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Phenol peak at RNA isolation with Trizol method - (Sep/16/2011 )


I have been trying to isolate total RNA from barley leaves. Although I had very little amount of leaf samples in the beginning, I was doing fine until addition of isopropanol step. Whenever I add isopropanol to the colorless upper phase, after shaking and waiting a few seconds, I face that my samples are turning to yellow color. And at the end of the experiment, my pellets stay yellow. I know that for solving this problem, we need to use fresh bottle of isopropanol (Mine expired at May 2010). But, I want to know that is it isopropanol that leads to phenol peaks in all my samples, or am I doing something wrong in or after EtOH washing. I have kept the pellets waiting for around 45 minutes after removing all the EtOH I can, but still it didn't change the fact that we had phenol peaks all the time. So, can this problem be solved if I use a new bottle of isopropanol, or may there be another point I should be careful?

Thanks for your help


I'd suggest doing a chloroform extraction of your colorless sample prior to adding isopropanol. This will bring most of the phenol into the chloroform phase.
I don't think isopropanol ever goes bad, so that is not the problem.