The QIAGEN Fast Cycling PCR Kit is provided in a master mix format, which includes all of the components necessary for fast and specific amplification on every cycler (see table). PCR products obtained from genomic DNA and cDNA, including complex templates, up to 3.5 kb can be reliably amplified. High specificity is ensured due to HotStarTaq Plus DNA Polymerase. Significant time savings of up to 75% can be achieved using a novel, proprietary PCR buffer formulation that significantly reduces the time required to form the polymerase, primer, and template complex. This allows the polymerase to extend the primers during the PCR annealing step thereby dramatically reducing the duration of annealing steps in every PCR cycle. PCR cycling times can be reduced from over 1 hour (for 35 cycles) to less than 20 minutes (see figure " Significant time savings"). Fast-ramping cyclers can achieve a total PCR time of just 30 minutes (see figure " Time savings of up to 75%").
Everything required for reliable fast-cycling PCR
|HotStarTaq Plus DNA Polymerase ||Highly specific products ( up to 3.5 kb) |
|Fast Cycling PCR Buffer (including Q-Bond Molecule) ||High speed |
Minimal optimization required
|CoralLoad Dye ||Immediate gel-loading of PCR products|
Improved pipetting visualization
|Q-Solution ||Facilitates amplification of GC-rich templates|
|QIAGEN Fast Cycling PCR Master Mix ||Convenient and fast reaction setup|
High specificity and sensitivity
HotStarTaq Plus DNA Polymerase, provided in the master mix, ensures unrivaled hot-start PCR performance. HotStarTaq Plus DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer dimers at low temperatures (see figure " Specific and reliable results") and also enables convenient room-temperature reaction setup. 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.
Unique fast-cycling PCR buffer
Novel Fast Cycling PCR Buffer facilitates amplification of specific PCR products with significantly reduced cycling time. Based on the original QIAGEN PCR Buffer, this new formulation enables a high ratio of specific-to-nonspecific primer binding during the short annealing step of every PCR cycle. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR 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 dramatically reduced and often not required.
The innovative fast-cycling buffer also contains the novel Q-Bond Molecule, which dramatically increases the binding affinity of DNA polymerase to single-stranded DNA, thereby facilitating the reduction of annealing time to just 5 seconds (see figure " Mechanism of fast cycling during annealing"). The unique buffer formulation and optimized DNA polymerase concentration also enables a significant reduction in the denaturation and extension times. Fast Cycling PCR Buffer has been developed to eliminate the need for optimization of individual primer–template systems, resulting time and cost savings. Specific amplification is maintained over a wide range of temperatures and Mg2+ concentrations, without the need for time-consuming optimization.
The QIAGEN Fast Cycling PCR Kit is supplied with CoralLoad Dye, which enables direct loading of the reaction onto an agarose gel without the need to add a gel-loading buffer (see figure " Ready-to-load PCR reactions"). CoralLoad Dye 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. The dye ensures improved pipetting visibility and enables direct loading of PCR products onto a gel for enhanced convenience.
The QIAGEN Fast Cycling PCR Kit also provides Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or GC-rich 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.