Bio-Rad Aurum™ Total RNA 96 Kit - Manual (Page 15)

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5. Add 700 µl of low stringency wash solution to each well of the RNA
binding plate. Gradually increase the negative pressure between –17 to
–23 inHg over a 5–10 sec period by slowly closing the vacuum regulator.
After all wells have emptied, open the vacuum regulator until the gauge
reads approximately 0 inHg.
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) 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. Gradually increase the negative pressure between –17 to
–23 inHg over a 5–10 sec period by slowly closing the vacuum regulator.
After all wells have emptied, open the vacuum regulator until the gauge
reads approximately 0 inHg.
9. Add 700 µl of low stringency wash solution to each well of the RNA
binding plate. Gradually increase the negative pressure between –17 to
–23 inHg over a 5–10 sec period by slowly closing the vacuum regulator.
After all wells have emptied, continue to apply vacuum for an additional
4 min to purge the wells of residual wash solution. When completed,
open the vacuum regulator until the gauge reads approximately 0 inHg.
10. Set up the Aurum™ Vacuum Manifold for elution according to “Manifold
Elution Setup” on page 8.
11. Pipet 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. Gradually increase the negative
pressure between –17 to –23 inHg over a 5–10 sec period by slowly
closing the vacuum regulator. Continue to apply vacuum for 5 min. Open
the vacuum regulator and turn off the vacuum source.
† Note: Pipet 40 µl when isolating total RNA from small amounts of
starting material (<10 mg of tissue or 500,000 cells).