RNA isolation, dilution question - (Feb/03/2014 )
I have a basic and probably silly question. I am isolating RNA with TRIzol. I have performed the basic steps and resolve the pellet in 50 uL water. I ve measured the concentration and it was 3313 ng/uL (Around 165 ug). 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 unite enzyme in 1000 uL reaction mix. Is this logic true and if it is how I am going to do the dilution?
So, you want a 1000 ul reaction with 100 ug of RNA. Since your RNA is at 3.3 ug/ul, you will need 100 ug/3.3 ug/ul = 30 ul of your RNA solution. You will also need 100 ul of 10x DNAseI buffer, and 100 units of DNAse. You'll need to add about 830 ul of water (make sure it is nuclease free).