Description

The qScript® XLT One-Step RT-PCR Kit is a convenient and highly sensitive system for amplification of RNA templates up to 2 kb. cDNA synthesis and PCR amplification are carried out in the same tube without opening between incubation steps. This system has been optimized to deliver maximum RT-PCR efficiency, sensitivity, and specificity. qScript XLT is an engineered M-MLV reverse transcriptase with reduced RNase H activity and improved activity and stability at higher temperatures. The use of higher temperatures (48 to 55°C) for the cDNA synthesis step of one-step RT-PCR provides higher specificity for primer annealing and disruption of RNA secondary structure that can interfere with cDNA synthesis. The enzyme is supplied as a mixture with ribonuclease inhibitor protein to protect the integrity of RNA templates in crude lysates or samples where RNase contamination may limit assay sensitivity. This reagent leverages PCR-inhibitor neutralizing ToughMix® technology and is highly resistant to template impurities commonly encountered with clinical, environmental or plant specimens. Ultra pure AccuStart® II antibody hotstart is blended with a highly processive Taq DNA polymerase and 3′ exonuclease proof-reading Taq DNA polymerase for improved PCR fidelity and maximum amplifiable length. High-avidity monoclonal antibodies provide an extremely stringent automatic hot-start that minimizes the potential for primer-dimer and other non-specific PCR artifacts without compromising polymerase activities. Highly specific amplification is crucial to successful RT-PCR as non-specific product(s) can compete with amplification of the target sequence and impair PCR efficiency. The proprietary reaction buffer has been specifically formulated to maximize activities of both the reverse transcriptase and thermostable DNA polymerase while minimizing the potential for primer-dimer and other non-specific PCR artifacts.

Features & Benefits

• Obtain high-fidelity cDNA from large, complex RNA (≤ 2kb) in a seamless, 1-step process.
• Approximately 30% of PCR products will contain a single non-templated, 3′-adenine for TA cloning.

Contents

• 25X concentrated One-Step RT master mix containing: qScript XLT reverse transcriptase ribonuclease inhibitor protein (RIP).
• 2X concentrated One-Step HiFi PCR ToughMix containing:
  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP.
  • AccuStart II Taq DNA Polymerase with added 3′-exonuclease proofreading activity
  • Proprietary enzyme stabilizers and performance-enhancing additives.
  • Titrated reference dye (if applicable).
• GelTrack® Loading Dye: 50X concentrated mixture of RT-PCR compatible, blue and yellow electrophoretic mobility dyes. (Does not contain density reagent)
• Nuclease-free water

Storage & Shipping

Kit components are stable for up to one year when stored in a constant temperature freezer at -20°C. For convenience, the One-Step ToughMix may be stored unfrozen at 4°C for up to 6 months. Repeated freezing and thawing of the ToughMix will not affect product performance.

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