Help! RNA isolation from yeast

Geoff Birrell gbirrell at stanford.edu
Tue Jul 31 23:55:22 EST 2001


I have had success using the hot phenol method, clean up with
phenol:chloroform, then chloroform. My yields were much higher than with
Trizol and ratios generally above 1.8.

Geoff


Julia Molitor wrote:

> Dear netters,
>
> I am trying to set up a good RNA isolation protocol from S. cerevisiae
> for microarray analysis.
>
> Ï tried the hot phenol method first, but the prep was pretty dirty. Now
> I am using the Trizol method in combination with glass beads. There is
> only little RNA degradation visible on gel, but the prep is contaminated
> with plasmidial DNA (apparently) and has a high protein content (OD
> 260/280= 1.1-1.4).
> When resuspending the pellet in water, I always have very poor
> solubility (20 ul for a prep of 8xe6 cells). Do I have to increase the
> volume? And why do I see bands at 1 and 1.5kb approx. when running on a
> standart TAE gel, when I was told that ribosomal bands appear at 1.8 and
> 3.5kb?
>
> What can you advise to get better results? -I am pretty under time
> pressure and I cannot afford to buy a kit (and wait 4 weeks until it
> gets to the end of the world...)
>
> Thank you
>
> Julia Molitor
> M. Sc. Biotechnology
> Center for Biochemical Engineering
> and Biotechnology
> Department of Chemical Engineering
> University of Chile
> Beauchef 861
> Santiago
> Chile
>
> ---




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