HotStarTaq Master Mix Kit

どのようなPCRアプリケーションでも特異性の高い増幅を実現

  • 至適化なしに高いPCR特異性を実現
  • 反応セットアップが室温で可能
  • 即使用可能なマスターミックスフォーマットでピペッティング操作が減少

HotStarTaq Master Mixには、HotStarTaq DNA Polymerase、至適化が最小限で済む画期的なQIAGEN PCR Buffer、およびdNTPが含まれています。マスターミックスには全ての成分が含まれているので、ピペッティングステップとコンタミのリスクが減少し、スループット数と再現性が増加します。

Product Cat. no. List price:
HotStarTaq Master Mix Kit (250 U)
3 x 0.85 ml HotStarTaq Master Mix (contains 250 units HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP)and 2 x 1.7 ml RNase-Free Water
203443
224,00 €
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HotStarTaq Master Mix Kit (1000 U)
12 x 0.85 ml HotStarTaq Master Mix (contains 1000 units HotStarTaq DNA Polymerase, PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP) and 8 x 1.7 ml RNase-Free Water
203445
730,00 €
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Hot StarTaq Master Mix Kit (2500 U)
1 x 25 ml HotStarTaq Master Mix (contains 2500 units HotStarTaq DNA Polymerase PCR Buffer with 3 mM MgCl2, and 400 µM of each dNTP) and 1 x 50 ml RNase-Free Water
203446
1.647,00 €
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The HotStarTaq Master Mix Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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HotStarTaq procedure.|Superior performance.|Higher specificity with different primer–template systems.|Effect of hot start on RT-PCR performance.|Highly sensitive single-cell PCR.|Tolerance to variable temperatures and magnesium concentrations.|Specific amplification in multiplex PCR.|Increased specificity of primer annealing.|
The HotStarTaq procedure is fast and easy for maximum convenience.|A 497 bp fragment was amplified from 50 copies of an HIV-pol-gene construct which had been added to 1 µg human genomic DNA. Different hot-start enzymes were employed: HotStarTaq DNA Polymerase from QIAGEN (HotStarTaq); hot-start enzyme from Supplier AII (Hot-start enzyme); Taq-antibody mixture from Supplier L (Antibody-mediated); enzyme without hot start from Supplier R (No hot start). Specific PCR product is indicated by the arrow. Equal volumes of the reaction were analyzed on a 2% agarose gel. M: markers.|Three different primer–template systems were amplified under the same conditions with either Taq DNA polymerase from Supplier R (R) or with HotStarTaq DNA Polymerase (H). System 1: A 1.1 kb fragment of a D-IgI homolog was amplified from human genomic DNA. System 2: A 296 bp fragment from the chromosomal region correlated with X-linked juvenile retinoschisis was amplified from human genomic DNA. System 3: A 214 bp fragment of the β-actin gene was amplified from cDNA synthesized from total RNA. M: markers.|A 1.1 kb fragment of the human interleukin 1 receptor (type II) gene was amplified from cDNA. Amplification reactions were prepared in triplicate using Taq DNA polymerase and buffer from Supplier L (No hot start); antibody-mediated hot start using enzyme and buffer from Supplier L (Antibody-mediated); and HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq). M: markers.|A 500 bp fragment of the murine p53 gene was amplified from single cells isolated by flow cytometry and directly sorted into individual PCR tubes. Reactions were prepared in parallel using HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq), a hot-start enzyme and buffer from Supplier AII (Hot-start enzyme), or antibody-mediated hot start and buffer from Supplier L (Antibody-mediated). M: markers.|[A] PCR amplification at the indicated annealing temperatures using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human cystic fibrosis gene was amplified. M: markers. [B] Tolerance to Variable Magnesium Concentration. PCR amplification at the indicated Mg2+ concentrations using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human prion protein gene was amplified. M: markers.|Fragments from the murine p53 gene were amplified from genomic DNA in multiplex PCR. Parallel reactions were prepared using standard reaction conditions and an enzyme from Supplier R (No hot start) or using HotStarTaq Master Mix Kit from QIAGEN (HotStarTaq Master Mix). M: markers.|Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.|
Performance

HotStarTaq Master Mix Kitは全てのロットで、低コピー数のターゲットを増幅する実験によりPCR特異性の厳密さや再現性をチェックするなど、広範囲の品質管理テストを受けています。HotStarTaq Master Mix Kitは他社のキットに比べて高性能で、特異性と感度の高いホットスタートPCRを実現します(図"Higher specificity with different primer–template systems異なるプライマー/テンプレートシステムにおける高特異性"、"卓越した性能 " および表)。キットに付属の画期的なPCRバッファーは、様々なPCR条件において特異性を実現し、至適化は最小限に抑えられます(図 "幅広い至適アニーリング温度" および"異なったマグネシウム濃度への適応")。

高い特異性と簡便な操作を実現するHotStarTaq Master Mix Kitは、複雑なゲノムテンプレートあるいはcDNAテンプレート(図“RT-PCR性能へのホットスタートの影響”)、複数のプライマーペア(図“Multiplex PCRにおける特異的な増幅”)、増幅困難なサンプルあるいは低コピーのターゲット(図“高感度のシングルセルPCR”)などとの使用に適しています。これはまた、遺伝子スクリーニングのような多数サンプルを増幅するプロジェクトに最適です。

ホットスタート法の比較 
HotStarTaq DNA Polymerase ホットスタート酵素AII 抗体利用 マニュアル ワックスバリア
特異的な増幅 ++ + + +/– +/–
PCR至適化の不要性 ++ +/– +/–
取り扱いの簡便さ ++ ++ +
HotStarTaq DNA Polymerase 詳細データ

濃度:5 units/µl
組み換え酵素:Yes
基質アナログ:dNTP、ddNTP、dUTP、biotin-11-dUTP、DIG-11-dUTP、 fluorescent-dNTP/ddNTP
エクステンション速度:72℃ で2~4 kb/min 
半減期:97℃で10分、94℃で60分 
増幅効率:≥105
5'–>3' エキソヌクレアーゼ活性:Yes
余分なA付加:Yes
3'–>5' エキソヌクレアーゼ活性:No
ヌクレアーゼの混入:No
RNasesの混入:No
プロテアーゼの混入:No
自己プライマー活性:No 

Principle

HotStarTaq Master Mixは即使用可能なマスターミックスで、HotStarTaq DNA Polymerase、QIAGEN PCR Buffer、dNTPsが入っています。Taq DNA Polymeraseを修飾したHotStarTaq DNA PolymeraseはホットスタートPCR において高い特異性を実現します。

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymeraseは、常温では不活性状態でポリメラーゼ活性はありません。この特性により、PCR セットアップや最初のPCR サイクル中の低温での非特異的なプライマーのアニーリングやプライマーダイマーの形成を回避できます(図"ホットスタートPCRで最高のパフォーマンスを実現"および"異なるプライマー/テンプレートシステムにおける高特異性")。HotStarTaq DNA Polymeraseは、95℃、15 分間のインキュベーションステップで活性化され、このステップは既存のサーマルサイクリングのプログラムに容易に導入できます。

QIAGEN PCR Buffer

QIAGEN PCR Buffer は、各PCRサイクルでアニーリングステップ中にプライマー特異的な結合の割合を非特異的な結合に対して高めることにより、各PCRサイクルの特異的な増幅を維持します(図“プライマーのアニーリングの特異性増大”)。このバッファーはユニークな配合比のKClと (NH4)2SO4を含み、従来のPCRバッファーに比べ、幅広いアニーリング温度やMg2+濃度の範囲で厳密で特異的なプライマーアニーリングを実現します。従って、異なるアニーリング温度あるいはMg2+濃度を用いて行なうPCRの至適化は最小限ですみ、または不要なこともあります(図 "幅広い至適アニーリング温度"および"異なったマグネシウム濃度への適応")。

Procedure
HotStarTaq Master Mix Kitは簡便なマスターミックスフォーマットですので、操作は容易です。HotStarTaq DNA Polymeraseは、95℃、15 分間のインキュベーションステップで活性化され、このステップは既存のサーマルサイクリングのプログラムに容易に導入できます。マスターミックスを用いて、反応セットアップを室温で迅速かつ容易に行なえます。 25 µlのHotStarTaq Master Mixを各PCRチューブにピペットでいれ、RNaseフリー水(キットに付属)で希釈したプライマーとテンプレートDNAを25 µl添加します(図"HotStarTaq操作手順")。ピペッティングステップは最小限に抑えられ、エラーとコンタミのリスクが減少し、スループット数と再現性が増加します。キットに付属の至適化済みのプロトコールにより、PCRセットアップを迅速かつ容易に行なえます。
Applications

HotStarTaq Master Mix Kitは、増幅などの高度なアプリケーションを含む様々なアプリケーションに最適です: 

  • 複雑なゲノムテンプレート
  • 複雑なcDNAテンプレート(例;RT-PCR)
  • 低コピーのターゲット(例;シングルセルPCR)
  • マルチプレックスPCR

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Kit Handbooks
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HotStarTaq DNA Polymerase; HotStarTaq Master Mix Kit - For highly specific hot-start PCR without optimization  
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User-Developed Protocols
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As starting material, 5 g soil was mixed with different amounts of Bacillus subtilis cells. Sensitivity was 5 x 103 cells/5g soil.
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Safety Data Sheets
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Highly specific PCR results with both manual and automated PCR setup
HotStarTaq procedure.

The HotStarTaq procedure is fast and easy for maximum convenience.

Superior Performance in Hot-Start PCR
Superior performance.
A 497 bp fragment was amplified from 50 copies of an HIV-pol-gene construct which had been added to 1 µg human genomic DNA. Different hot-start enzymes were employed: HotStarTaq DNA Polymerase from QIAGEN (HotStarTaq); hot-start enzyme from Supplier AII (Hot-start enzyme); Taq-antibody mixture from Supplier L (Antibody-mediated); enzyme without hot start from Supplier R (No hot start). Specific PCR product is indicated by the arrow. Equal volumes of the reaction were analyzed on a 2% agarose gel. M: markers.
Higher Specificity with Different Primer–Template Systems
Higher specificity with different primer–template systems.
Three different primer–template systems were amplified under the same conditions with either Taq DNA polymerase from Supplier R (R) or with HotStarTaq DNA Polymerase (H). System 1: A 1.1 kb fragment of a D-IgI homolog was amplified from human genomic DNA. System 2: A 296 bp fragment from the chromosomal region correlated with X-linked juvenile retinoschisis was amplified from human genomic DNA. System 3: A 214 bp fragment of the β-actin gene was amplified from cDNA synthesized from total RNA. M: markers.
Effect of Hot Start on RT-PCR Performance
Effect of hot start on RT-PCR performance.
A 1.1 kb fragment of the human interleukin 1 receptor (type II) gene was amplified from cDNA. Amplification reactions were prepared in triplicate using Taq DNA polymerase and buffer from Supplier L (No hot start); antibody-mediated hot start using enzyme and buffer from Supplier L (Antibody-mediated); and HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq). M: markers.
Single-Cell PCR
Highly sensitive single-cell PCR.
A 500 bp fragment of the murine p53 gene was amplified from single cells isolated by flow cytometry and directly sorted into individual PCR tubes. Reactions were prepared in parallel using HotStarTaq DNA Polymerase and PCR Buffer from QIAGEN (HotStarTaq), a hot-start enzyme and buffer from Supplier AII (Hot-start enzyme), or antibody-mediated hot start and buffer from Supplier L (Antibody-mediated). M: markers.
Wide Annealing-Temperature Window
Tolerance to variable temperatures and magnesium concentrations.
[A] PCR amplification at the indicated annealing temperatures using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human cystic fibrosis gene was amplified. M: markers. [B] Tolerance to Variable Magnesium Concentration. PCR amplification at the indicated Mg2+ concentrations using QIAGEN PCR Buffer and Taq DNA Polymerase (QIAGEN). The same PCR was performed in parallel using PCR buffer and Taq DNA polymerase from another supplier (Supplier AII). The single-copy human prion protein gene was amplified. M: markers.
Specific Amplification in Multiplex PCR
Specific amplification in multiplex PCR.
Fragments from the murine p53 gene were amplified from genomic DNA in multiplex PCR. Parallel reactions were prepared using standard reaction conditions and an enzyme from Supplier R (No hot start) or using HotStarTaq Master Mix Kit from QIAGEN (HotStarTaq Master Mix). M: markers.
NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing
Increased specificity of primer annealing.
Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.