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I have a basic and probably silly question. I am isolating RNA with TRIzol. I have performed the basic steps and dissolved the pellet in 50 uL water. I ve measured the concentration and it was 3313 ng/uL (Around 165 ug total yield.). Now my next step is DNase treatment. It says 1 unit of enzyme for 1 ug of RNA in 10 ul reaction mix. I would like to dilute my RNA to 100 ug and use 100 units of enzyme in a 1000 uL reaction mix.

Is this logic true, and if it is how I am going to do the dilution?

Thanks!

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  • $\begingroup$ Short answer: Yes. :-) $\endgroup$
    – Chris
    Commented Feb 3, 2014 at 20:42

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If I understand correctly what you mean by "I would like to dilute my RNA to 100 ug":

Your RNA preparation is at 3.3 μg μl-1.

To add 100 μg to a reaction you need to add 100/3.3 = 30 μl

Then make the total volume up to 1000 μl with enzyme and buffer as appropriate.

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  • $\begingroup$ Thank you. That was exactly what I did but then I thought that while I am voluming up with buffer, enzyme and RNase-free water, concentration would change again? I mean I add 30 uL water more to my 50 uL stock, but then for the DNase reaction, I ve added more. So would that be a problem? $\endgroup$
    – golgicik
    Commented Feb 3, 2014 at 21:44
  • $\begingroup$ The final RNA concentration will be 100ng/ul. So add up all the single components (30ul of RNA, the buffer you need for 1000ul, the enzyme) and fill up with clean water to 1000ul. $\endgroup$
    – Chris
    Commented Feb 3, 2014 at 21:57
  • $\begingroup$ As long as you add 50 micrograms of RNA to the reaction tube and then add other components to a final total volume of 1000 micro litres then everything will be as you wish. Dnase isn't particularly choosy about conditions anyway - as long as there is some Mg2+ about. $\endgroup$
    – Alan Boyd
    Commented Feb 3, 2014 at 21:57
  • $\begingroup$ I might have added much RNA though. :) My friend was using just 1 units of enzyme for 100 ug RNA and it was also working. I just followed the manufacturers instructions. I hope it will be fine! Thank you for the answers @AlanBoyd and Chris. $\endgroup$
    – golgicik
    Commented Feb 3, 2014 at 22:06

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