Description
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.
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 A II 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: ≥10 5 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, MgCl 2, 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 (NH 4) 2 SO 4, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg 2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg 2+ 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.
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.
