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RNeasy Mini Handbook 10/2019
57
Resuspend the cells in 2 ml freshly prepared Buffer Y1 containing lyticase or
zymolase. Incubate for 10–30 min at 30°C with gentle shaking to generate
spheroplasts. Spheroplasts must be handled gently.
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.
Centrifuge for 5 min at 300 x
g
to pellet the spheroplasts. Carefully remove and
discard the supernatant.
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.
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 1 volume (usually 350 µl) of 70% ethanol 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.
1b. Enzymatic lysis of ≤2 x 10
7
freshly harvested cells (do not use more than 2 x 10
7
cells):
Harvest the cells in a 12 ml or 15 ml centrifuge tube 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 medium will affect digestion of the cell wall.
Resuspend the cells in 100 µl freshly prepared Buffer Y1 containing lyticase or
zymolase. Incubate for 10–30 min at 30°C with gentle shaking to generate
spheroplasts. Spheroplasts must be handled gently.
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