How to get rid of DNA contamination in RNA prep - (Aug/22/2013 )
I´ve a problem with contaminating gDNA in my RNA extractions. I follow a Hot SDS-Hot Phenol protocol and I end up with approx. 30 -50 % DNA regarding the total amount of nucleic acids (This is an estimation by interpreting a reducing agarose gel). I´ve checked and double checked the pH of the Phenol, which is around 5. Furthermore I add NaOAc to decrease pH during the extraction - so I don´t think it´s the pH. But what can it be then? I´ve done up to 4 Phenol extractions with a single sample getting the same result. Furthermore I´ve tried to do the extraction with Phenol:CHCl3:IAA (25:24:1). However, there it seems that there is even more DNA in it in the end.
TRIZOL extraction works better, though. However the overall RNA yield is much lower and TRIZOL is expensive. So I´d rather like to stick to the Phe:CHCl3 protocol which gives good quality RNA when analyzed with agilent Bioanalyzer.
I´ve read now dozens of RNA extraction protocols, but I cannot really find a solution for my problem.
Can anybody help?
thanks for every response
Probably not the answer you're looking for but I suggest going with Trizol. You say the results are better but the RNA yield is lower. Perhaps the RNA yield only appears lower because there is no contaminating DNA.
thank you for your reply! In a direct comparison on a gel the RNA yield with TRIZOL is considerably lower. However, my problem is that you really need large quantities of TRIZOL, which is quite expensive and still there is visible DNA contamination on the gel. If if have too I will use TRIZOL, however I won´t if there is any other way.
Is it possible to "overload" the phenol:CHCl3 System? After lysis with SDS the solution becomes extremely viscous. I have doubled the amounts of all reagents in the original protocol, however without success so far. Even if not, after 3-4 extractions the DNA should be washed out, shouldn´t it?
Use RNAzol which costs half. I prefer it. Both(RNAzol and TRIZol) are of inventions of Chomczynski.
200 ml = 203.00 $
compare with TRIZol.
200 ml = 416.00 $
Make Trizol yourself:
1- Saturated Phenol with Tris.HCl.pH 4.8 to 5
2- DTT 1 %
3- Guanidine Isothiocyanate 4 Mol
You can also try adding Triton-x-100 or NP-0 from 0.1 to 0.5 %.