QIAGEN Fast Cycling PCR Kit

모든 thermal cycler를 이용하여 빠르고 특이성이 높은 PCR을 수행할 수 있습니다

Features

  • 초고속 hot-start PCR — 최대 75%의 시간절약
  • 어떤 thermal cycler에도 적합함— 모든 실험실에 적용 가능
  • High specificity and sensitivity — optimization 필요 없이 성공적인 결과를 얻을 수 있음
  • 쉽고 편리하게 사용 — 선택적인 PCR buffer, primer를 다시 제작하지 않아도 됨
  • No need to redesign primers for fast PCR

Product Details

The fast-cycling PCR master mix contains HotStarTaq Plus DNA Polymerase for highly specific and sensitive PCR, and a unique buffer formulation for extremely short denaturation, annealing, and extension steps. The proprietary PCR buffer significantly reduces the time required to form the polymerase, primer, and template complex, reducing the total PCR cycling time from approximately 1.5 hours to just 20 minutes. Optional CoralLoad Dye (also included with the kit) contains gel-tracking dyes for convenient analysis. In addition, Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC rich) templates, is also provided.

Performance

The QIAGEN Fast Cycling PCR Kit combines the high performance of HotStarTaq Plus DNA Polymerase with a significantly shortened PCR cycling time on any thermal cycler. Unlike other fast-cycling chemistries, which often result in amplification failure, HotStarTaq Plus DNA Polymerase in combination with the QIAGEN Fast Cycling PCR Buffer, promotes specific primer–template annealing and simultaneously reduces nonspecific annealing (see figure " Specific and reliable results"). Amplification of nonspecific products, primer dimer formation, and background smear are minimized in every PCR cycle. Time savings of up to 75% can be achieved using a novel, proprietary PCR buffer formulation. PCR cycling times can be reduced from over 1 hour (for 35 cycles) to less than 20 minutes. Fast-ramping cyclers can achieve a total PCR time of just 30 minutes (see figure " Time savings of up to 75%"). Suboptimal PCR can be improved with Q-Solution, a novel additive also provided with the kit.
See figures

Principle

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
Kit componentFeatures
HotStarTaq Plus DNA Polymerase Highly specific products ( up to 3.5 kb) 
High sensitivity 
Room-temperature setup
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.

CoralLoad Dye

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.

Q-Solution

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.

See figures

Procedure

The QIAGEN Fast Cycling PCR 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. 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 Dye, also provided with the kit, ensures improved pipetting visibility and enables direct loading of PCR products onto a gel for enhanced convenience.

PCR cycling times can be reduced from over 1 hour (for 35 cycles) to less than 20 minutes (see figure " Significant time savings" and table "PCR cycling times for different fragment lengths"). Fast-ramping cyclers can achieve a total PCR time of just 30 minutes (see figure " Time savings of up to 75%"). The time savings afforded by the QIAGEN Fast Cycling PCR Kit make it highly suitable for increased PCR throughput — purchase of additional and expensive lab equipment is not required.

Significant time savings

The QIAGEN Fast Cycling PCR Kit allows increased PCR throughput using existing lab equipment. It can be used with any thermal cycler, including fast ramping cyclers, without loss of specificity (see figure " Time savings of up to 75% "). In addition, unlike other PCR chemistries, there is no need to modify or change primers or annealing temperatures. Each kit component has been specifically designed to provide convenient and reliable fast-cycling PCR (see figure " Easy and convenient procedure" and see table).

PCR cycling times for different fragment lengths
Fragment length (bp) QIAGEN Fast Cycling procedure (min) Standard cycling procedure (min)Time savings (%)
200 15 68 78
500 20 68 71
1000 29 85 66
3000 63 155 59
See figures

Applications

The QIAGEN Fast Cycling PCR Kit enables increased PCR throughput on any thermal cycler and is suitable for highly sensitive and specific PCR, as well as general PCR, and for use with complex genomic and cDNA templates. The kit highly suited for use with standard, as well as fast-ramping thermal cyclers.

Supporting data and figures

Specifications

FeaturesSpecifications
Real-time or endpointEndpoint
ApplicationsKOPCR, RT-PCR, Complex genomic templates, very low copy targets
Sample/target typeGenomic DNA and cDNA
With/without hotstartWith hotstart
Reaction typePCR amplification
MastermixYes
Single or multiplexSingle
Enzyme activity5' -> 3' exonuclease activity

Resources

Certificates of Analysis (1)

FAQ

Do you have a protocol for polyacrylamide gel analysis of oligonucleotides?
Yes, please follow the Supplementary Protocol 'Polyacrylamide_gel_analysis_of_oligonucleotides' (PCR03).
FAQ ID -961
How do you achieve fast cycling, yet still deliver the same performance in PCR as that achieved with standard cycling?

Our unique multiplex PCR buffer system with ammonium and potassium ions and Factor MP has been further optimized in QuantiFast and Rotor-Gene Kits. We have also discovered Q-Bond, a buffer component which supports the rapid formation of the polymerase–primer–template complex, leading to reduced annealing times.

FAQ ID -1430
What is Q-Bond used in the QIAGEN Fast Cycling PCR and QuantiFast Kits?

The Q-Bond Molecule, present in the PCR Buffer of the QIAGEN Fast Cycling PCR Kit and the QuantiFast Kits, dramatically increases the binding affinity of DNA polymerase to single-stranded DNA, thereby facilitating the reduction of annealing time to just a few seconds. It is a non-protein PCR component. Unfortunately, all further information on this molecule is proprietary.

FAQ ID -1554