QuantiTect SYBR® Green PCR Kits

For real-time PCR and two-step RT-PCR using SYBR Green

  • High PCR specificity with integrated hot start
  • Reliable quantification of low-abundance transcripts
  • Accurate quantification over several logs of template
  • Available with or without uracil-N-glycosylase (UNG)
  • No need to optimize reaction and cycling conditions

The QuantiTect SYBR Green PCR Kit provides highly specific quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using SYBR Green I detection. The combination of a hot start and a unique qPCR buffer system ensures highly specific and sensitive real-time quantification of gDNA and cDNA targets. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C.

Quantity Product Cat. no. Price
 
 
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Quantity Product Cat. no. Price Sum
 
QuantiTect SYBR Green PCR Kit (40)
Trial kit for 40 x 50 µl reactions: 1 ml 2x QuantiTect SYBR Green PCR Master Mix, 2 ml RNase-Free Water
204141
$117.00
$0.00
 
QuantiTect SYBR Green PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect SYBR Green PCR Master Mix, 2 x 2 ml RNase-Free Water
204143
$426.00
$0.00
 
QuantiTect SYBR Green PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect SYBR Green PCR Master Mix, 20 ml RNase-Free Water
204145
$1,708.00
$0.00
 
QuantiTect SYBR Green PCR +UNG Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect SYBR Green PCR Master Mix, 100 µl UNG Solution, 2 x 2 ml RNase-Free Water
204163
$495.00
$0.00

QuantiTect SYBR® Green PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Two-step RT-PCR.|Highly specific amplification.|Highly sensitive quantification on the ABI PRISM 7700.|Specific quantification over a wide linear range.|Specific and sensitive quantification.|High specificity in real-time PCR.|Specific primer annealing.|
The QuantiTect SYBR® Green PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and template to the ready-to-use PCR master mix, and start the reaction on any real-time cycler.|In comparison with other DNA polymerases, only HotStarTaq DNA Polymerase in combination with a unique buffer specifically amplified a 497 bp fragment (from 50 copies of an HIV-pol-gene construct in a background of 1 µg human genomic DNA). M: Markers.|Template ranging from 1 x 106 to 5 copies of a plasmid containing a human CFTR gene fragment (in a background of 50 ng maize genomic DNA with no CFTR gene) was amplified on the ABI PRISM 7700. CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride channel. NTC: No template control.|Dilutions of human leukocyte cDNA were analyzed using the QuantiTect Primer Assay for human BAX and the indicated kits on the Rotor-Gene 3000. Highly specific quantification with the QuantiTect Kit and Assay is demonstrated by the single peak in melting curve analysis and the flat curve for the NTC reaction. NTC: No template control. Norm. Fluoro.: Normalized fluorescence.|Dilutions of human leukocyte cDNA were analyzed using the QuantiTect Primer Assay for human IL8 and the indicated kits on the Applied Biosystems 7500. Only the combination of QuantiTect Kit and Assay provided highly specific quantification, as demonstrated by the single peak in melting curve analysis and the flat curve for the NTC reaction. NTC: No template control.|SYBR® Green-based real-time PCR with UNG pretreatment was carried out using either [A] the QuantiTect SYBR® Green PCR +UNG Kit or [B] a kit and UNG from Supplier R. Reactions were run in duplicate on the LightCycler 480 using 10-fold dilutions of human leukocyte cDNA (100 ng to 100 pg) and a QuantiTect Primer Assay for Myc (a protooncogene). Melting curve analysis (see insets) revealed higher PCR specificity with the QuantiTect Kit than with the kit from Supplier R.|A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|
Performance

QuantiTect SYBR Green PCR Kits allow specific quantification over a wide linear range (see figure "Specific quantification over a wide linear range"). HotStarTaq DNA Polymerase provides the most stringent hot start compared with other polymerases, further increasing the specificity of the reaction (see figure "Highly specific amplification"). The unique composition of PCR buffer and HotStarTaq DNA Polymerase enable quantification of even low-abundance transcripts — as few as 5 copies of a target can be accurately detected (see figure "Highly sensitive quantification"). When used in combination with the QuantiTect Reverse Transcription Kit and QuantiTect Primer Assays, the QuantiTect SYBR Green PCR Kit delivers sensitive and reliable results (see figure "Specific and sensitive quantification").

The combination of the specially optimized UNG solution and the proven PCR master mix in the QuantiTect SYBR Green PCR +UNG Kit ensures effective elimination of carried-over PCR products together with reliable quantification of target sequences (see figure "High specificity in real-time PCR").

Principle

QuantiTect SYBR Green PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification cDNA targets using SYBR Green I. 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 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 DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

QuantiTect SYBR Green PCR master mix also contains dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR, which ensures that any contaminating PCR products do not affect subsequent PCR reactions.

Components of 2x QuantiTect SYBR Green PCR Kit
ComponentFeatures Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect SYBR Green PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
dNTP mix Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions Eliminates contamination from carryover of PCR products by optional UNG treatment
SYBR Green I dye Yields a strong fluorescent signal upon binding double-stranded DNA Highly sensitive quantification
ROX dye For normalization of fluorescent signals on Applied Biosystems and, optionally,  Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers
Procedure

QuantiTect SYBR Green PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and DNA template to the ready-to-use PCR master mix, and start the reaction (see flowchart "Two-step RT-PCR"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions. 

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.

Highly specific results in gene expression analysis are guaranteed when QuantiTect SYBR Green PCR Kits are used in combination with QuantiTect Primer Assays. These are genomewide, bioinformatically validated primer sets for detecting transcripts from human, mouse, rat, and many other species. QuantiTect Primer Assays can be easily ordered online at GeneGlobe. 

Applications

The QuantiTect SYBR Green PCR Kit is for use in gene expression analysis of cDNA targets and quantitative gDNA analysis. QuantiTect 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.

Feature
Specifications
Applications Real-time quantification of genomic DNA or cDNA targets
Reaction type Real-time PCR and two-step RT-PCR
Real-time or endpoint Real-time
Sample/target type DNA, cDNA
Single or multiplex Single
SYBR Green I or sequence-specific probes SYBR Green I
Thermal cycler All real-time cyclers (e.g. LC, RG, ABI)
With or without ROX With ROX

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Kit Handbooks
5
For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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PCR産物のキャリーオーバーを除去するためにQuantiTect® PCR Kitsと併用
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For use with QuantiTect PCR Kits to eliminate carryover of PCR products
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SYBR Green I を用いたリアルタイム定量PCRおよび2ステップRT-PCR用
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For quantitative, real-time PCR and two-step RT-PCR using SYBR Green I
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