What is the suggested protocol for generating long, full-length cDNA transcripts (>5 kb)?
EnzScript first-strand reactions containing up to 1 µg total RNA primed with a GSP or poly oligo(dT) and incubated at 42–50°C for up to 60 minutes is recommended for generating long (>5 kb) transcripts. In general, RNase H (ENZ Y9220) treatment of cDNA reactions (5 U, incubation at 37°C for 20 minutes, heat kill at 65°C for 10 minutes) prior to PCR is recommended when amplification of long transcripts is desired (Kitabayashi, M., et al. (2003) Biosci. Biotechnol. Biochem. 67:2474). In a two-step reverse transcription PCR application, the cDNA added as template for the PCR is generally 1–2 µl (5–10%) of the first-strand reaction. VeraSeq Ultra DNA Polymerase (ENZ P7520L) is recommended for PCR when amplifying long transcripts (up to 12 kb).