HotStarTaq Plus Master Mix Kit

For fast and highly specific amplification in all applications
  • Fast 5-minute enzyme activation time
  • Fewer pipetting steps reduces the risk of contamination
  • High PCR specificity without the need for optimization
  • Optional ready-to-load buffer additive for easier handling
HotStarTaq Plus Master Mix contains HotStarTaq Plus DNA Polymerase, the unique QIAGEN PCR Buffer that minimizes the requirement for optimization, and dNTPs. The HotStarTaq Plus Master Mix Kit provides the same unrivalled highly specific and sensitive PCR as the HotStarTaq Master Mix Kit, but combined with a fast 5-minute enzyme activation time. In addition, CoralLoad Concentrate, containing two gel-tracking dyes, is also provided for improved pipetting visualization and immediate gel-loading of PCR products.
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HotStarTaq Plus Master Mix Kit (250)
For 250 x 20 μl reactions: 3 x 0.85 ml HotStarTaq Plus Master Mix (contains 250 units of HotStarTaq Plus DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 μM of each dNT), 1 x 0.55 ml CoralLoad Concentrate, 2 x 1.9 ml RNase-Free Water
203643
$219.00
HotStarTaq Plus Master Mix Kit (1000)
For 1000 x 20 μl reactions: 12 x 0.85 ml HotStarTaq Plus Master Mix (contains 1000 units of HotStarTaq Plus DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 μM of each dNTP), 4 x 0.55 ml CoralLoad Concentrate, 8 x 1.9 ml RNase-Free Water
203645
$789.00
HotStarTaq Plus Master Mix Kit (2500)
For 2500 x 20 μl reactions: 25 ml HotStarTaq Plus Master Mix (contains 2500 units of HotStarTaq Plus DNA Polymerase total, PCR Buffer with 3 mM MgCl2, and 400 μM of each dNTP), 5.5 ml CoralLoad Concentrate, 2 x 20 ml RNase-Free Water
203646
$1,831.00
The HotStarTaq Plus Master Mix Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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CoralLoad Concentrate.|HotStarTaq Plus procedure.|Highest specificity.|Higher specificity with different primer–template systems.|Effect of hot start on RT-PCR performance.|Specific amplification in multiplex PCR.|Highly sensitive single-cell PCR.|Increased specificity of primer annealing.|
[A] CoralLoad Concentrate contains 2 gel-tracking dyes for improved pipetting visibility during PCR setup, enabling immediate gel loading of PCR products for [B] easy visualization of DNA migration.|The HotStarTaq Plus procedure is fast and easy for maximum convenience.|PCR was performed with HotStarTaq Plus DNA Polymerase, HotStarTaq DNA Polymerase, and Taq DNA Polymerase from QIAGEN, and 3 hot-start PCR enzymes from the indicated suppliers. Parallel reactions were performed following the suppliers' recommendations, using 50 ng human genomic DNA. A 1.5 kb fragment of the human CFTR gene was amplified in 35 PCR cycles. M: markers.|Three different primer-template systems were amplified under the same conditions with either Taq DNA polymerase from Supplier R (R) or with HotStarTaq DNA Polymerase (H). System 1: A 1.1 kb fragment of a D-IgI homolog was amplified from human genomic DNA. System 2: A 296 bp fragment from the chromosomal region correlated with X-linked juvenile retinoschisis was amplified from human genomic DNA. System 3: A 214 bp fragment of the β-actin gene was amplified from cDNA synthesized from total RNA. M: markers. Note: Data are shown for HotStarTaq DNA Polymerase; identical sensitivity and specificity were obtained with HotStarTaq Plus DNA Polymerase.|A 1.1 kb fragment of the human interleukin 1 receptor (type II) gene was amplified from cDNA. Amplification reactions were prepared in triplicate using Taq DNA polymerase and buffer from Supplier L (No hot start); antibody-mediated hot start using enzyme and buffer from Supplier L (Antibody-mediated); and HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq). M: markers. Note: Data are shown for HotStarTaq DNA Polymerase; identical sensitivity and specificity were obtained with HotStarTaq Plus DNA Polymerase.|Fragments from the murine p53 gene were amplified from genomic DNA in multiplex PCR. Parallel reactions were prepared using standard reaction conditions and an enzyme from Supplier R (No hot start) or using HotStarTaq Master Mix Kit from QIAGEN (HotStarTaq Master Mix). M: markers. Note: Data are shown for HotStarTaq Master Mix Kit; identical sensitivity and specificity were obtained with HotStarTaq Plus Master Mix Kit.|Single-cell PCR of a 500 bp fragment of the murine p53 gene was carried out in duplicate using HotStarTaq and HotStarTaq Plus DNA Polymerase. Both enzymes showed equally high specificity and sensitivity. M: markers. Note: Data are shown for HotStarTaq Plus DNA Polymerase; identical sensitivity and specificity were obtained with the HotStarTaq Plus Master Mix Kit.|Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.|
Performance

Each lot of HotStarTaq Plus 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 Plus Master Mix Kit outperforms kits from other suppliers and ensures high specificity and superior performance in hot-start PCR (see figures "Highest specificity" and "Higher specificity with different primer–template systems", and table). The innovative PCR buffer provided with the kit ensures specificity over a wide range of PCR conditions, minimizing the need for optimization. CoralLoad Concentrate, also included with the kit, ensures greater convenience by improving pipetting visibility and allowing direct loading of PCR products onto a gel. CoralLoad Concentrate can be added to the PCR without affecting amplification sensitivity or specificity. PCR fragments amplified in the presence of CoralLoad Concentrate have been successfully tested for cloning and restriction digestion without prior purification.

The combination of high specificity and easy handling makes the HotStarTaq Plus Master Mix Kit suitable for use with complex genomic or cDNA templates (see figure "Effect of hot start on RT-PCR performance"), multiple primer pairs (see figure "Specific amplification in multiplex PCR), and templates isolated from difficult sources or very low-copy targets (see figure "Highly sensitive single-cell PCR"). The HotStarTaq Plus Master Mix Kit is also suitable for projects such as genetic screening, in which large numbers of samples are amplified.

Comparison of hot-start methods 
HotStarTaq Plus DNA Polymerase HotStarTaq DNA Polymerase Hot-start enzyme from Supplier AII Supplier R Supplier I (antibody-mediated) Manual Wax  barrier
Specific amplification ++ ++ + ++ + +/– +/–
Minimal PCR optimization ++ ++ +/– +/– +/–
Easy to use +++ ++ ++ + +
Speed of activation ++ + ++ ++ ++
HotStarTaq Plus 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 

Principle

HotStarTaq Plus Master Mix is a ready-to-use mixture of HotStarTaq Plus DNA Polymerase, QIAGEN PCR Buffer, MgCl2, and dNTPs. HotStarTaq Plus DNA Polymerase provides the unrivaled performance of HotStarTaq DNA Polymerase with a shortened activation time of just 5 minutes.

HotStarTaq Plus DNA Polymerase, a modified form of QIAGEN Taq DNA Polymerase, is supplied in an inactive state that 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 "Highest specificity" and "High specificity with different primer–template systems"). HotStarTaq Plus DNA Polymerase is activated by a short 5-minute incubation at 95°C, which can be easily 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. 

CoralLoad Concentrate

The HotStarTaq Plus Master Mix Kit contains CoralLoad Concentrate (see figure "CoralLoad Concentrate"), which contains a gel-loading reagent and 2 gel-tracking dyes. CoralLoad Concentrate improves pipetting visibility and facilitates estimation of DNA migration distance and optimization of agarose gel run time. When using CoralLoad Concentrate, PCR products can be directly loaded onto an agarose gel without prior addition of loading buffer.

Procedure
HotStarTaq Plus Master Mix Kit is supplied in a convenient master mix format for maximum ease of use. HotStarTaq Plus DNA Polymerase is activated by a 5-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs. Room-temperature reaction setup using the master mix is fast and easy — simply pipet 10 µl HotStarTaq Plus Master Mix into each PCR tube and add 10 µl of primers and template DNA diluted in the RNase-free water provided with the kit (see figure "HotStarTaq Plus procedure"). Pipetting steps are minimized, reducing the possibility of errors and contamination, and ensuring increased throughput and reproducibility. The kit includes a streamlined, optimized protocol for fast and easy PCR setup. CoralLoad Concentrate, also provided with the kit, ensures improved pipetting visibility and enables direct loading of PCR products onto a gel for enhanced convenience.
Applications

The HotStarTaq Plus Master Mix Kit is highly 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
Feature
Specifications
Applications PCR, RT-PCR, Complex genomic templates, very low-copy targets
Enzyme activity 5' -> 3' exonuclease activity
Mastermix Yes
Reaction type PCR amplification
Real-time or endpoint Endpoint
Sample/target type Genomic DNA and cDNA
Single or multiplex Single
With/without hotstart With hotstart

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For highly specific hot-start PCR without optimization  
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至適化不要で特異性の高いホットスタートPCR
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