficiency can be reduced by the presence of impurities in nucleic acid preparations or in biological samples. These PCR inhibitors are completely removed when template is prepared using QIAGEN Kits for nucleic acid purification. Please refer to the Brochure "Maximizing PCR and RT-PCR success" for additional information.

The optimal primer–template ratio has to be determined empirically. If too little template is used, primers may not be able to find their complementary sequences. Too much template may lead to an increase in mispriming events. Generally, no more than 1 ug of template DNA should be used per PCR reaction. As an initial guide, spectrophotometric and molar conversion values for different nucleic acid templates are listed below.

Spectrophotometric conversions for nucleic acid templates

1 A₂₆₀ unit*	Concentration (ug/ml)
Double-stranded DNA	50
Single-stranded DNA	33
Single-stranded RNA	40

*Absorbance at 260 nm = 1

Molar conversions for nucleic acid templates

Nucleic Acid	Size	pmol/ug	Molecules/ug
1 kb DNA	1000 bp	1.52	9.1 x 10¹¹
pUC 19 DNA	2686 bp	0.57	3.4 x 10¹¹
pTZ18R DNA	2870 bp	0.54	3.2 x 10¹¹
pBluescript II DNA	2961 bp	0.52	3.1 x 10¹¹
Lambda DNA	48,502 bp	0.03	1.8 x 10¹⁰
Average mRNA	1930 nt	1.67	1.0 x 10¹²
Genomic DNA
Escherichia coli	4.7 x 10⁶*	3.0 x 10^-4	1.8 x 10⁸**
Drosophila melanogaster	1.4 x 10⁸*	1.1 x 10^-5	6.6 x 10⁵**
Mus musculus (mouse)	2.7 x 10⁹*	5.7 x 10^-7	3.4 x 10⁵**
Homo sapiens (human)	3.3 x 10⁹*	4.7 x 10^-7	2.8 x 10⁵**

* Base pairs per haploid genome

** For single-copy genes

FAQ ID -74

PCR products that will be cloned using the QIAGEN PCR Cloning Kit should be generated using a thermostable DNA Polymerase without proofreading activity, such as Taq DNA Polymerase. Such polymerases attach a single A overhang to their reaction products, which can hybridize to the U overhang of the pDrive Cloning Vector. For efficient addition of an A overhang during the PCR procedure, we recommend a final extension step for 10 min at 72°C as described in the standard protocols of the Taq PCR- and HotStarTaq PCR handbook.

FAQ ID -165

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

Yes, please follow either of the User-Developed Protocols:

FAQ ID -921

Yes. Please see Table 3 in our brochure Maximizing PCR and RT-PCR success. We tested the effects of different inhibitory substances in a number of PCR systems. We also analyzed the effect of including different volumes of reverse transcription (RT) reaction mixtures in PCR. Please see the table below for a list of commonly encountered template impurities and their inhibitory effects on PCR.

Impurities showing inhibitory effects on PCR

Substance	Inhibitory concentration
SDS	>0.005% (w/v)
Phenol	>0.2% (v/v)
Ethanol	>1% (v/v)
Isopropanol	>1% (v/v)
Sodium Acetate	≥5 mM
Sodium Chloride	≥25 nM
EDTA	≥0.5 mM
Hemoglobin	≥1 mg/ml
Heparin	≥0.15 i.U./ml
Urea	>20 mM
RT reaction mixture	≥15%

FAQ ID -818

The DNA yield obtained in a PCR reaction depends on the size of the amplicon, design of the primers, starting amount of template and primers, amplification efficiency, reaction volume, numbers of PCR cycles etc. Therefore it is really difficult to predict what yield to expect. Nevertheless, in our experience, approximately 1 µg is a good guess for most cases.

FAQ ID -750

Taq DNA Polymerase and HotStarTaq DNA Polymerase are compatible with cycle sequencing. However, our buffer system is not optimized for this purpose. Optimization of reaction conditions is therefore required when using these Polymerases for cycle sequencing. Unfortunately, we do not have any protocols for this application. An initial activation of the enzyme is necessary if HotStarTaq DNA Polymerase is used.

FAQ ID -741

To determine the optimal annealing temperature for a PCR assay, a Temperature Gradient experiment should be performed. To do this, you will set up several PCR reactions in duplicate for the same primer/template combination, using the same PCR chemistry, and subject each of the reactions to a slightly different annealing temperature within a specified range. If a thermal cycler with a temperature gradient function can be used, you can simply program a temperature range for adjacent wells in the cycling block. If no cycler with a gradient function exists in your lab, you will either have to perform duplicate reactions at different temperatures in different machines (if available), or back to back in the same machine.

FAQ ID -288