As we know that 1 ug/ul of RNA would yeild 1 ug/ul of cDNA. I have to perform in vitro transcription of cDNA into mRNA and how much amount of cDNA will result up to 50ug (0.05mg) of mRNA?
Hi Mamuna,
This would depend on a few factors, like the kits you use, the length of reading frame, incubation time, etc. With T7 HiScribe CleancapKit, a 20-uL reaction yields me between 1500-2500 ng/uL or 1.2 kb mRNA in 50-uL elution after DNAseI treatment and column purification, so 1 ug of template therefore, yields on average, 100 ug of mRNA and to get 50 ug mathematically you would need half of the DNA volume. Please go with manufacturer's recommendations to get the best results though and have a go with incubating the reaction longer if you don't have enough of the template. Good luck!
Maria Victorova Thank you very Much for such comprehensive answer, this has quite made the procedure clear for me. As I haven't got any kit yet, I had ambiguity regarding the yield size.
Hi Mamuna, I would also recommend to do optimisation reactions with different concentrations of template. The incubation time is usually btween 2-5 hours depending on the reading frame length. Good luck!
Hi Maria, Thank you for the suggestion, I am sure this will benefit my experiment.
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