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

12
Bacteria
Follow steps B1–B4, then continue with step 1 of “All Starting Sample Types”
on page 13.
B1. Transfer up to the equivalent of 3 OD • ml bacterial culture into a 2 ml
capped microcentrifuge tube (provided). Centrifuge at maximum speed
for 1 min. Decant the supernatant and blot the tube with paper towels.
B2. Add 100 µl of 500 µg/ml lysozyme in TE (10 mM Tris, 1 mM EDTA,
pH 7.5) to each tube. Pipet up and down to resuspend the pellet
thoroughly. Incubate at room temperature for 5 min.
B3. Add 350 µl of lysis solution (already supplemented with
1% b-mercapto-ethanol) to each tube. Pipet up and down several times
to mix thoroughly.
B4. Add 250 µl of 70% isopropanol (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.
Yeast
Follow steps C1–C6, then continue with step 1 of “All Starting Sample Types”
on page 13.
C1. Prepare 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. Transfer up to the equivalent of 3 OD • ml yeast culture into a 2 ml capped
microcentrifuge tube (provided). Centrifuge at maximum speed for 1 min.
Decant the supernatant, and blot the tube with paper towels.
C3. Add 1 ml of 50 units/ml lyticase in lyticase dilution buffer equilibrated to
30°C to each tube. Pipet up and down to resuspend the yeast pellet
completely. Incubate for 10 min.
C4. Centrifuge the tube at 5,000 rpm for 5 min. Decant the supernatant and
gently blot the tube on paper towels.
C5. Add 350 µl of lysis solution (already supplemented with
1% b-mercapto-ethanol) to each tube. Pipet up and down several times
to mix thoroughly.
C6. 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.
4110133B:4110133A.qxd 1/16/2009 2:35 PM Page 12