Loading ...
Loading ...
Loading ...
58
RNeasy Mini Handbook 10/2019
Depending on the yeast strain used, the incubation time, amount of enzyme, and
composition of Buffer Y1 may vary. For optimal results, follow the guidelines of
the enzyme supplier. Complete spheroplasting is essential for efficient lysis.
Add 350 µl Buffer RLT and vortex vigorously to lyse the spheroplasts. If insoluble
material is visible, centrifuge for 2 min at full speed, and use only the supernatant
in subsequent steps.
Note: Ensure that β-ME is added to Buffer RLT before use (see “Things to do
before starting”).
Add 250 µl ethanol (96–100%) to the homogenized lysate, and mix well by
pipetting. Do not centrifuge. Proceed immediately to step 2.
Precipitates may be visible after addition of ethanol. This does not affect the
procedure.
1c. Mechanical disruption of cells (do not use more than 5 x 10
7
cells):
Add approximately 600 µl of acid-washed glass beads to a tube that fits the
TissueLyser or other bead mill (see page 22 for details).
Harvest the cells by centrifuging at 1000 x
g
for 5 min at 4°C. Decant the
supernatant, and carefully remove any remaining media by aspiration. If the
centrifuge will be used later in this procedure, heat it to 20–25°C.
Note: Incomplete removal of the supernatant will inhibit lysis and dilute the lysate,
affecting the conditions for binding of RNA to the RNeasy membrane. Both effects
may reduce RNA yield.
Loosen the cell pellet thoroughly by flicking the tube. Add 600 µl Buffer RLT, and
vortex to resuspend the cell pellet. Add the sample to the acid-washed glass
beads.
Note: Ensure that β-ME is added to Buffer RLT before use (see “Things to do
before starting”).
Loading ...
Loading ...
Loading ...