cDNA/Reverse Transcriptase Tips

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  • ProtoScript® II Reverse Transcriptase (NEB #M0368) is capable of generating cDNA of more than 10kb, at temperatures up to 48°C. We recommend 42°C for routine reverse transcription.

    You can increase the yield of long cDNA products by doubling the amount of enzyme and dNTP.

    For most RT-PCR reactions, RNase H treatment is not required; but, for some difficult amplicons or sensitive assays, add 2 units of E.coli RNase H to the reaction and incubate at 37°C for 20 minutes.

    Streamline your reaction setup by using the ProtoScript II First Strand cDNA Synthesis Kit (NEB #E6560). This kit combines ProtoScript II Reverse Transcriptase, a thermostable M-MuLV (RNase H) Reverse Transcriptase, and recombinant RNase Inhibitor in an enzyme Master Mix, along with a separate Reaction Mix containing dNTPs. Additionally, the kit contains two optimized reverse transcription primer mixes.

    Intact RNA of high purity is essential for generating cDNA for cloning applications.

    Total RNA or mRNA can be used in the reverse transcription reaction.  Total RNA is generally sufficient for cDNA synthesis reactions. However, if desired, mRNA can be easily obtained using a polyA Spin mRNA Isolation Kit (NEB #S1560) or Magnetic mRNA Isolation Kit (NEB #S1550).

    The amount of RNA required for cDNA cloning depends on the abundance of the transcript-of-interest. In general 1 ng to 1 μg total RNA or 0.1 ng to 100 ng mRNA are recommended.