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

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4. Add 700 µl of low stringency wash solution to the RNA binding column
and close the vacuum regulator dial until the gauge indicates –17 to
–23 inHg. Continue to apply the vacuum until the low stringency wash
solution has passed through the column. Open the vacuum regulator until
the gauge indicates 0 inHg.
5. 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 provided) to the vial
and pipetting up and down briefly to mix.
6. For each column processed, mix 5 µl of reconstituted DNase I with
75 µl of DNase dilution solution in a 1.5 ml microcentrifuge tube (not
provided). Scale up proportionally if processing more than one column.
Add 80 µl of diluted DNase I to the membrane stack at the bottom of
each column. Allow the digest to incubate at room temperature for
15 min (25 min for animal tissue).
7. Add 700 µl of high stringency wash solution to the RNA binding column
and close the vacuum regulator dial until the gauge indicates –17 to
–23 inHg. Continue to apply the vacuum until the high stringency wash
solution has passed through the column. Open the vacuum regulator until
the gauge indicates 0 inHg.
8. Add 700 µl of low stringency wash solution to the RNA binding column
and close the vacuum regulator dial until the gauge indicates –17 to
–23 inHg.
9. Transfer the RNA binding column to a 2 ml capless tube (provided).
Centrifuge for an additional 2 min to remove residual wash solution.
10. Transfer the RNA binding column to a 1.5 ml capped microcentrifuge tube
(provided). Pipet 80 µl (or 40 µl)
† of the elution solution onto the
membrane stack at the bottom of the RNA binding column and allow 1 min
for the solution to saturate the membranes. Centrifuge for 2 min to elute
the total RNA.
† 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 can be used immediately in downstream
applications. Alternatively, the total RNA can be aliquoted stored at –20°C
or at –80°C for later use.
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