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RNeasy Mini Handbook 10/2019
35
Protocol: Purification of Total RNA from Animal
Cells Using Vacuum/Spin Technology
This protocol requires the RNeasy Mini Kit.
Determining the correct amount of starting material
See “Determining the correct amount of starting material”, page 27.
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).
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.
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