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60
RNeasy Mini Handbook 10/2019
Optional: If performing optional on-column DNase digestion (see “Eliminating genomic
DNA contamination”, page 26), follow Appendix D (page 82), steps 1–4, after
performing this step.
3. Add 700 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 4.
Note: After centrifugation, carefully remove the RNeasy spin column from the collection
tube so that the column does not contact the flow-through. Be sure to empty the collection
tube completely.
Skip this step if performing optional on-column DNase digestion (page 69).
4. Add 500 µl Buffer RPE 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
the flow-through.
Reuse the collection tube in step 5.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE
before use (see “Things to do before starting”).
5. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge
for 2 min at ≥8000 x
g
(≥10,000 rpm) to wash the spin column membrane.
The long centrifugation dries the spin column membrane, ensuring that no ethanol is
carried over during RNA elution. Residual ethanol may interfere with downstream
reactions.
Note: After centrifugation, carefully remove the RNeasy spin column from the collection
tube so that the column does not contact the flow-through. Otherwise, carryover of
ethanol will occur.
* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety
information.
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