iScript™ All-in-One RT Master Mix (gDNA Removal)

iScript™ 5X All-In-One RT Master Mix is a convenient, ready-to-use formulation for first-strand cDNA synthesis, including genomic DNA (gDNA) removal, all in one tube. This powerful formulation addresses the common challenge of genomic DNA contamination, ensuring accurate RNA detection without compromising reverse transcription or cDNA synthesis. cDNA synthesis will be simple, reliable, and reproducible.

The optimized 5X RT Master Mix includes ABP novel iScript™ IV Reverse Transcriptase (RT), RNase Inhibitor, temperature-sensitive DNase, dNTPs, and a carefully balanced mix of Oligo(dT)s and Random Primers.  The iScript™ IV Reverse Transcriptase features genetic modifications that abolish RNase H activity to achieve thermal stability, allowing the RNA template to remain intact during reverse transcription for enhanced cDNA yield and integrity. By eliminating the need for multiple separate reagents, this all-in-one mix offers a straightforward, reliable, and reproducible workflow that consistently delivers high-quality cDNA, even from challenging or low-quality RNA samples—ideal for a wide range of downstream molecular biology applications.

Special Features

• Easy one-step setup reduces pipetting and sample handling.
• Removes contaminating gDNA with temperature-sensitive DNase.
• High-quality cDNA yield for downstream applications.

Applications

• cDNA synthesis for PCR.
• Construction of cDNA libraries.
• Generation of probes for hybridization.

General Protocol for First-Strand cDNA Synthesis

RT reactions should be assembled in a RNase-free environment. The use of clean pipettes designated for PCR and aerosol resistant barrier tips are recommended.

     1.  Thaw template RNA and all reagents on ice. Mix each solution by vortexing, and centrifuge briefly to collect residual liquid from the sides of the tubes.

     2.  Prepare the following reaction mixture in a tube on ice:
          

    3.  Mix the components well and collect by brief centrifugation.

    4.  Incubate the tube at 37°C for 5 minutes, followed by 50°C for 15 minutes.

    5.  Stop the reaction by heating at 90°C for 1 minute. Chill on ice. The newly synthesized first-strand cDNA is ready for immediate downstream applications, or for long-term storage at -20°C.