Maxima H Minus First Strand cDNA Synthesis Kit, with dsDNase, Each - FAQs

View additional product information for Maxima H Minus First Strand cDNA Synthesis Kit - FAQs (K1652, K1651, K1682, K1681)

6 product FAQs found

How can I inactivate Thermo Scientific dsDNase?

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

When should I choose regular RevertAid RT or Maxima RT vs. RevertAid H-minus RT or Maxima H-minus RT?

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. We also recommended using RNase H-minus RTs for template-independent addition of C nucleotides.

Does dsDNase cleave ssDNA?

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.

Can dsDNase be added directly to a reverse transcription reaction to remove genomic DNA?

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

When using the Maxima or Maxima H Minus First Strand cDNA Synthesis kits with dsDNase, can the genomic DNA elimination step be omitted?

Yes. dsDNase buffer is not needed for subsequent reverse transcription reaction. If RNA sample is not contaminated with gDNA, dsDNase treatment may be omitted.

Is it preferable to use elevated temperature in the RT reaction when using Maxima reverse transcriptases?

cDNA synthesis at higher temperatures ensures successful transcription of RNA with high levels of secondary structure, reducing issues of primer access to template. Therefore, we do recommend to use RT enzymes with high thermostability, e.g. Maxima and Maxima H Minus Reverse Transcriptases, which provide higher yields of full-length cDNA, better sensitivity, and successful transcription of GC-rich templates.