Bio-Rad Aurum™ Total RNA Mini Kit - Manual (Page 15)

Section 7
Vacuum Protocol
Important: Please read Section 5, “Before Using the Aurum™ Total RNA
Mini Kit” and Section 6 "Vacuum Manifold Setup and Use With the Column
Adaptor Plate (CAP)," before proceeding.
This procedure requires the Aurum vacuum manifold and column adaptor
plate (catalog # 732-6470), or any vacuum manifold with luer fittings. Vacuum
filtration steps should be carried out at –17 to –23 inHg for optimum
performance. Centrifugation steps can be performed on 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) and
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 "All Starting Sample Types" on
page 13.
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.
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