For fast, real-time PCR and two-step qRT-PCR using SYBR Green
Specific and sensitive detection of even low-copy targets
Optimized master mix for reliable results without optimization
5-minute enzyme activation and fast cycling conditions
Accurate detection of a wide range of template amounts
Universal protocol for all standard and fast cyclers
The QuantiFast SYBR Green PCR Kit delivers fast and specific quantification of gDNA or cDNA targets by real-time PCR or two-step RT-PCR using SYBR Green I detection. Q-bond technology and an optimized, ready-to-use master mix enable shorter real-time PCR run times, not only on fast cyclers with short ramping times, but also on standard cyclers. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. For convenience, the master mix can be stored at 2–8°C.
For 4000 x 25 µl reactions: 2 x 25 ml 2x QuantiFast SYBR Green PCR Master Mix (contains ROX dye), 20 ml RNase-Free Water
The QuantiFast SYBR® Green PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Significantly reduced PCR times.
The QuantiFast SYBR® Green PCR Kit reduces total PCR run time by up to 60% in real-time two-step RT-PCR on standard cyclers (40 cycles without melting curve analysis; comparison with standard QIAGEN real-time PCR kits). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000.
Resolution of small differences in copy number.
The QuantiFast SYBR® Green PCR Kit was used to detect the Y-chromosome-specific single-copy gene SRY in genomic DNA from a male donor using the Mastercycler ep realplex. [A] Curves for 1000 down to 1.5 copies can be clearly distinguished from each other. [B] A plot of copy number (log) versus CT value demonstrates high linearity.
Faster results without compromising sensitivity.
Expression of MYC (a proto-oncogene) in human leukocytes was analyzed by real-time two-step RT-PCR on the Applied Biosystems 7500 Fast System. Duplicate reactions were run using 10-fold cDNA dilutions (10 ng to 10 pg). [A] Fast cycling mode with the QuantiFast Kit gave similar CT values to [B] standard cycling mode with the QuantiTect Kit. In contrast, a kit for standard cycling from Supplier AII gave worse CT values not only in [C] fast cycling mode, but also in [D] recommended standard cycling mode.
Sensitive two-step RT-PCR.
Expression of MYC (a proto-oncogene) in human leukocytes was analyzed using the iCycler iQ and [A] the QuantiFast SYBR® Green PCR Kit or [B] a kit from Supplier Bv. Duplicate reactions were run using 10-fold cDNA dilutions (100 ng to 0.1 ng). The instrument-dedicated kit from Supplier BV was used according to the fast cycling protocol. The QuantiFast Kit gave lower CT values, indicating greater sensitivity.
Specific primer annealing.
A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.
Fast primer annealing.
[A] Q-Bond in QuantiFast Buffer increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.
Performance
The QuantiFast SYBR Green PCR Kit delivers highly specific and sensitive results, outperforming other real-time PCR kits used in fast cycling mode (see figure "Sensitive two-step RT-PCR"). PCR run times are reduced by up to 60% (see figure "Significantly reduced PCR times"), allowing you to get results faster without compromising PCR performance (see figure "Faster results without compromising sensitivity"). You can also greatly increase your sample throughput or efficiently share a cycler with other users.
The QuantiFast SYBR Green PCR Kit enables accurate quantification of targets over several log dilutions of template. Even small differences in the amount of low-copy targets can be clearly distinguished (see figure "Resolution of small differences in copy number").
Principle
The QuantiFast SYBR Green PCR Kit delivers highly sensitive and specific results over a wide dynamic range on both standard and fast cyclers. The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. A specially developed fast PCR buffer contains the novel PCR additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing"). A balanced combination of K+ and NH4+ ions in the PCR buffer promotes specific primer annealing and enables high PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, HotStarTaq Plus DNA Polymerase requires only 5 minutes at 95°C for activation and provides a stringent hot start, preventing the formation of nonspecific products.
Components of 2x QuantiFast SYBR Green PCR Kit*
Component
Features and benefits
Benefits
HotStarTaq Plus DNA Polymerase
5 min activation at 95ºC
Set up of qPCR reactions at room temperature
QuantiFast SYBR Green PCR Buffer
Balanced combination of NH4+ and K+ ions
Specific primer annealing ensures reliable PCR results
Unique Q-Bond additive
Faster PCR run times, enabling faster results and more reactions per day
SYBR Green I dye
Yields a strong fluorescent signal upon binding to double-stranded DNA
Highly sensitive amplification
ROX dye
Normalizes fluorescent signals on Applied Biosystems and, optionally, Agilent instruments
Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler
* Also contains a dNTP mix (dATP, dCTP, dGTP, dTTP).
Procedure
The QuantiFast SYBR Green PCR Kit is a ready-to-use master mix that eliminates the need for optimization of reaction and cycling conditions. Simply add primers and DNA template to the ready-to-use PCR master mix, and start the reaction. Follow the protocol in the handbook to get fast and reliable results on any real-time cycler.
For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit. The kit provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.
We also recommend QuantiTect Primer Assays for gene expression analysis using SYBR Green. QuantiTect Primer Assays are bioinformatically validated primer sets for any gene from human, mouse, rat, and many other species. Assays can be easily ordered online at the GeneGlobe Web portal.
Applications
The QuantiFast SYBR Green PCR Kit is for use in gene expression analysis of cDNA targets and quantitative gDNA analysis. QuantiFast SYBR Green PCR Kits are compatible with all available real-time cyclers, including instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene SYBR Green PCR Kit, which has been specially developed for fast cycling on these instruments.
Features
Specifications
Applications
SYBR Green-based, real-time PCR, two-step RT-PCR
Reaction type
Real-time and two-step RT-PCR
Real-time or endpoint
Real-time
Sample/target type
cDNA, DNA
Single or multiplex
Single
SYBR Green I or sequence-specific probes
SYBR Green I
Thermal cycler
Applied Biosystems, Bio-Rad, Cepheid, QIAGEN, Eppendorf, Roche, and Agilent
For 200 x 20 µl reactions: 4 x 100 µl 7x gDNA Wipeout Buffer, 4 x 50 µl Quantiscript Reverse Transcriptase, 4 x 200 µl 5x Quantiscript RT Buffer, 4 x 50 µl RT Primer Mix, 4 x 1.9 ml RNase-Free Water
Nonsterile polypropylene tubes (0.85 ml maximum capacity, less than 0.7 ml storage capacity, 0.4 ml elution capacity); 2304 in racks of 96; includes cap strips
For 100 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 200 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 1 ml), dNTP Mix (1 x 200 µl, 10 mM each), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml)
