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RNeasy Mini Handbook 10/2019
83
provided. To avoid loss of DNase I, do not open the vial. Inject RNAse-free water into
the vial using an RNAse-free needle and syringe. Mix gently by inverting the vial. Do not
vortex.
For long-term storage of DNase I, remove the stock solution from the glass vial, divide it
into single-use aliquots, and store at −30 to −15°C °C for up to 9 months. Thawed
aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after
thawing.
Procedure
Prepare and load samples onto the RNeasy spin column as indicated in the individual
protocols. Instead of performing the first wash step, follow steps 1–4 below.
1. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge
for 15 s at ≥8000 x
g
(≥10,000 rpm) to wash the spin column membrane. Discard the
flow-through.*
Reuse the collection tube in step D4.
2. Add 10 µl DNase I stock solution (see above) to 70 µl Buffer RDD. Mix by gently
inverting the tube. Centrifuge briefly to collect residual liquid from the sides of the tube.
Buffer RDD is supplied with the RNAse-Free DNase Set.
Note: DNase I is especially sensitive to physical denaturation. Mixing should only be
carried out by gently inverting the tube. Do not vortex.
3. Add the DNase I incubation mix (80 µl) directly to the RNeasy spin column membrane,
and place on the benchtop (20–30°C) for 15 min.
Note: Be sure to add the DNase I incubation mix directly to the RNeasy spin column
membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the
O-ring of the spin column.
* Flow-through contains Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety information.
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