Bio-Rad Aurum™ Total RNA 96 Kit - Manual (Page 14)

10
Yeast
Follow steps C1–C5, then continue with step 1 of “All Starting Cell Types”
on page 10. 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. Set up the Aurum™ Vacuum Manifold and one RNA binding plate
according to “Manifold Wash Setup” instructions on page 7.
2. With the vacuum turned off and the regulator open, transfer the lysate
from each sample to a well of the RNA binding plate.
3. Turn the vacuum on and gradually increase the negative pressure
between –17 to –23 inHg over a 5–10 sec period by slowly closing
the vacuum regulator. Once all wells have emptied, open the vacuum
regulator fully. Check that the regulator gauge reads approximately 0 inHg.
Note: Gradual application of negative pressure is required to ensure
uniform flow of lysates through all 96 wells of the RNA binding plate.
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.