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Q1 – How long should I incubate for the RT step?
We recommend that for simple, non-complex RNA of 1 kb or less that a 5 min incubation is a good starting point. For a more complex or “dirty” starting RNA template or for longer target cDNA libraries, we recommend between 20 min and 60 min incubation times.
Q2 – Can I use the Scriptase II cDNA Kit with a high amount of RNA (2.5 µg per 20 µl)?
We recommend a max of 1 µg. Reason: the highly expressed genes will over proportionally be represented in such high concentration. Meaning the chances of finding a high-concentrated mRNA is much higher than a low concentrated one. If you stick to 1 µg, the amount of enzyme to mRNA will be much higher, guaranteeing the complete RT of all mRNA.