Bio-Rad Aurum™ Total RNA 96 Kit - Manual (Page 13)

9
Section 7
Vacuum Protocol
Please read Section 5, “Before Using the Aurum™ Total RNA 96 Kit” and
Section 6, “Vacuum Manifold Setup and Use With 96-Well Plates” 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 Sample Types” share a common protocol.
Cultured Cell Lines
Follow steps A1–A3, then continue with step 1 of “All Starting Cell Types”
on page 10.
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 10. 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.
B2. Add 350 µl of lysis solution (already supplemented with 1%
b-mercaptoethanol) to each sample and pipet up and down several
times to mix thoroughly.
B3. Add 250 µl of 70% isopropanol (not supplied) to each sample and pipet
up and down to mix thoroughly. Make sure that no bilayer is visible and
that the viscosity is substantially reduced.