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I am using a RNA which is in vitro transcripted before I started my project. It turned out it is not prepared properly and has DNA contamination.

Instead of perform the in vitro transcription again, I want to try to clean it with DNase treatment and LiCl precipitation afterwards. Do you think would that work?

And second thing, in vitro transcipted RNA is in different dilutions, so I have a little bit of stock (maybe 5 uL) which is around 200 ng/uL and I have other dilutions 1 and 0.1 ng/uL. I would like to clean all of them. Do you think if I just mix all of the dilutions with the stock and determine the concentration and do the rest? Is it appropriate?

Thanks!

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DNase treatment is actually one of the optional steps if you want to get rid of the original DNA template: https://www.neb.com/protocols/2013/04/02/standard-rna-synthesis-e2050

If you know, your the contamination happened after the transcription, DNase treatment should obviously help.

Concerning the aliquots: your stock is 200 times more concentrated than the aliquots (so to get equal amounts of RNA as in 1 µl of the stock, you'd need to take 200 µl of the 1 ng/µl aliquot). I don't think it's worth mixing them (and the efficiency of RNA precipitation will likely be decreased), otherwise just make another transcription.

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  • $\begingroup$ Thanks @har-wradim. You are right about the aliquots, I will just try to clean the stock even though it is not that much. I think the contamination is due to original DNA template. Do you think it would still help? $\endgroup$
    – golgicik
    Feb 4, 2014 at 18:58
  • $\begingroup$ It should, that's precisely the purpose of the optional DNase treatment step. For many tasks the presence of the DNA template is not a problem though. $\endgroup$
    – alephreish
    Feb 4, 2014 at 20:31
  • $\begingroup$ I am mixing it with another RNA and performing qPCR. So for me, I need to see if I get the exact amount of RNA that I put after certain steps. And with -RT reaction of this RNA (without RT enzyme) it gives signal. That's why I am not sure if the signal that I get from my experiment belongs to my RNA input or DNA template contamination. $\endgroup$
    – golgicik
    Feb 4, 2014 at 20:47

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