
11
5. Add700µloflowstringencywashsolutiontoeachwelloftheRNA
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.
ReconstitutetheDNaseIbyadding250µl10mMTris,pH7.5(not
supplied) to the vial. Pipet up and down briefly to mix.
7. Foreachwell(ofa96-wellplate)processed,mix2.5µlofreconstituted
DNaseIwith77.5µlofDNasedilutionsolution.Forone96-wellplate,
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
ifprocessingmoreorlessthanonefullplateatatime.Add80µldiluted
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. Add700µlofhighstringencywashsolutiontoeachwelloftheRNA
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. Add700µloflowstringencywashsolutiontoeachwelloftheRNA
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. Pipet80µl(or40µl)
†
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
closingthevacuumregulator.Continuetoapplyvacuumfor5min.Open
the vacuum regulator and turn off the vacuum source.
†
Note: Pipet40µlwhenisolatingtotalRNAfromsmallamountsof
starting material (<10 mg of tissue or 500,000 cells).
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