TopTaq Master Mix Kit

For highly reliable end-point PCR with unrivaled ease of use

  • Ready-to-use master mix with minimal pipetting steps
  • Fridge storage eliminates thawing time
  • Reliable high-yield PCR performance
  • Convenient room-temperature handling
  • Optimized protocol suitable for all PCR assays
The TopTaq Master Mix Kit offers maximum convenience by combining all the benefits of specific TopTaq DNA Polymerase with the advantage of a ready-to-use master mix. It contains TopTaq DNA Polymerase, the unique TopTaq PCR Buffer that minimizes the requirement for optimization, and dNTPs. The unique TopTaq Stabilizer included in the enzyme storage buffer allows storage of TopTaq Master Mix at 4°C and reaction setup at room temperature, without time-consuming thawing of reagents. Providing all components in a ready-to-use master mix reduces pipetting steps, which lowers the risk of contamination. High yields of PCR product are achieved, even after storing the TopTaq Master Mix for extended periods of time at 25°C or 4°C.
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TopTaq Master Mix Kit (250)
For 200 x 50 µl reactions: 2x TopTaq Master Mix containing 250 units of TopTaq DNA Polymerase in total, 10x CoralLoad Concentrate, and RNase-Free Water
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The TopTaq 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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Reliable high-yield PCR, independent of storage conditions.|Convenient CoralLoad Concentrate.|TopTaq procedure.|High yields of PCR product without optimization|Increased specificity of primer annealing.|Stabilization at 4°C.|
PCR was performed using TopTaq Master Mix stored at –20°C, 4°C, and 25°C for 4 months. Two different genes from human genomic DNA (prp, 750 bp and HugL, 1200 bp) were amplified according to the standard optimized protocol (duplicates shown). The results show high yields of specific PCR product. [A] Gel image produced by the QIAxcel System. Markers: GelPilot 1 kb Plus Marker and QX Alignment Marker 15 bp/10 kb. [B] The QIAxcel System fully automates sensitive, high-resolution capillary electrophoresis of up to 96 samples per run.|[A] CoralLoad Concentrate contains two gel-tracking dyes, enabling immediate gel loading of PCR products for [B] easy visualization of DNA migration.|Due to the unique proprietary TopTaq Stabilizer contained in the enzyme storage buffer, TopTaq DNA Polymerase is the first PCR kit that can be stored at 4°C. TopTaq Stabilizer binds to TopTaq DNA Polymerase, preventing polymerase inactivation. Template DNA and primers are also prevented from binding to the polymerase at low temperatures. During the initial denaturation step, the TopTaq Stabilizer freely dissociates from the polymerase without compromising polymerase activity. This added convenience allows immediate reaction setup without the use of ice — saving the time usually required for reagent thawing.|[A] TopTaq DNA Polymerase was used to amplify 14 different PCR products from mammalian genomic DNA ranging in size from 100 bp to 2 kb according to the standard preoptimized protocol and using identical cycling conditions. [B] The same four targets (shown in lanes 11–14) were amplified under identical cycling conditions using DNA polymerases from the indicated suppliers. TopTaq DNA Polymerase (QIAGEN) resulted in high yields of specific PCR products, regardless of fragment size.|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.|[A] TopTaq DNA Polymerase is stable at 4°C or room temperature due to the presence of low-molecular-weight molecules (TopTaq Stabilizer) on the surface of the polymerase. [B] At higher temperatures, the TopTaq Stabilizer completely dissociates from the polymerase without compromising polymerase activity.|
Performance
TopTaq Master Mix Kit is used for standard and specialized end-point PCR applications without the need for optimization. High yields of PCR product are achieved, even after storing TopTaq Master Mix for 4 months at 25°C or 4°C, demonstrating the fact that TopTaq Master Mix offers a very robust and reliable amplification system (see figure Reliable high-yield PCR). TopTaq Stabilizer binds to TopTaq DNA Polymerase, preventing polymerase inactivation during long-term storage. With its balanced potassium and ammonium ions, the unique PCR buffer included in the master mix promotes specific primer–template annealing and simultaneously reduces nonspecific annealing. TopTaq DNA Polymerase outperformed DNA polymerases tested from other suppliers and ensures high yields of specific products, even when using low template amounts, and independent of fragment size (see figure High PCR product yields without optimization).
Principle

TopTaq Master Mix Kit has been developed by QIAGEN to provide highly reliable end-point PCR with unrivaled ease of use. Until the development of the TopTaq PCR system, all PCR enzymes required storage at –20°C. Due to the unique proprietary TopTaq Stabilizer contained in the enzyme storage buffer, TopTaq Master Mix Kit is the first PCR kit that can be stored at 4°C. This ready-to-use master mix format ensures greater convenience. High yields of specific products are achieved due to TopTaq DNA Polymerase, even when amplifying fragments of different sizes (see figure "High PCR product yields without optimization").

TopTaq PCR Buffer

The innovative TopTaq PCR Buffer facilitates the amplification of specific PCR products. During the annealing step of every PCR cycle, the buffer greatly increases the ratio of specific primer binding over 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 temperatures and Mg2+ concentrations than conventional PCR buffers (see figure "Increased specificity of primer annealing"). The need to optimize PCR by varying the annealing temperature or the Mg2+ concentration is dramatically reduced, or often not required.

TopTaq Stabilizer

TopTaq Stabilizer binds to TopTaq DNA Polymerase, preventing polymerase inactivation during long-term storage (see figure "Stabilization at 4°C"). Template DNA and primers are also prevented from binding to the polymerase at low temperatures. During the initial denaturation step, TopTaq Stabilizer freely dissociates from the polymerase without compromising polymerase activity.  

CoralLoad Concentrate

For enhanced convenience, TopTaq Master Mix Kit includes CoralLoad Concentrate. CoralLoad Concentrate contains a gel-loading reagent and two gel-tracking dyes. This unique reagent 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 (see figure "Convenient CoralLoad Concentrate").

Procedure
The TopTaq Master Mix Kit offers all of the benefits of specific TopTaq DNA Polymerase combined with the advantage of a ready-to-use master mix. With the TopTaq Master Mix Kit, separate pipetting of individual components is not required and the risk of contamination and pipetting variability is minimized, making PCR setup quick and easy. During the initial denaturation step, the TopTaq Stabilizer freely dissociates from the polymerase without compromising polymerase activity. This added convenience allows immediate reaction setup without the use of ice — saving the time usually required for reagent thawing (see flowchart "TopTaq procedure"). 
Reliable high-yield PCR without the need for optimization

The optimized protocol suitable for all PCR assays eliminates the need for lengthy optimization of experimental parameters for individual primer–template systems, resulting in substantial time and cost savings. High yields of specific PCR product are achieved, even after storing the TopTaq Master Mix for 4 months at 25°C or 4°C (see figure "Reliable high-yield PCR").

Applications
The TopTaq Master Mix Kit is highly suited to all end-point PCR applications.
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Feature
Specifications
Applications PCR, RT-PCR, DNA Fingerprinting
dNTP's included Yes (in Master Mix)
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
Storage conditions Fridge storage and room temperature setup
With/without hotstart Without Hotstart

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Kit Handbooks
1
TopTaq PCR Handbook - (EN)
For standard and specialized end-point PCR applications without the need for optimization
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