Trizol RNA/DNA extraction - (Jun/29/2009 )
Has anyone done simultaneous extraction of RNA and DNA using Trizol? In my hand the RNA part was fine but nothing pelleted in the DNA protocol. Here are a few things I noticed in the DNA isolation protocol.
1. The manual says it is important to completely remove the aqueous phase (which was used for RNA isolation) before you start. In doing so, I think I had to remove almost all interphase (insoluble) material. I am using very small amount of tissue so there wasn't much visible interphase in the first place.
2. The manual says to centrifuge at "no more than 2,000 xg". I thought it was a rather slow spin for precipitating nucleic acids. I even asked Invitrogen technical support and they said it was not a typo. Is this (slow spin) what you do? Does it work for you?
namekuji on Jun 29 2009, 11:06 PM said:
this spin is not to pellet the nucleic acid, it is to separate the aqueous from the organic phase.
mdfenko on Jun 30 2009, 07:09 AM said:
No no you don't understand. This is AFTER the separation of the aqueous and organic phases. You remove the aqueous phase which contains RNA, then you add 100% ethanol to the organic phase and spin at 2000 g, which is supposed to pellet DNA.
I used an Ambion kit and a Qiagen kit for extracting DNA and RNA from one FFPE tissue sample but these kits use first phenol/chloroform then silica columns for the isolation. They work quit good I must say.