Storage Condition

-20℃

Components

Description

This kit is a high-quality, efficient, and convenient one-step cDNA synthesis system. It contains thermostable M-MLV GIII Reverse Transcriptase, reaction buffer, RNase inhibitor, dNTPs, Oligo(dT)₂₀VN, and random primers. Simply add RNA template and water to initiate the reaction. The resulting cDNA is suitable for downstream qPCR experiments.

To eliminate genomic DNA contamination, the kit includes dsDNase, which specifically digests dsDNA or DNA strands in RNA-DNA hybrids. It is heat-labile and inactivated during reverse transcription. This avoids EDTA addition and saves time, ensuring higher accuracy in gene expression quantification.

Protocol

1. For RNA with low genomic DNA (Recommended)

1. On ice, prepare 20 μl mix:
   • Template RNA: 50 ng – 1 μg
   • MasterMix: 4 μl
   • dsDNase: 1 μl
   • Nuclease-Free Water: to 20 μl
2. Incubate 37℃ 2 min (remove gDNA).
3. Incubate 55℃ 15 min.
4. Incubate 85℃ 5 min (terminate).
5. Place on ice or store at -20℃.

2. For RNA with high genomic DNA

• On ice, prepare 10 μl mix: RNA (50 ng–1 μg), dsDNase (1 μl), 10× Buffer (1 μl), Water to 10 μl.
• Incubate 37℃ 2–5 min.
• Incubate 65℃ 2 min to inactivate dsDNase; chill on ice.

• On ice, prepare 20 μl mix: 10 μl product from above, MasterMix (4 μl), Water to 20 μl.
• Incubate 50℃ 15 min (25℃ 10 min optional pre-step if no poly(A) tail).
• Incubate 85℃ 5 min (terminate).
• Store at -20℃ (≤1 week) or -80℃ (long-term).

Notice

• Premix includes Oligo(dT)₂₀VN + random primers, suitable for poly(A) RNA, rRNA, tRNA, and prokaryotic RNA.
• Not suitable for small RNAs like miRNA.
• Random primers initiate at any RNA position; not recommended for full-length cDNA cloning.
• For full-length cDNA of eukaryotes, use RTase III Primer Flexible All-in-One Mix (EG24102).