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RNeasy Mini Handbook 10/2019
55
The following values may be used as a rough guide. An
S. cerevisiae
culture containing
1–2 x 10
7
cells per milliliter, diluted 1 in 4, gives an OD600 value of approximately 0.25
with a Beckman DU
®
-7400 spectrophotometer or 0.125 with a Beckman DU-40
spectrophotometer. These correspond to calculated OD values of 1 or 0.5, respectively, for
1–2 x 10
7
yeast cells per milliliter.
Important points before starting
If using the RNeasy Kit for the first time, read “Important Notes” (page 19). If working
with RNA for the first time, read Appendix A (page 73).
Yeast cells should be harvested in log-phase growth.
Important: If performing enzymatic lysis (step 1a or 1b), use only freshly harvested cells.
If performing mechanical disruption, cell pellets can be stored at −90 to −65°C for later
use or used directly in the procedure. Homogenized cell lysates from step 1c can be
stored at −90 to −65°C for several months. Frozen lysates should be incubated at 37°C
in a water bath until completely thawed and salts are dissolved. Avoid prolonged
incubation, which may compromise RNA integrity. Proceed to step 2.
Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and
then place at room temperature.
Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not compatible
with disinfecting reagents containing bleach. See page 7 for safety information.
After enzymatic lysis or mechanical disruption, perform all steps of the procedure at
room temperature. During the procedure, work quickly.
After harvesting the cells, perform all centrifugation steps at 20–25°C in a standard
microcentrifuge. Ensure that the centrifuge does not cool below 20°C.
Things to do before starting
β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. Add 10 µl β-ME per
1 ml Buffer RLT. Dispense in a fume hood and wear appropriate protective clothing.
Buffer RLT containing β-ME can be stored at room temperature for up to 1 month.
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