Taq DNA Polymerase

用于常规和特殊的PCR

Features

  • QIAGEN PCR缓冲体系最大限度地减少了PCR条件的优化
  • 独特的即用型PCR缓冲液,操作更快速简单
  • Q-Solution可帮助扩增GC含量高的模板
  • 提供多种规格的包装方便使用
Taq DNA Polymerase (250 U)

Cat. No. / ID: 201203

250 units Taq DNA Polymerase, 10x PCR Buffer, 10x CoralLoad PCR Buffer, 5x Q-Solution, 25 mM MgCl2
Enzyme
Taq DNA Polymerase
Taq DNA Polymerase Kit
Size
Taq DNA Polymerase (250 U)
Taq DNA Polymerase (1000 U)
Taq DNA Polymerase (5000 U)
Taq DNA Polymerase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。

Product Details

QIAGEN PCR Buffer中的Taq DNA Polymerase最大限度的减少了使用时对PCR条件的优化,新型的辅助剂Q-Solution则有助于实现“困难”模板(比如,GC含量高的模板)的高效扩增。CoralLoad PCR Buffer(包含两种胶示踪染料)使PCR产物可以直接上样电泳。

Performance

Taq DNA Polymerase可在多种PCR条件下进行灵敏的PCR反应,无需耗时的优化过程(参见" Tolerance of different primer Tm Values" 和" Specific amplification of long PCR products")。每批Taq DNA Polymerase都受到全面的质量控制检测,包括严格的PCR特异性和可重复性分析,从人类基因组DNA扩增低拷贝的目的基因(参见" Lot-to-lot reproducibility")。试剂盒提供的QIAGEN PCR Buffer和CoralLoad PCR Buffer具有独特的组成,可在多种PCR条件下进行高度特异性的PCR,无需优化(参见" Wide annealing-temperature window" 和" Tolerance to variable magnesium concentration")。此外,CoralLoad PCR Buffer使得PCR产物可直接上样到琼脂糖凝胶,更易于操作,更快获得结果。试剂盒中提供的Q-Solution可进一步提高PCR的性能(参见" Amplification of difficult templates")。

Taq DNA Polymerase的规格

浓度: 5 单位/µl 
重组酶: 是
底物类似物: dNTP、ddNTP、dUTP、biotin-11-dUTP、DIG-11-dUTP和fluorescent-dNTP/ddNTP
延伸速率: 72°C 2–4 kb/min 
半衰期: 97°C 10 min;94°C 60 min
扩增效率: ≥105
5'–>3'外切酶活性: 有
额外添加A: 有
3'–>5'外切酶活性: 无
核酶污染: 无
RNases污染: 无
蛋白酶污染: 无
自引发活性: 无

See figures

Principle

Taq DNA Polymerase是一种高品质重组酶,适合常规和专门的PCR应用(参见" Tolerance of different primer Tm Values" 和" Specific amplification of long PCR products")。

QIAGEN PCR Buffer

研发的新型QIAGEN PCR Buffer可节省时间和精力,减少所需PCR优化。QIAGEN PCR Buffer含有KCl和(NH4)2SO4 。独特的缓冲液便于扩增特异性PCR产物。在每个PCR循环的退火步骤,缓冲液使引物结合具有高特异性比率。KCl和(NH4)2SO4独特比例结合,使PCR缓冲液与常规PCR缓冲液相比,可在更宽范围的退火温度和Mg2+浓度下提供严格的引物退火条件。极大减少通过改变退火温度或Mg2+浓度的PCR优化过程,有时甚至不需要(参见" Wide annealing temperature window" 和" Tolerance to variable magnesium concentration")。

CoralLoad PCR Buffer

CoralLoad PCR Buffer具有QIAGEN PCR Buffer的所有优点。此外,还可直接将PCR反应液上样到琼脂糖凝胶,无需再单独添加凝胶上样缓冲液。与常规QIAGEN PCR Buffer一样,CoralLoad PCR Buffer具有相同的PCR特异性和最少的反应优化。另外,缓冲液还含有两种标记染料:一种橙色染料和一种红色染料,便于估计DNA迁移距离和优化琼脂糖凝胶电泳时间(参见" CoralLoad PCR Buffer")。缓冲液提高了移液可视性,使PCR产物可直接上样到凝胶,提高了便利性。

Q-Solution

Q-Solution通过修饰DNA的熔解行为,便于扩增GC含量高的模板或含有高度二级结构的模板。使用这种独特的试剂常常能完成或改进不理想的PCR(参见" Amplification of difficult templates")。与DMSO和其他PCR添加剂不同,Q-Solution可在多种引物-模板体系中使用特定工作浓度,而不会产生毒性作用。

See figures

Procedure

Taq DNA Polymerase可在多种应用中进行高度特异性的PCR,减少PCR参数的优化过程。该试剂盒简化的、易于操作的流程使PCR反应体系构建更加简单。CoralLoad PCR Buffer也增加便利性和易操作性。PCR产物可直接上样到凝胶中,无需添加上样染料。可提升PCR性能的Q-Solution确保成功处理GC含量高的模板。

Applications

Taq DNA Polymerase适用于常规和特殊的应用,包括:

  • 常规PCR
  • RT-PCR
  • 筛选 
  • 基于PCR的DNA指纹图谱分析(VNTR、STR和RAPD)

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsPCR, RT-PCR, DNA fingerprinting
dntpsincludedNo
realtimeorendpointEndpoint
reactiontypePCR amplification
singleormultiplexSingle
withwithouthotstartWithout hotstart
enzymeactivity5' -> 3' exonuclease activity
mastermixNo
sampletargettypeGenomic DNA and cDNA

Resources

产品介绍与指南 (3)
Second edition — innovative tools
Addressing critical factors and new solutions
快速启动实验方案 (1)
补充实验方案 (1)
试剂盒操作手册 (1)
For standard and specialized PCR applications with minimal optimization

Publications

Increased expression of matrix metalloproteinase-9 in the eutopic endometrial tissue of women with endometriosis.
Collette T; Maheux R; Mailloux J; Akoum A;
Hum Reprod; 2006; 21 (12):3059-67 2006 Jul 31 PMID:16880228
Kaposi sarcoma herpesvirus-encoded vFLIP and vIRF1 regulate antigen presentation in lymphatic endothelial cells.
Lagos D; Trotter MW; Vart RJ; Wang HW; Matthews NC; Hansen A; Flore O; Gotch F; Boshoff C;
Blood; 2006; 109 (4):1550-8 2006 Oct 17 PMID:17047149
Progesterone receptor polymorphism +331G/A is associated with a decreased risk of deep infiltrating endometriosis.
van Kaam KJ; Romano A; Schouten JP; Dunselman GA; Groothuis PG;
Hum Reprod; 2006; 22 (1):129-35 2006 Aug 18 PMID:16920727
Haplotype analysis of the DQA genes in sheep: evidence supporting recombination between the loci.
Hickford JG; Zhou H; Fang Q;
J Anim Sci; 2006; 85 (3):577-82 2006 Nov 22 PMID:17121973
Role of RelGsu in stress response and Fe(III) reduction in Geobacter sulfurreducens.
DiDonato LN; Sullivan SA; Methé BA; Nevin KP; England R; Lovley DR;
J Bacteriol; 2006; 188 (24):8469-78 2006 Oct 13 PMID:17041036

FAQ

How is "Touchdown PCR" used to increase PCR specificity?

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
What should the starting template DNA quality and quantity be for PCR?

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
What kind of PCR products can be cloned with the QIAGEN PCR Cloning Kit?

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
Why do I get smeared PCR products?

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

 

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
Does QIAGEN sell Q-Solution separately?
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 PolymeraseQIAGEN Multiplex PCR-, and the QIAGEN OneStep RT-PCR Kits.
FAQ ID -204
Do CoralLoad dyes supplied in various QIAGEN PCR Kits interfere with downstream applications?

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
Can Taq DNA Polymerase use RNA as a template, and generate false positives in "no-RT" controls?
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
Is Q-Solution required for PCR with QIAGEN's PCR kits?

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 HotStarTag Plus DNA Polymerase, HotStar HiFidelity Polymerase, Taq DNA Polymerase, HotStarTaq DNA Polymerase,  and the QIAGEN Multiplex PCR Handbooks.

FAQ ID -380
How can I avoid primer-dimer formation during PCR amplification?
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
How can I tell if I have primer-dimers in my PCR reaction?
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
What makes QIAGEN's 10x Taq and HotStarTaq DNA Polymerase PCR buffer superior?
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
How comparable is CoralLoad gel loading dye contained in various QIAGEN PCR Kits to Sigma Red?

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
Have you tested the effect of inhibitors on PCR performance?

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
What is the fidelity of TopTaq DNA Polymerase?

The error rate of TopTaq DNA Polymerase is very similar to that of standard Taq DNA Polymerase: approximately 2-3x 10e-5 (per base, per cycle).

 

 

 

 

FAQ ID -1739
What is the composition of the QIAGEN 10x PCR Buffer in Taq- and HotStarTaq DNA Polymerase Kits?

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
How much DNA is obtained in the average PCR reaction?

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
Can QIAGEN's Taq- and HotstarTaq DNA Polymerases be used for cycle sequencing?
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
What is the largest PCR amplicon that can be amplified with the HotStar HiFidelity Polymerase Kit?

In QIAGEN labs, we have amplified PCR products up to 5 kb from complex genomic DNA, and up to 10 kb from less complex lambda phage DNA with the HotStar HiFidelity Polymerase Kit, following standard protocols in the HotStar HiFidelity PCR Handbook.

For targets larger than 5 kb of complex genomic DNA, and larger than 10 kb of less complex DNA, we recommend to follow the protocol 'Amplification of Long PCR Products' in the HotStar HiFidelity PCR Handbook. The protocol uses a mixture of HotStar HiFidelity DNA Polymerase and Taq, or HotStar Taq Plus DNA Polymerase, and allows much longer fragments to be generated. In-house we have tested fragments up to 13 kb from complex genomic DNA or up to 30 kb from less complex lambda phage DNA using this protocol.

 

 

FAQ ID -1047
How can one determine the optimal annealing temperature for a specific PCR assay?

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