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RNeasy Mini Handbook 10/2019
39
4. Add 1 volume of 70% ethanol to the homogenized lysate, and mix well by pipetting.
Important: Do not centrifuge.
Note: The volume of lysate may be less than 350 µl or 600 µl due to loss during
homogenization.
Note: When purifying RNA from certain cell lines, precipitates may be visible after
addition of ethanol. This does not affect the procedure.
5. Transfer 700 µl of each sample from step 4, including any precipitate that may have
formed, to each RNeasy spin column on the vacuum manifold.
6. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off the vacuum
and ventilate the vacuum manifold.
Make sure that the vacuum manifold is assembled correctly before loading. The
flow-through is collected in the QIAvac 24 Plus, the QIAvac 24 base, or the QIAvac 6S
waste tray.* If a spin column clogs, switch off the vacuum, ventilate, and try again. If it
still clogs, continue with “Protocol: Purification of Total RNA from Animal Cells Using
Spin Technology”, page 25.
Note: Be sure to switch off the vacuum and ventilate the manifold between pipetting steps
to maintain uniform conditions for each sample.
7. If necessary, repeat steps 5 and 6 with the remaining volume (approx. 500 µl) of each
sample.
The flow-through is collected in the QIAvac 24 Plus, the QIAvac 24 base, or the QIAvac
6S waste tray.*
8. Add 700 µl Buffer RW1 to each RNeasy spin column.
9. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off the vacuum
and ventilate the vacuum manifold.
The flow-through is collected in the QIAvac 24 Plus, the QIAvac 24 base, or the QIAvac
6S waste tray.*
* 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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