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 to 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 (2).  In a two-step RT PCR application, the cDNA added as template into the PCR reaction is generally 1 to 2 µl (5 to 10%) of the first-strand reaction. VeraSeq Ultra DNA Polymerase (ENZ P7520L) is recommended for PCR when amplifying long transcripts (up to 12 kB).

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