Bio-Rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit - Manual (Page 21)

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4. Add 0.2 ml of chloroform to the lysate, then cover and shake
vigorously for 15 sec. Do not vortex!
5. Incubate for 5 min at room temperature while periodically
mixing the sample.
6. Centrifuge at 12,000 x g for 15 min at 4°C.
Following centrifugation, the mixture will separate into three phases: an
upper, colorless aqueous phase, a white interphase, and a lower, red
organic phase. RNA will be exclusively in the aqueous phase, while DNA
and proteins remain in the interphase and organic phase. The volume of
the aqueous phase should be approximately 600 µl, or 60% of the volume
of PureZOL used in the initial disruption.
If removal of contaminating DNA is a requirement, prepare the DNase I
enzyme by following steps a-b below while centrifuging the samples for phase
separation:
a. DNase I is provided as a lyophilized powder. If the DNase has already
been reconstituted, skip to step b. Otherwise, reconstitute the DNase I
by adding 250 µl of 10 mM Tris, pH 7.5 (not provided) to the vial and
mix by pipetting up and down briefly. Do not vortex! See Section 4,
Materials and Equipment Required (Not Provided in the Kit), on how to
prepare 10 mM Tris, pH 7.5.
b. For each column to be processed, mix 5 µl of reconstituted DNase I
with 75 µl of DNase dilution solution in a 1.5 ml microcentrifuge tube.
Scale up proportionally if processing more than one column. Once
diluted with DNase dilution solution, do not refreeze for later use.
7. Without disturbing the interphase, immediately transfer the
aqueous phase to a 2.0 ml microcentrifuge tube.
Note: It is crucial that none of the interphase or organic phase is
transferred with the aqueous phase. It is recommended that some of the
aqueous phase be left behind to avoid the risk of contaminating the RNA
with contaminants such as phenol, which can interfere with downstream
applications.
8. Add an equal volume (approximately 600 µl) of 70% ethanol (not
provided) to the tube and mix thoroughly by pipetting up and
down.
9. Attach an Aurum total RNA binding mini column to a luer fitting
of the column adaptor plate on the Aurum vacuum manifold or
to a compatible vacuum manifold. Refer to Figure 2 for setup. The
vacuum source should be turned off and the vacuum regulator should be
completely open.
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