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66
RNeasy Mini Handbook 10/2019
8. 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
flow-through.
Reuse the collection tube in step 9.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE
before use (see “Things to do before starting”).
9. 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.
10. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and
discard the old collection tube with the flow-through. Close the lid gently, and centrifuge
at full speed for 1 min.
Perform this step to eliminate any possible carryover of Buffer RPE, or if residual
flow-through remains on the outside of the RNeasy spin column after step 9.
11. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30–50 µl
RNAse-free water directly to the spin column membrane. Close the lid gently, and
centrifuge for 1 min at ≥8000 x
g
(≥10,000 rpm) to elute the RNA.
12. If the expected RNA yield is >30 µg, repeat step 11 using another 30–50 µl RNAse-free
water, or using the eluate from step 11 (if high RNA concentration is required). Reuse the
collection tube from step 11.
If using the eluate from step 11, the RNA yield will be 15–30% less than that obtained
using a second volume of RNAse-free water, but the final RNA concentration will be
higher.
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