Bio-Rad Aurum™ Total RNA 96 Kit - Manual (Page 16)

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Note: Gradual application of negative pressure is required to prevent
sample spraying and cross-contamination.
The eluted total RNA samples in the sample collection plate can be
used immediately in downstream applications. Alternatively, the sample
collection plate can be sealed with sealing tape and stored at –20°C or
–80°C for later use.
Section 8
Spin Protocol
Please read Section 5, “Before Using the Aurum™ Total RNA 96 Kit” before
proceeding.
Except for the first few steps that are specific for the starting sample types
(A, for cultured cell lines; B, for bacteria; and C, for yeast), the remaining
procedures within “All Starting Cell Types” share a common protocol.
Cultured Cell Lines
Follow steps A1–A3, then continue with step 1 of “All Starting Cell Types” on
page 13.
A1. For nonadherent cell cultures, rinse the cells with PBS and transfer up
to 1 x 10 6 cells into each well of a 96-well microplate (not provided).
Centrifuge the plate at 300 x g for 5 min then aspirate the supernatant
from each well.
For adherent cell cultures, rinse the wells (each containing up to
1 x 10
6 cells) of the growth vessel once with PBS and aspirate.
A2. Add 150 µl of lysis solution (already supplemented with
1% b-mercaptoethanol) to each well and pipet up and down several times
to lyse cells thoroughly.
A3. Add 150 µl of 70% ethanol (not supplied) to each well and 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–B3, then continue with step 1 of “All Starting Cell Types”
on page 13. If starting with a grow block of bacterial culture (maximum 1
OD/ml/well), centrifuge the grow block at 1,500 x g for 10 min. Decant the
supernatant, and blot the block with paper towels.
B1. Resuspend up to 8 x 10 8 bacterial cells per well (1 OD
/
ml/well), adding
100 µl of 500 µg/ml lysozyme in TE (10 mM Tris, 1 mM EDTA, pH 7.5).
Incubate at room temperature for 5 min.