ppt'n of RNA from sucrose gradient--isopropanol or ethanol??

[Nick Theodorakis]

Mano <mdep at musica.mcgill.ca> wrote:
>Hi all,
>    I am isolating RNA from sucrose gradients of polysome
profiles.  I
>pheno/chloroform extract twice, then 1 time with chloroform and
then
>precipitate with 0.85 volumes of isopropanol.  Recently, I used
phenol
>buffered with citrate at pH 4.3 and had poor recovery.  I went
back to
>water buffered-phenol and had recovery.  However, I think that
using
>isopropanol is not efficient enough.  Do you guys think that I
should
>precipitate using salt and 2.5 vol ethanol instead??  Thanks.
>
>Mano
>
>

When I did those, I used to worry that the increased viscocity
from the sucrose would keep the RNA from pelleting efficiently.
So I would either dilute it a bit and/or use ethanol (because of
the increase in volume, you'd get a decrease in sucrose conc.)
I would also spin for longer (say, 30 min instead of the 10-15
you would usually do in a microfuge). I also added about 10 ug
of tRNA carrier to each fraction.

You also probably don't need so many extractions; except for the
top couple of fractions, there's not so much protein in the
polysome fractions.

Have fun.

Nick


Nick Theodorakis

nicholas_theodorakis at urmc.rochester.edu
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