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50
RNeasy Mini Handbook 10/2019
3b. Disruption using a mortar and pestle followed by homogenization using a
QIAshredder homogenizer:
Immediately place the weighed (fresh, frozen, or RNAprotect stabilized) 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.
Add the appropriate volume of Buffer RLT (see Table 8). 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.
3c. Disruption using a mortar and pestle followed by homogenization using a needle
and syringe:
Immediately place the weighed (fresh, frozen, or RNAprotect stabilized) 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.
Add the appropriate volume of Buffer RLT (see Table 8), and homogenize by
passing the lysate at least 5 times through a blunt 20-gauge needle fitted to an
RNAse-free syringe. Proceed to step 4.
3d. Disruption and homogenization using the TissueLyser: See the
TissueLyser
Handbook
. Then proceed to step 4.
4. Centrifuge the lysate for 3 min at full speed. Carefully remove the supernatant by
pipetting, and transfer it to a new microcentrifuge tube (not supplied). Use only this
supernatant (lysate) in subsequent steps.
In some preparations, very small amounts of insoluble material will be present after the
3 min centrifugation, making the pellet invisible.
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