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32
RNeasy Mini Handbook 10/2019
Note: Incomplete homogenization leads to significantly reduced RNA yields and can
cause clogging of the RNeasy spin column. Homogenization with a rotor–stator or
QIAshredder homogenizer generally results in higher RNA yields than with a syringe
and needle.
3a. Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collection
tube, and centrifuge for 2 min at full speed. Proceed to step 4.
3b. Homogenize the lysate for 30 s using a rotor–stator homogenizer. Proceed to
step 4.
3c. Pass the lysate at least 5 times through a blunt 20-gauge needle (0.9 mm diameter)
fitted to an RNAse-free syringe. Proceed to step 4.
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 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 6.
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.
* 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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