Cat. No. / ID: 203603
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 DNA Polymerase outperformed kits tested 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. Suboptimal PCR can be improved with Q-Solution, also provided with the kit (see figure " Amplification of difficult templates"). Together, these components ensure specific amplification in a range of applications (see figure " Effect of hot start on RT-PCR performance" and " Highly sensitive single-cell PCR").
|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 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 " Higher 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.
HotStarTaq Plus DNA Polymerase is supplied with CoralLoad PCR Buffer, which has all of the advantages of QIAGEN PCR Buffer but can also be used to directly load the PCR reaction onto an agarose gel without the need to add a gel loading buffer. 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, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA, is also provided with HotStarTaq DNA Polymerase. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or GC-rich templates (see figure " Amplification of difficult templates"). Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed. Adding Q-Solution to the PCR does not compromise PCR fidelity.
HotStarTaq Plus DNA Polymerase 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|
|With/without hotstart||With hotstart|
|Sample/target type||Genomic DNA and cDNA|
|Enzyme activity||5' -> 3' exonuclease activity|
|Reaction type||PCR amplification|
|Single or multiplex||Single|
|Real-time or endpoint||Endpoint|
No. HotStarTaq Plus DNA Polymerase is supplied with conventional QIAGEN PCR Buffer and CoralLoad PCR Buffer in separate vials. Both buffers minimize nonspecific amplification products, primer–dimers, and background.
CoralLoad PCR Buffer has all of the advantages of QIAGEN PCR Buffer but can also be used to directly load the PCR reaction onto an agarose gel without the need to add a gel loading buffer.
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.
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.
Yes. Do not increase the activation time for HotStarTaq Plus DNA Polymerase beyond the recommended 5 minutes, as PCR product yield may be reduced and less specific amplification may result. Please follow the instructions in the HotStarTaq Plus PCR Handbook closely, and use the cycling parameters presented in the handbook to ensure optimal results.
The activation time for HotStarTaq Plus DNA Polymerase used in the QuantiFast SYBR Green PCR Kits is longer than that for QuantiFast Probe PCR Kits. This is due to differences in buffer composition. Buffer components such as salts and additives influence the time required for enzyme activation.
Yes. Separated dyes are visible on an agarose gel down to a volume of 2 µl of PCR product generated with HotStarTaq Plus DNA Polymerase and CoralLoad PCR buffer.
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.