Bio-Rad PureZOL™ RNA Isolation Reagent - Manual (Page 12)

top layer. Carryover of the solid debris can cause column clog-
ging and affect RNA sample purity.
3. Add 0.2 ml of chloroform per 1 ml of PureZOL used in step
1, then cover and shake vigorously for 15 seconds. Do not
vortex.
4. Incubate for 5 minutes at room temperature while
periodically mixing the sample.
5. Centrifuge at 12,000 x g for 15 minutes at 4°C.
Following centrifugation, the mixture will separate into 3 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 or 60% of the volume of PureZOL used in the
initial disruption.
6. Without disturbing the interphase, immediately transfer the
aqueous phase to a new RNase-free tube.
Note: It is crucial that none of the interphase or organic phase
be transferred with the aqueous phase. Some of the aqueous
phase should be left behind to avoid the risk of contaminating
the RNA with contaminants such as phenol, which can interfere
with downstream applications.
7. Add 0.5 ml of isopropyl alcohol per 1 ml of PureZOL used
in step 1. Mix thoroughly and then incubate at room
temperature for 5 minutes.
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