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RNeasy Mini Handbook 10/2019
59
Vortex and agitate the sample at top speed in the TissueLyser or other bead mill
with cooling until cells are completely disrupted.
Most small-capacity bead mills do not have a cooling mechanism and therefore
require the user to stop the bead mill regularly and cool the sample on ice. The
time required for cell disruption and the length and frequency of the cooling
intervals may vary depending on the type of bead mill used. Please refer to the
supplier’s instructions.
Note: Do not replace bead-milling with vortexing, as this significantly reduces
RNA yield.
Remove the sample from the TissueLyser or bead mill, and allow the beads to
settle. Transfer the lysate (usually 350 µl) to a new microcentrifuge tube (not
supplied). Centrifuge for 2 min at full speed, and transfer the supernatant to a
new microcentrifuge tube (not supplied). Use only the supernatant in subsequent
steps.
Add 1 volume of 70% ethanol to the homogenized lysate, and mix well by
pipetting. Do not centrifuge. Proceed to step 2.
Note: The volume of lysate may be less than 350 µl due to loss during
homogenization.
Note: Precipitates may be visible after addition of ethanol. This does not affect the
procedure.
2. Transfer the sample (usually 700 µl), 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 3.
If the sample volume exceeds 700 µl, centrifuge successive aliquots in the same RNeasy
spin column. Discard the flow-through after each centrifugation.*
* 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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