HotStarTaq DNA Polymerase

모든 applications에 최적의 amplication이 가능합니다

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HotStarTaq DNA Polymerase (250 U)

Cat. No. / ID: 203203

250 units HotStarTaq DNA Polymerase, 10x PCR Buffer, 5x Q-Solution, 25 mM MgCl₂

SGD 315.00

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수량

250 U

1000 U

5000 U

25,000 U

수량

HotStarTaq DNA Polymerase은/는 분자생물학 분야에 사용하기 위한 것입니다. 이 제품은 질병의 진단, 예방, 또는 치료용이 아닙니다.

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특징

Hot start tag 사용으로 non-specific amplification을 줄임
Optimization 최소화 — 최소의 시간과 비용
쉬운 사용방법 — 상온에서 간단하게 reaction setup이 가능함

제품 세부 정보

HotStarTaq DNA Polymerase uses a chemically mediated hot start that, unlike, antibody-mediated systems, leads to complete inactivation of the polymerase until the initial heat activation step at the start of PCR. HotStarTaq DNA Polymerase is supplied with the unique QIAGEN PCR Buffer, which minimizes nonspecific amplification products, primer dimers, and background. Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC rich) templates, is also provided.

성능

Each lot of HotStarTaq 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 DNA Polymerase 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 figures " Wide annealing temperature window" and " Tolerance to variable magnesium concentration"). Suboptimal PCR can be improved with Q-Solution, also provided with the kit (see figure " Amplification of difficult templates"). Together, these components ensure specific amplification in a range of applications (see figure " Effect of hot start on RT-PCR performance" and " Highly sensitive single-cell PCR").

Comparison of hot-start methods
	HotStarTaq DNA Polymerase	Hot-start enzyme from Supplier A_II	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

그림 참조

Superior performance.

Tolerance to variable temperature and magnesium concentrations.

Amplification of difficult templates.

Effect of hot start on RT-PCR performance.

Highly sensitive single-cell PCR.

원리

HotStarTaq DNA Polymerase, a modified form of Taq DNA Polymerase, provides high specificity in hot-start PCR. The kit includes an innovative dual-cation PCR buffer, Q-Solution, and MgCl₂.

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").

Q-Solution

Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA, is also provided with HotStarTaq DNA Polymerase. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or with GC-rich templates (see figure " Amplification of difficult templates"). Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed. Adding Q-Solution to the PCR does not compromise PCR fidelity.

그림 참조

Superior performance.

Increased specificity of primer annealing.

Tolerance to variable temperature and magnesium concentrations.

Amplification of difficult templates.

절차

HotStarTaq DNA Polymerase is supplied with a streamlined, optimized protocol for fast and easy PCR setup. HotStarTaq DNA Polymerase is activated by a 15-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs. Reactions can be set up at room temperature, ensuring greater convenience and ease of use (see figure "HotStarTaq procedure").

응용 분야

HotStarTaq DNA Polymerase is 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

HotStarTaq procedure.

Higher specificity with different primer–template systems.

Specifications

Features	Specifications
Applications	PCR, RT-PCR, Complex genomic templates, very low-copy targets
With/without hotstart	With hotstart
Reaction type	PCR amplification
Sample/target type	Genomic DNA and cDNA
Real-time or endpoint	Endpoint
Enzyme activity	5' -> 3' exonuclease activity
Mastermix	No
Single or multiplex	Single

리소스

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

Addressing critical factors and new solutions

Publications

MALDI-TOF mass spectrometry for multiplex genotyping of CYP2B6 single-nucleotide polymorphisms.

Blievernicht JK; Schaeffeler E; Klein K; Eichelbaum M; Schwab M; Zanger UM;

Clin Chem; 2006; 53 (1):24-33 2006 Nov 2 PMID:17082249

Age-related urinary excretion of BK polyomavirus by nonimmunocompromised individuals.

Zhong S; Zheng HY; Suzuki M; Chen Q; Ikegaya H; Aoki N; Usuku S; Kobayashi N; Nukuzuma S; Yasuda Y; Kuniyoshi N; Yogo Y; Kitamura T;

J Clin Microbiol; 2006; 45 (1):193-8 2006 Nov 8 PMID:17093017

Transcriptional regulation of human CYP2A13 expression in the respiratory tract by CCAAT/enhancer binding protein and epigenetic modulation.

Ling G; Wei Y; Ding X;

Mol Pharmacol; 2006; 71 (3):807-16 2006 Dec 5 PMID:17148654

Seizures and enhanced cortical GABAergic inhibition in two mouse models of human autosomal dominant nocturnal frontal lobe epilepsy.

Klaassen A; Glykys J; Maguire J; Labarca C; Mody I; Boulter J;

Proc Natl Acad Sci U S A; 2006; 103 (50):19152-7 2006 Dec 4 PMID:17146052

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation.

Candiloro IL; Mikeska T; Dobrovic A;

Epigenetics; 2011; 6 (4):500-7 2011 Apr 1 PMID:21364322

FAQ

Touchdown PCR uses a cycling program with varying annealing temperatures. It is a useful method to increase the specificity of PCR. The annealing temperature in the initial cycle should be 5–10°C above the Tm of the primers. In subsequent cycles, the annealing temperature is decreased in steps of 1–2°C/cycle until a temperature is reached that is equal to, or 2–5°C below, the Tm of the primers. Touchdown PCR enhances the specificity of the initial primer–template duplex formation and hence the specificity of the final PCR product.

To program your thermal cycler for touchdown PCR, you should refer to the manufacturer’s instructions. For additional hints and tips for successful PCR, review the Appendix Sections in our PCR Kit handbooks, and our Brochures and Application Guides for PCR and RT-PCR.

FAQ ID -75

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

Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:

too much starting template
Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.
carry-over contamination
If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
enzyme concentration too high
When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.
too many PCR cycles
Reduce the number of cycles in steps of 3 cycles.
Mg2+ concentration not optimal
Perform PCR with different final concentrations of Mg2+ from 1.5–5.0 mM (in 0.5 mM steps) using the 25 mM MgCl2 solution provided (see table below):

Final Mg2+ concentration in reaction (mM)	1.5	2.0	2.5	3.0	3.5	4.0	4.5	5.0
Required volume of 25 mM MgCl2 per reaction (ul)	0	2	4	6	8	10	12	14

Primer concentration not optimal or primers degraded
Repeat the PCR with different primer concentrations from 0.1–0.5 µM of each primer (in 0.1 µM steps). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel.
Primer design not optimal

Review your primer design, and design new primers

For additional information on optimization of PCR results, please refer to the Appendix sections of the Taq PCR and HotStarTaq DNA Polymerase Handbook, and our comprehensive Brochure Critical Factors for Successful PCR.

FAQ ID -87

No, we do not sell Q-Solution separately. It is available only as a component of the Taq DNA Polymerase, Taq PCR Core, HotStarTaq DNA Polymerase, QIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.

FAQ ID -204

CoralLoad dyes supplied in PCR Kits such as, e.g., Taq, HotStarTaq, and TopTaq DNA Polymerase and TopTaq Master Mix do not interfere with most downstream enzymatic applications.

However, for reproducible results, purification of PCR products using the QIAquick or MinElute PCR Purification Kits prior to enzymatic manipulation is recommended.

FAQ ID -1745

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

FAQ ID -961

Taq DNA Polymerase has an intrinsic RNA-dependent DNA polymerase activity (reverse transcriptase activity). However, this activity is very low and is only present under buffer conditions that are completely different from those present during PCR. Therefore, "no-RT" controls would not give false positive results due to reverse transcription activity of the Taq polymerase.

FAQ ID -523

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

Prerequisites for avoiding primer-dimer formation during PCR include the design of optimal primer pairs, and the use of appropriate primer concentrations. Complementarity of two or three bases at the 3' ends of primer pairs and complementary sequences within a primer sequence and between the primer pair should be avoided. Reduce the primer concentration to the lowest amount at which product amplification can be achieved by conducting test runs with primer concentration gradients.

FAQ ID -544

In quantitative (real-time) PCR, primer-dimers will appear as a peak with a Tm lower than the Tm of the specific product. This peak will also appear in the no-template control (NTC). In non-quantitative endpoint PCR, primer dimer will appear as a more or less faint smear on an agarose gel, below the product band of interest.

FAQ ID -552

No. Neither the PCR Buffer nor the Enzyme Storage Buffer contain Triton.

FAQ ID -327

The QIAGEN 10x Taq and HotStarTaq DNA Polymerase PCR buffer contains a uniquely balanced combination of KCl and (NH4)2SO4. It provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers.

FAQ ID -566

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

No, the initial activation time of 15 minutes at 95°C is crucial. Enzyme activation will be incomplete when using shorter activation times, resulting in inefficient PCR product amplification.

FAQ ID -565

QIAGEN's 10x PCR Buffer provided in the Taq DNA Polymerase, Taq PCR Core, and HotStarTaq DNA Polymerase Kits contains:

Tris-Cl
KCl
(NH₄)₂SO₄
15 mM MgCl₂ ; at pH 8.7 (20°C).

Note that further details on the composition of the 10x PCR Buffer are proprietary.

FAQ ID -606

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