Bio-Rad Aurum™ Total RNA 96 Kit - Manual (Page 18)

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5. Add 700 µl of low stringency wash solution to each well of the RNA
binding plate. Centrifuge for 2 min at 1,500 x g. Discard the low
stringency wash solution from the waste tray and replace the RNA binding
plate on top of the same waste tray.
6. The RNase-free DNase I is provided as a lyophilized powder. Reconstitute
the DNase I by adding 250 µl 10 mM Tris, pH 7.5 (not supplied, see note
below) to the vial. Pipet up and down briefly to mix.
7. For each well (of a 96-well plate) processed, mix 2.5 µl of reconstituted
DNase I with 77.5 µl of DNase dilution solution. For one 96-well plate,
mix the entire contents of one vial of reconstituted DNase I with 7.75 ml
DNase dilution solution in a 15 ml sterile conical tube. Scale proportionally
if processing more or less than one full plate at a time. Add 80 µl diluted
DNase I to the membrane at the bottom of each well of the RNA binding
plate. Cover the plate with sealing tape and allow the digestion to
incubate at room temperature for 10 min.
8. Add 700 µl of high stringency wash solution to each well of the RNA
binding plate. Centrifuge for 2 min at 1,500 x g. Discard the high
stringency wash solution from the same waste tray and replace the RNA
binding plate on top of the tray.
9. Add 700 µl of low stringency wash solution to each well of the RNA
binding plate. Centrifuge for 2 min at 1,500 x g. Discard the low
stringency wash solution from the waste tray and replace the RNA binding
plate on top of the same tray. Centrifuge for an additional 2 min at
1,500 x g.
10. Place the RNA binding plate on top of the provided collection plate.
11. Pipette 80 µl (or 40 µl) † of the elution solution onto the membrane stack
at the base of each well of the RNA binding plate and allow 1 min for the
solution to saturate the membranes. Centrifuge for 2 min at 1,500 x g.
† Note: Pipet 40 µl when isolating total RNA from small amounts of
starting material (<10 mg of tissue or 500,000 cells).
The eluted total RNA samples in the sample collection plate can be
used immediately in downstream applications. Alternatively, the sample
collection plate can be sealed with sealing tape and stored at –20°C or
–80°C for later use.