ISOLATE
RNA Kits
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7.2 total rna isolation from eukaryotic cells
Before you start:
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Before using for the first time, add 96-100% ethanol to the Wash Buffers AR
and BR as indicated on the bottles and mix.
•
If any of the buffers form precipitates upon storage, re-dissolve by gently
warming. Cool to room temperature before use.
•
Cells grown in cell culture vessels can be lysed directly in the vessel or
trypsinized to detach from the vessel. Once homogenized in the Lysis Buffer,
the sample can be stored at -20ºC for several months.
•
For information on how to work with RNA, read Hints and Tips on page 19.
1. Harvest up to a maximum of 5 x 10
6
cells.
Cells grown in suspension
•
Centrifuge appropriate number of cells at 300 x g for 5 minutes. Remove all
supernatant by aspiration taking care not to disturb the pellet.
Cells grown in a monolayer
•
Remove the cell culture medium completely by aspiration. Incomplete removal
of the medium will inhibit lysis of the cells and compromise the efficiency of
RNA extraction. Proceed directly to step 2.
2. add 450µl lysis buffer R to the sample. Resuspend the sample completely
by pipetting up and down a few times. Incubate the sample for 3 minutes at
room temperature.
Note: No cell clumps should be visible after the lysis step to maximize RNA yield.
3. Place spin column R1 into a 2ml collection Tube. carefully transfer the
supernatant from the sample to spin column R1. centrifuge at 10,000 x g
(12,000rpm) for 2 minutes. Discard spin column R1 and save THe fIlTRaTe
(for optional Dnase digestion see Hints and tips, Page 20).
Note: Ensure that there is no lysate remaining on Spin Column R1. If required, centrifuge the
Spin Column again until all liquid has passed through the membrane.
4. add 1 volume of 70% ethanol to the filtrate and mix well by pipetting.
Transfer immediately to spin column R2 placed in a 2ml collection Tube.
centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard the filtrate and
place spin column R2 in a new collection Tube.
Note: Ensure that there is no lysate remaining on Spin Column R2. If required, centrifuge the
