Bio-Rad Aurum™ Total RNA Fatty and Fibrous Tissue Kit User Manual
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10. Pipet 700 µl of the RNA sample into the RNA binding mini
column. Turn the vacuum on and adjust to –17 to –23 inHg by closing
the vacuum regulator. Continue to apply vacuum until all of the RNA
sample passes through the column. Open the vacuum regulator until the
gauge indicates 0 inHg.
11. Repeat step 11 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 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 passes through the column. Open the vacuum regulator
until the gauge indicates 0 inHg.
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.
14. Add 700 µl of high stringency wash solution to the RNA
binding mini 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 passes through the column. Open
the vacuum regulator until the gauge indicates 0 inHg.
15. Add 700 µl of low stringency wash solution (already
supplemented with ethanol) 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.
16. Transfer the RNA binding mini column to a 2.0 ml capless tube
(provided). Centrifuge for 2 min at >12,000 x g to remove the
residual wash solution.
17. Transfer the RNA binding column to a 1.5 ml microcentrifuge
tube (provided).
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