DNAse treatment in same step of RNA extraction - (Mar/07/2006 )

Hello !

Does anyone know a protocol to do DNAse I to remove DNA contaminant in the same step of RNA extraction ?

I actually wanted to try DNAse I treatment in the water phase after phenol/chloroforme (before RNA precipitation) but im not sur if the enzyme will work with residual phenol and chloroforme even if I bring the water constitution to the buffer of DNAse I. (tris-kcl, etc)

thanks for your hint,

hi
re do a chloroform step (removes all residual phenol) or ethanol precipitate and treat

Fred! You are the TRIzol king! I was just looking through your old posts wrt to redoing the chloroform step, and here you mention it again today...so good for me..

Ok, my problem is that my A260 readings seem to be greatly exaggerated, when comparing quantitation at A260 vs. using the Agilent 2100 Bioanalyzer. When I measured control pure RNA from another company, the A260 readings and bioanalyzer readings are very close; therefore, I concluded that I have phenol contamination that exaggerates my A260 and I want to do an additional chloroform extraction. (Note: my bioanalyzer readings show no indication of DNA contamination, that is why I think it is phenol.)

I found one protocol that said something about adding 1:1 phenol/chloroform to the aqueous RNA but it is not clear when. My question is, after adding chloroform to the homogenate in TRIzol and removing the aqueous phase, do I simply add another 200 µL to the aqueous phase, shake, spin, and once again remove the aqueous phase? Or do I need to prepare a 1:1 phenol/chloroform solution and add THAT to the aqueous phase I just extracted?

Sorry if I provided too much info for a simple question. Thanks a lot in advance!

Hi,

I just wanted to add that I have started doing an additional chloroform extraction step (just adding 200 ul chloroform again) to the aqueous phase during a tri-reagent (same as trizol) RNA extraction.
I do this step directly after the addition of chloroform, spin, and then taking off the aqueous phase.

I was having problems with the cDNA, and now it seems to be working better.. My 260/280 readings are much higher.

I had no idea that this is also good for DNA removal. I was still doing a DNAse treatment after ethanol precipitation. Maybe its not necessary after all?

dear soluene... Thanks for the nickname... "TriKing"...
Well. After adding the chloroform to tri solution, spin, i pipett the aqueous phase (generally 400µl) and add the same volume of chloroform (so roughly 400µl). Spin 5' 10000rpm and pipette the aqueous phase. Then ethanol precipitate.

I add that my chloroform is stabilized by isoamyl alcohol. So i add 400µl of a 24:1 chlorofom:IAA solution.

Thank you so much Fred! Your experience with RNA has helped me immensely in the past few weeks!