Qiagen RNeasy: Early precipitate??
qiagen at kaiwan.com
Wed Mar 20 12:54:38 EST 1996
Jim Hill <jah1 at st-andrews.ac.uk> wrote:
>I am attempting to purify RNA from herring embryos. After homogenization in
>the lysis buffer I add the 70% EtOH and a precipitate forms. This precipitate
>blocks the spin column if the entire solution is applied to it so I have been
>spinning down the precipitate and applying the supernatant to the spin
>column. Am I blundering? Is the RNA likely to be in the supernatant or the
>pellet?? I am currently unable to check the product of the purification on a
>gel since I'm sampling in the field.
>Any efforts to lay my mind at rest on this issue would be greatly appreciated.
>Dr James A. Hill,
>Gatty Marine Laboratory,
>University of St. Andrews,
>East Sands, St. Andrews, Fife.
>KY16 8LB. U.K.
I am sorry to have to tell you, but the precipitate that formed in your
lysate after the addition of ethanol indead contains your RNA. The formation
of this precipitate is quite normal and should not be removed.
To avoid clogging, please try the following recommendations, all of which
will result in more efficient lysis and a less viscous lysate:
use extra Lysis Buffer RLT
(RNeasy kit contains enough for 800 µl RLT per prep)
use syringe/needle or QIAshredder after homogenization to shear genomic DNA
spin homgenized lysate BEFORE the addition of ethanol to pellet
insoluble debris RNA will stay in supernatant
To discuss in more detail, please give our QIAGEN UK Tech Service Department
a call at 01306-740325.
Brigitte Steude Masone
Temporary e-mail address: qiagen at Kaiwan.com
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