Bio-Rad Aurum™ Total RNA 96 Kit - Manual (Page 17)

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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.
Yeast
Follow steps C1–C5, then continue with step 1 of “All Starting Cell Types”
on page 13. If starting with a grow block of yeast culture (maximum 2 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.
C1. Prepare 100 ml lyticase dilution buffer:
1 M sorbitol
0.1 M EDTA, pH 7.4
0.1% (v/v) b-mercaptoethanol
Equilibrate the buffer at 30°C before use.
C2. Resuspend up to 2 x 10 7 yeast cells per well (2 OD/ml/well), adding
1 ml of 50 units/ml lyticase in lyticase dilution buffer equilibrated to 30°C
to each well. Incubate for 10 min.
C3. Centrifuge at 1,500 x g for 5 min. Decant the supernatant.
C4. 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.
C5. Add 350 µl of 70% ethanol (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.
All Starting Cell Types
1. Place an RNA binding plate on top of a waste tray or a pipet tip box lid.
2. Transfer the lysate from each well of the 96-well plate into a
corresponding well of the RNA binding plate.
3. Centrifuge for 2 min. Discard the filtrate from the waste tray and replace
the RNA binding plate on top of the same waste tray.
4. The low stringency wash solution is provided as a 5x concentrate. Add
4 volumes (240 ml) 95–100% ethanol to the low stringency
wash solution concentrate before initial use.