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RNeasy Mini Handbook 10/2019
51
5. Add 1 volume of 70% ethanol* to the cleared lysate, and mix immediately by pipetting.
Do not centrifuge. Proceed immediately to step 6.
Note: The volume of lysate may be less than 350 µl or 600 µl due to loss during
homogenization and centrifugation in steps 3 and 4.
Note: Precipitates may be visible after addition of ethanol. This does not affect the
procedure.
6. Transfer up to 700 µl of the sample, including any precipitate that may have formed, to
an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently,
and centrifuge for 15 s at ≥8000 x
g
(≥10,000 rpm). Discard the flow-through.
†
Reuse the collection tube in step 7.
If the sample volume exceeds 700 µl, centrifuge successive aliquots in the same RNeasy
spin column. Discard the flow-through after each centrifugation.
†
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.
7. 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 8.
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).
* Using 50% ethanol (instead of 70% ethanol) may increase RNA yields from liver samples.
†
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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