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.
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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
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204143
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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
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204145
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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
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204163
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QuantiTect SYBR® Green PCR Kits は分子生物学実験用です。疾病の診断、治療または予防の目的には使用することはできません。
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.
| 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.
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Feature
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Specifications
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Applications
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Real-time quantification of genomic DNA or cDNA targets
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Reaction type
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Real-time PCR and two-step RT-PCR
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Real-time or endpoint
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Real-time
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Sample/target type
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DNA, cDNA
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Single or multiplex
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Single
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SYBR Green I or sequence-specific probes
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SYBR Green I
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Thermal cycler
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All real-time cyclers (e.g. LC, RG, ABI)
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With or without ROX
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With ROX
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For use with QuantiTect PCR Kits to eliminate carryover of PCR products
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詳細を表示
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For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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詳細を表示
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For quantitative, real-time PCR and two-step RT-PCR using SYBR Green I
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詳細を表示
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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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画像
2ステップRT-PCR
QuantiTect SYBR® Green PCR Kitを用いると、手間と時間のかかる反応条件の至適化を行なう必要がない。即使用可能なPCRマスターミックスにテンプレートとプライマーを添加するだけで、ほとんどのリアルタイムサイクラーを用いて反応を開始できる。
特異性の高い増幅
他社のDNAポリメラーゼと比較した結果、ユニークなバッファーと組み合わせたHotStarTaq DNA Polymeraseのみが497 bp フラグメントを特異的に増幅した(1 µgのヒトゲノムDNAに添加した50 コピーのHIV-pol-遺伝子コンストラクトからのフラグメント)。M:マーカー。
ABI PRISM 7700で非常に感度の高い定量
トウモロコシから精製したゲノムDNA 50 ng(トウモロコシはCFTR遺伝子を含んでいない)を含む反応液にヒトCFTR遺伝子フラグメントを組み込んだプラスミドをテンプレートとして5~1 x106コピー数添加して、ABI PRISM 7700を用いて増幅した。CFTR(cystic fibrosis transmembrane conductance regulator)はcAMP依存性塩素イオンチャンネルである。NTC:No Template Control。
幅広い範囲で直線性のある高感度定量
ヒト白血球cDNAの希釈液からヒトBAX用のQuantiTect Primer Assayと表記のキットを用いてRotor-Gene 3000で解析した。融解曲線分析において単一ピークが検出され、NTC反応では蛍光強度の上昇が見られないことは、QuantiTect Primer Assayによる特異的な検出を実証している。NTC:No Template Control。Norm.Fluoro.:正規化された蛍光。
特異性と感度の高い定量
ヒト白血球cDNAの希釈液からヒトIL8用のQuantiTect Primer Assay と表記のキットをApplied Biosystems 7500を用いて解析した。融解曲線分析において単一ピークが検出され、NTC反応で蛍光強度の上昇が見られないことにより、QuantiTect SYBR® Green PCR KitとQuantiTect Primer Assayの組み合わせが特異的な検出であることを実証している。NTC:No Template Control。
高感度のリアルタイムPCR
UNG 前処理とSYBR® GreenベースのリアルタイムPCR は、[A]QuantiTect SYBR Green PCR +UNG Kit あるいは[B] R 社のキットとUNG を用いて行なった。ヒト白血球cDNA の10倍段階希釈液(100 pg ~100 ng)とMyc(がん原遺伝子)用QuantiTect Primer Assay を用いてLightCycler 480 上でduplicateで反応を行なった。融解曲線解析(挿入図)は、R 社よりもQuantiTect Kit を用いた方がPCR 特異性の高いことを示した。
特異的なプライマーアニーリング
バランスの取れたKCl および(NH4)2SO4の組み合わせは、プライマーのPCRテンプレートへの特異的なアニーリングを促進する。K+は二本鎖DNAのリン酸基に結合し、プライマーのアニーリングを安定化する。NH4+は、ミスマッチな塩基対間の水素結合を不安定化する。
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