Taq DNA Polymerase

For standard and specialized PCR applications

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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

Quantity

250 U

1000 U

5000 U

25,000 U

Inquire

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

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특징

QIAGEN PCR Buffer for minimal optimization
빠르고 편리함을 제공하는 CoralLoad PCR loading buffer가 들어있음
혁신적인 Q-Solution으로 GC-rich templates 또는 extensive secondary structure template도 증폭가능
쉽고 간편한 조작을위해 format선택이 가능함

제품 세부 정보

Taq DNA Polymerase is supplied with the unique QIAGEN PCR Buffer that minimizes the need for optimization of PCR parameters, as well as Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC rich) templates. In addition, CoralLoad PCR Buffer (containing two gel-tracking dyes) is also provided, enabling immediate loading of PCR products.

성능

Taq DNA Polymerase outperformed kits tested from other suppliers and delivers robust PCR performance in a wide range of PCR conditions, without the need for time-consuming optimization (see figures " Tolerance of different primer T_m Values" and " Specific amplification of long PCR products"). Every lot of Taq 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 from human genomic DNA (see figure " Lot-to-lot reproducibility"). The unique formulation of QIAGEN PCR Buffer and CoralLoad PCR Buffer, also provided with the kit, enable highly specific PCR in a variety of PCR conditions with minimal optimization requirements (see figures " Wide annealing-temperature window" and " Tolerance to variable magnesium concentration"). In addition, CoralLoad PCR Buffer enables immediate loading of PCR products onto an agarose gel for even easier handling and faster results. Suboptimal PCR can be improved using Q-Solution, a PCR additive, also provided with the kit (see figure " Amplification of difficult templates").

Taq 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

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.

원리

Taq DNA Polymerase is a high-quality recombinant enzyme that is suitable for general and specialized PCR applications (see figures " Tolerance of different primer T_m Values" and " Specific amplification of long PCR products").

QIAGEN PCR Buffer

Innovative QIAGEN PCR Buffer has been developed to save time and effort by reducing the need for PCR optimization. QIAGEN PCR Buffer contains both KCl and (NH₄)₂SO₄ (see figure " Increased specificity of primer annealing"). This unique buffer facilitates the amplification of specific PCR products. During the annealing step of every PCR cycle, the buffer allows a high ratio of specific-to-nonspecific primer binding. Owing to a uniquely balanced combination of KCl and (NH₄)₂SO₄, the PCR 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 dramatically reduced and often not required (see figures " Wide annealing temperature window" and " Tolerance to variable magnesium concentration").

CoralLoad PCR Buffer

CoralLoad PCR Buffer has all the advantages of QIAGEN PCR Buffer. In addition, it can also be used to directly load the PCR reaction onto an agarose gel — separate addition of a gel loading buffer is not required. CoralLoad PCR Buffer provides the same high PCR specificity and minimal reaction optimization as the conventional QIAGEN PCR Buffer. Additionally, it contains two marker dyes — an orange dye and a red dye — that facilitate estimation of DNA migration distance and optimization of agarose gel run time (see figure " CoralLoad PCR Buffer"). The buffer ensures improved pipetting visibility and enables direct loading of PCR products onto a gel, for enhanced convenience.

Q-Solution

Q-Solution facilitates amplification of GC-rich templates or templates with a high degree of secondary structure by modifying the melting behavior of DNA. Use of this unique reagent often enables or improves suboptimal PCR (see figure " Amplification of difficult templates"). Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primer–template system and is not toxic.

그림 참조

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.

절차

Taq DNA Polymerase ensures highly specific PCR for a range of applications — with minimal optimization of PCR parameters. The streamlined, easy-to-follow protocol provided with the kit simplifies PCR setup. For added convenience and easier handling, CoralLoad PCR Buffer is provided. PCR products can be directly loaded onto a gel without the addition of a loading dye. To ensure success with GC-rich templates, the PCR enhancer Q-Solution is included.

응용 분야

Taq DNA Polymerase is used for standard and specialized applications, including:

General PCR
RT-PCR
Screening
PCR-based DNA fingerprinting (VNTR, STR, and RAPD)

지원되는 데이터 및 수치

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
Applications	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

리소스

Second edition — innovative tools

Addressing critical factors and new solutions

PCR 実験における重要項目と新技術

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

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 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

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