Bio-Rad Aurum™ Total RNA Mini Kit - Manual (Page 22)

18
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.
Centrifuge for 30 sec. Discard the high stringency wash solution from the
wash tube and replace the column in the same wash tube.
8. Add 700 µl of low stringency wash solution to the RNA binding column.
Centrifuge for 1 min (for cultured, bacterial, or yeast cells) or 30 sec (for
animal or plant tissue). Discard the low stringency wash solution from the
wash tube and replace the column in the same wash tube.
9. 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). Pipette 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 stored at –20°C or at
–80°C for later use.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 18