Help! RNA isolation from yeast

Julia Molitor jmolitor at cec.uchile.cl
Tue Jul 31 17:01:38 EST 2001


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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