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RNeasy Mini Handbook 10/2019
29
Cell pellets can be stored at −90 to −65°C for later use or used directly in the procedure.
Determine the number of cells before freezing. Frozen cell pellets should be thawed
slightly so that they can be dislodged by flicking the tube in step 2. Homogenized cell
lysates from step 3 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. If any
insoluble material is visible, centrifuge for 5 min at 3000–5000 x
g
. Transfer supernatant
to a new RNAse-free glass or polypropylene tube, and continue with step 4. 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.
Perform all steps of the procedure at room temperature. During the procedure, work
quickly.
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
If purifying RNA from cell lines rich in RNAses, we recommend adding
β-mercaptoethanol (β-ME) 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.
Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of
ethanol (96–100%) as indicated on the bottle to obtain a working solution.
If performing optional on-column DNase digestion, prepare DNase I stock solution as
described in Appendix D (page 82).
Procedure
1. Harvest cells according to step 1a or 1b.
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