Maxima First Strand cDNA Synthesis Kit for RT-qPCR

dsDNase can be inactivated by incubating the sample at 55°C for 5 min in the presence of 10 mM DTT.

It is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning. RNase H degrades RNA from RNA-DNA duplexes, which can result in truncated cDNA during reverse transcription of long mRNA. It is also recommended to use RNase H-minus RTs for template-independent addition of C nucleotides. In contrast, reverse transcriptases with intrinsic RNase H activity are often favored in qPCR applications.

All Thermo Scientific reverse transcriptases possess intrinsic TdT activity although at varying degrees depending upon the reaction conditions. For addition of template-independent C nucleotides (as for SMART and RACE experiments), this specific TdT activity can be induced by Mn2+. We would recommend Maxima H- or RevertAid H- minus RTs for this purpose.

No, dsDNase cleaves only double-stranded DNA. However, if ssDNA forms double-stranded structures, it will act as a target for dsDNase. Because of this, long and complex ssDNA may be partially degraded. Therefore we recommend additional dsDNase inactivation step for RT-PCR amplification of targets 3 kb or longer. The inactivation step should be performed by 5 min incubation at 55 degrees C in the presence of 10 mM DTT.

No. RT reaction composition inhibits dsDNase activity, and genomic DNA removal may be incomplete.