Taq DNA Polymerase
- QIAGEN PCR缓冲体系最大限度地减少了PCR条件的优化
Cat. No. / ID: 201203
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"）。
浓度: 5 单位/µl
延伸速率: 72°C 2–4 kb/min
半衰期: 97°C 10 min；94°C 60 min
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。
Taq DNA Polymerase适用于常规和特殊的应用，包括：
|applications||PCR, RT-PCR, DNA fingerprinting|
|enzymeactivity||5' -> 3' exonuclease activity|
|sampletargettype||Genomic DNA and cDNA|
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.
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)|
*Absorbance at 260 nm = 1
Molar conversions for nucleic acid templates
|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|
|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
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.
Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:
too much starting templateCheck the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.
carry-over contaminationIf 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 highWhen using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.
too many PCR cyclesReduce the number of cycles in steps of 3 cycles.
Mg2+ concentration not optimalPerform 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 degradedRepeat 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.
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.
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
|Sodium Acetate||≥5 mM|
|Sodium Chloride||≥25 nM|
|RT reaction mixture||≥15%|
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.
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.
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.