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
Edited by mike2k, 22 August 2013 - 07:03 AM.