Bio-Rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit - Manual (Page 27)

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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
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 ml) of 70% ethanol
(not provided) to the tube and mix thoroughly by pipetting up
and down.
9. Insert an RNA binding column into a 2.0 ml capless wash tube
(provided).
For steps 10–21, all centrifugation steps are performed at room
temperature.
10. Pipet up to 700 µl of the RNA sample into the RNA binding
mini column. Centrifuge for 60 sec at >12,000 x g. Remove the
RNA binding column from the wash tube, discard the filtrate from the
wash tube and replace the column into the same wash tube.
11. Repeat step 10 for the remainder of the sample.
The Aurum total RNA fatty and fibrous tissue kit supplies RNase-free
DNase I to be used to treat samples for complete removal of
contaminating genomic DNA. If removal of genomic DNA is not a
requirement, proceed directly to step 14. Otherwise, perform on-column
DNase I digest by proceeding to step 12.
12. Add 700 µl of low stringency wash solution (already
supplemented with ethanol) to the RNA binding column.
Centrifuge for 30 sec at >12,000 x g. Discard the low stringency
wash solution from the wash tube and replace the column into the same
wash tube.
13. Remove any contaminating genomic DNA from the RNA
sample.
a. Add 80 µl of the diluted DNase I to each column processed, making
sure to add the DNase to the center of the membrane stack at the
bottom of each column.
b. Allow the DNase digest to incubate at room temperature for 15 min.
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