Bio-Rad Aurum™ Total RNA Mini Kit - Manual (Page 19)

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Section 8
Spin Protocol
Important: Please read Section 5, “Before Using the Aurum™ Total RNA Mini
Kit,” before proceeding.
The Aurum total RNA mini kit can be used with any commercially available
microcentrifuge that can accommodate 1.5 and 2.0 ml tubes. All centrifugation
steps are performed at maximum speed (>12,000 x g) at room temperature.
Note: Except for the first few steps that are specific for the starting sample
types (A. for cultured cell lines, B. for bacteria, C. for yeast D. for animal and
plant tissue), the remaining procedures within "All Starting Sample Types"
share a common protocol.
Cultured Cell Lines
Follow steps A1–A3, then continue with step 1 of “All Starting Sample Types"
on page 17.
A1. For nonadherent cell cultures, rinse the cells with PBS and transfer up
to 2 x 10
6
cells into a 2 ml capped microcentrifuge tube (provided).
Centrifuge the tube at maximum speed for 2 min, decant the supernatant
from the tube, and blot the tube with paper towels.
For adherent cell cultures, rinse the growth vessel once with PBS and
aspirate. Proceed with lysis if the expected number of cells in the vessel
does not exceed 2 x 10
6
cells; otherwise, release the cells from the plate
and transfer up to 2 x 10
6
cells into a 2 ml capped microcentrifuge tube
(provided). Centrifuge the tube for 2 min. Decant the supernatant and blot
the tube with paper towels.
A2. Add 350 µl of lysis solution (already supplemented with 1% b-mercapto-
ethanol) to each tube or growth vessel. Pipet up and down several times
to lyse cells thoroughly.
A3. Add 350 µl of 70% ethanol (not supplied) to each tube. Pipet up and
down to mix thoroughly. Make sure that no bilayer is visible and that the
viscosity is substantially reduced.
Bacteria
Follow steps B1–B4, then continue with step 1 of “All Starting Sample Types”
on page 17.
B1. Transfer up to the equivalent of 3 OD • ml bacterial culture into a 2 ml
capped microcentrifuge tube (provided). Centrifuge at maximum speed for
1 min. Decant the supernatant and blot the tube with paper towels.
B2. Add 100 µl of 500 µg/ml lysozyme in TE (10 mM Tris, 1 mM EDTA,
pH 7.5) to each tube. Pipet up and down to resuspend the pellet
thoroughly. Incubate at room temperature for 5 min.
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