TaqDNA Polymerase

標準的および特殊なPCR 用

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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

PLN 971.00

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Quantity

250 U

1000 U

5000 U

25,000 U

数量

TaqDNA Polymeraseは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

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特徴

PCR条件の至適化が最小限で済むQIAGEN PCR Buffer
直接ゲルにロード可能なPCR バッファーでより迅速な操作
GC リッチなテンプレートの増幅を促進するQ-Solution
簡便で取り扱いやすいフォーマット

製品詳細

Taq DNA Polymeraseには、PCR条件の至適化が最小限で済む画期的なQIAGEN PCR Bufferと、増幅困難なテンプレート（GC リッチなど）の効率的な増幅を実現する画期的なQ-Solution が含まれています。さらに2 種類のマーカー色素を含むCoralLoad PCR Buffer も同梱されているので、PCR 産物を即座にゲルにアプライできます。

パフォーマンス

Taq DNA Polymeraseは、他メーカーの試験されたキットに比べて優れており、時間のかかる至適化を必要とせずに、様々なPCR条件で確固たるPCR性能を示します（図" T_m 値の異なるプライマーへの適応性" と" 長いPCR産物の特異的増幅"）。TaqDNA Polymeraseは、全てのロットで、低コピー数のヒトゲノムDNAをターゲットとした増幅実験により、PCR特異性の厳密さや再現性をチェックするなど、広範囲な品質管理テストを受けています（図 " ロット間での再現性"）。同様にキットに含まれているQIAGEN PCR BufferとCoralLoad PCR Bufferのユニークな組成によって、最小限の至適化に必要な事項と様々なPCR条件で、特異性の高いPCRが可能となります（図" 幅広い至適アニーリング温度"と " 異なったマグネシウム濃度への適応"）。さらに、CoralLoad PCR Bufferは、PCR産物をアガロースゲルに即座にロードすることを可能にするため、比較的簡単な操作で比較的早く結果が得られます。また、キット付属のPCR 添加物、Q－Solutionを使用すれば、不適切なPCR条件を改善することができます（図 " 増幅困難なテンプレートの増幅"）。

Taq DNA Polymeraseの詳細データ

濃縮：5 units/µl
組み換え酵素:はい
基質類似体： dNTP、ddNTP、dUTP、ビオチン-11-dUTP、DIG-11-dUTP、蛍光-dNTP/ddNTP
エクステンション率72℃ で2～4 kb/分
半減期：97℃で10分、94℃で60分
増幅効率：≥10⁵ 倍
5'->3' エキソヌクレアーゼ活性：あり
余分なA付加：あり
3'->5' エキソヌクレアーゼ活性：なし
混入しているヌクレアーゼ：なし
混入しているRNase：なし
混入しているプロテアーゼ：なし
自己プライミング活性:なし

図参照

Tolerance of different primer T_m values.

Specific amplification of long PCR products.

Lot-to-lot reproducibility.

A. Wide annealing temperature window. B. Tolerance of variable magnesium concentration.

Amplification of difficult templates with Q-Solution.

原理

Taq DNA Polymerase は、スタンダードなPCRから特殊なPCRまで適応できる高品質の組み換え酵素です（図 " T_m 値の異なるプライマーへの適応性" と" 長いPCR産物の特異的増幅"）。

QIAGEN PCR Buffer

革新的なQIAGEN PCR Bufferは、PCR至適化の操作を軽減することにより、時間と労力を節約するために開発されました。QIAGEN PCR Bufferは、KCl も (NH₄)₂SO₄ も含んでいます（図" プライマーのアニーリングにおける特異性が増加"）。このユニークなバッファーによって、特異的PCRの産物を増幅しやすくなります。PCR サイクルごとのアニーリングの間、非特異的なプライマー結合に対する特異的な結合の比率が、バッファーによって高く保たれます。このPCRバッファーは、KClと (NH₄)₂SO₄ のユニークな配合比により、従来のPCR バッファーに比べ幅広いアニーリング温度やMg²⁺濃度の範囲で厳密なプライマーアニーリング条件を実現します。異なるアニーリング温度あるいはMg²⁺濃度を用いて行なうPCRの至適化は、劇的に緩和され、多くの場合、不要となります（図 " 幅広い至適アニーリング温度"および" 異なったマグネシウム濃度への適応 "）。

CoralLoad PCR Buffer

CoralLoad PCR Bufferは、QIAGEN PCR Bufferの全ての特長を持っています。さらに、PCR反応液をアガロースゲルに直接ロードするために使用することができ、従って、ゲルローディングバッファーを別途加える必要はありません。CoralLoad PCR Bufferは従来のQIAGEN PCR Bufferと同程度の高いPCR特異性を持ち、反応の至適化は最小限に抑えられます。さらに、これに入っている2種類のマーカー色素（オレンジ色の色素と赤色の色素）により、DNAの移動距離の予測とアガロースゲルの泳動時間の至適化を容易に行なえます（図" CoralLoad PCR Buffer"）。本バッファーはピペッティングの目視化を改良し、PCR産物をゲルに直接ロードすることが可能なので、簡便性が増加します。

Q-Solution

Q-Solution は、DNAの溶解挙動を変えることで、GCリッチなテンプレート、または二次構造が複雑なテンプレートを増幅しやすくします。この試薬の添加により、しばしば最適条件ではないPCRが改善されたり、今まで得られなかったPCR産物の増幅が可能になったりします（図 " 増幅困難なテンプレートをQ-Solutionにより増幅"）。DMSO や他のPCR添加物と異なり、本溶液はどのようなプライマー／テンプレートシステムでも一定の濃度で作用し、毒性もありません。

図参照

Tolerance of different primer T_m values.

Specific amplification of long PCR products.

NH₄⁺ and K⁺ cations in QIAGEN PCR buffers increase specific primer annealing.

NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing.

A. Wide annealing temperature window. B. Tolerance of variable magnesium concentration.

CoralLoad PCR Buffer.

Amplification of difficult templates with Q-Solution.

操作手順

Taq DNA Polymeraseは、様々なアプリケーションで、PCRパラメータの至適化を最小限に抑えながら、特異性の高いPCRを確実にします。キット付属のプロトコールが簡潔で理解しやすいため、PCRセットアップが簡単です。利便性を加え、取り扱いをさらに容易にするために、CoralLoad PCR Bufferが含まれています。これによりローディング色素を加えずに、PCR産物をゲルに直接ロードすることができます。GCリッチなテンプレートで確実に成功するために、PCRを増進するQ-Solution が同梱されています。

アプリケーション

TaqDNA Polymeraseは以下のようなスタンダードな用途にも特殊な用途にも使用できます。

スタンダードな PCR
RT-PCR
スクリーニング
PCRによるDNAフィンガープリンティング（VNTR、STR、RAPD）

裏付けデータと数値

Lot-to-lot reproducibility.

A fragment of the single-copy gene for cystic fibrosis was amplified from 30 ng, 3 ng, and 300 pg human genomic DNA corresponding to 104, 103, and 102 copies of target template, respectively. Three different lots of QIAGEN Taq DNA Polymerase were used and equal volumes of the PCR product were analyzed on a 1% agarose gel. M: markers.

Specifications

Features	Specifications
ApplicationsJA	PCR, RT-PCR, DNA fingerprinting
dNTP's included	No
Real-time or endpoint	Endpoint
Reaction type	PCR amplification
Single or multiplex	Single
With/without hotstart	Without hotstart
Enzyme activity	5' -> 3' exonuclease activity
Mastermix	No
Sample/target type	Genomic DNA and cDNA

リソース

Second edition — innovative tools

Addressing critical factors and new solutions

Download Safety Data Sheets for QIAGEN product components.

For standard and specialized PCR applications with minimal optimization

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

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 HotStarTag 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

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

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

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 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

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

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

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