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64
RNeasy Mini Handbook 10/2019
If performing optional on-column DNase digestion, prepare DNase I stock solution as
described in Appendix D (page 82).
Procedure
1. Determine the amount of plant material. Do not use more than 100 mg.
Weighing tissue is the most accurate way to determine the amount.
2. Immediately place the weighed tissue in liquid nitrogen, and grind thoroughly with a
mortar and pestle. Decant tissue powder and liquid nitrogen into an RNAse-free,
liquid-nitrogen–cooled, 2 ml microcentrifuge tube (not supplied). Allow the liquid nitrogen
to evaporate, but do not allow the tissue to thaw. Proceed immediately to step 3.
RNA in plant tissues is not protected until the tissues are flash-frozen in liquid nitrogen.
Frozen tissues should not be allowed to thaw during handling. The relevant procedures
should be carried out as quickly as possible.
3. Add 450 µl Buffer RLT or Buffer RLC (see “Important points before starting”) to a
maximum of 100 mg tissue powder. Vortex vigorously.
A short 1–3 min incubation at 56°C may help to disrupt the tissue. However, do not
incubate samples with a high starch content at elevated temperatures, otherwise swelling
of the sample will occur.
Note: Ensure that β-ME is added to Buffer RLT or Buffer RLC before use (see “Things to do
before starting”).
4. Transfer the lysate to a QIAshredder spin column (lilac) placed in a 2 ml collection tube,
and centrifuge for 2 min at full speed. Carefully transfer the supernatant of the
flow-through to a new microcentrifuge tube (not supplied) without disturbing the
cell-debris pellet in the collection tube. Use only this supernatant in subsequent steps.
It may be necessary to cut off the end of the pipette tip to facilitate pipetting of the lysate
into the QIAshredder spin column. Centrifugation through the QIAshredder spin column
removes cell debris and simultaneously homogenizes the lysate. While most of the cell
debris is retained on the QIAshredder spin column, a very small amount of cell debris
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