KAPA SYBR® FAST One-Step qRT-PCR Master Mix (2X) Kit

The KAPA SYBR® FAST One-Step qRT-PCR Kit is an optimized solution for rapid, one-step real-time quantitative reverse transcription PCR (qRT-PCR). Designed for high sensitivity and specificity, this kit streamlines the detection and quantification of RNA targets in a single tube, reducing experimental variation and contamination risk. Compatible with multiple real-time PCR platforms, the kit ensures reliable performance across a wide range of applications.

Key Features:

1. One-Step, One-Tube Protocol:
   • Combines reverse transcription and qPCR in a single tube, simplifying workflows and minimizing contamination risks.
2. High Sensitivity and Specificity:
   • Engineered M-MuLV reverse transcriptase and DNA polymerase ensure robust amplification, even for low-copy RNA targets.
3. Wide Compatibility:
   • Suitable for use with SYBR® Green detection chemistry on platforms such as Applied Biosystems®, QuantStudio™, StepOne™, and Agilent Mx3000P series.
4. Broad Target Range:
   • Consistently high performance across various RNA concentrations and amplicon sizes, including GC- and AT-rich regions.
5. Rapid Cycling and Fluorescence:
   • Delivers higher fluorescence and earlier Cq values, enabling faster and more efficient reactions.
6. Stable and Convenient:
   • Components stable through 30 freeze-thaw cycles and resistant to light degradation, ensuring reliability.

Applications:

1. Gene Expression Analysis:
   • Quantifies RNA levels with high accuracy for understanding gene regulation.
2. Low-Copy Gene Detection:
   • Amplifies low-abundance RNA targets with high sensitivity.
3. miRNA and RNAi Research:
   • Supports studies on small RNA species and gene silencing mechanisms.
4. Microarray Validation:
   • Verifies gene expression patterns observed in microarray experiments.
5. Gene Knockdown Validation:
   • Confirms efficacy of RNA interference or CRISPR experiments.

Specifications:

Kit Components:

1. KAPA RT Mix:
   • Contains M-MuLV reverse transcriptase optimized for cDNA synthesis at 42°C.
2. KAPA SYBR® FAST DNA Polymerase:
   • Evolved enzyme for efficient qPCR amplification.
3. Reaction Buffer:
   • Proprietary buffer formulation includes MgCl₂, dNTPs, and SYBR® Green I dye for optimal reaction efficiency.
4. dUTP:
   • Ensures high-fidelity DNA synthesis and minimizes contamination risk.
5. MgCl₂:
   • Pre-mixed for convenience and reaction optimization.
6. SYBR® Green I Dye:
   • Fluorescent dye for real-time detection and quantification.

Recommended Protocol:

1. Preparation:
   • Thaw all components thoroughly and mix well before use. Protect light-sensitive reagents from prolonged exposure.
2. Reaction Setup:
   • Prepare a master mix containing:
     • KAPA RT Mix (reverse transcriptase)
     • KAPA SYBR FAST DNA Polymerase (2X Master Mix)
     • RNA template
     • Gene-specific primers
   • Adjust reaction volume to 20 µL (recommended).
3. Cycling Conditions:
   • Reverse Transcription: 5 min at 42°C.
   • RT Inactivation/Polymerase Activation: 3 min at 95°C.
   • Cycling (40 cycles):
     • Denaturation: 95°C for 3 sec.
     • Annealing/Extension: 60°C for 20 sec.
4. Data Acquisition:
   • Measure fluorescence at the end of each extension step for real-time quantification.

Advantages:

1. Rapid and Reliable:
   • Reduces reaction time with a fast, streamlined protocol.
2. High Efficiency:
   • Consistently high amplification efficiency ensures accurate RNA quantification.
3. Broad Applicability:
   • Suitable for diverse RNA types and experimental designs.
4. Easy Optimization:
   • Pre-mixed components and robust design simplify reaction setup and troubleshooting.

Storage and Stability:

• Store at -20°C. Protect from light to preserve fluorescent dye stability.
• Thawed reagents are stable at 4°C for short-term use (up to one week).

Product Details:

The KAPA SYBR® FAST One-Step qRT-PCR Kit is an indispensable tool for researchers requiring rapid, sensitive, and reliable RNA quantification. Its user-friendly format, high efficiency, and compatibility with various platforms make it an excellent choice for advanced molecular biology applications.