Cat. No. / ID: 203646
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 HotStarTaqPlus 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 HotStarTaqPlus Master Mix Kit is also suitable for projects such as genetic screening, in which large numbers of samples are amplified.
|HotStarTaqPlus DNA Polymerase||HotStarTaq DNA Polymerase||Hot-start enzyme from Supplier AII||Supplier R||Supplier I (antibody-mediated)||Manual||Wax barrier|
|Minimal PCR optimization||++||++||+/–||+/–||+/–||–||–|
|Easy to use||+++||++||++||+||+||–||–|
|Speed of activation||++||+||++||++||++||–||–|
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
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 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.
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.
The HotStarTaq Plus Master Mix Kit is highly suitable for a wide variety of applications, including challenging applications such as amplification of:
|Applications||PCR, RT-PCR, Complex genomic templates, very low-copy targets|
|Enzyme activity||5' -> 3' exonuclease activity|
|Sample/target type||Genomic DNA and cDNA|
|Single or multiplex||Single|
|Real-time or endpoint||Endpoint|
|With/without hotstart||With hotstart|
|Reaction type||PCR amplification|
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.
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.