17
ISOLATE
RNA Kits
P
la
nt
R
N
A
M
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it
2. centrifuge the lysate at maximum speed for 1 minute. Place spin column PR1
in a 2ml collection Tube. carefully transfer the supernatant from the sample to
spin column PR1. centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard
spin column PR1 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 PR1 filter. If required, centrifuge
Spin Column PR1 again until all liquid has passed through the membrane.
3. add 1 volume (usually 400µl) of 70% ethanol to the filtrate and mix well
by pipetting. Transfer immediately to a spin column PR2 placed in a 2ml
collection Tube. centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard
the filtrate and place spin column PR2 in a new collection Tube.
Note: Ensure that there is no lysate remaining on Spin Column PR2. If required, centrifuge Spin
Column PR2 again until all liquid has passed through the membrane.
4. add 500µl Wash buffer aPR to spin column PR2. centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place spin column PR2 in
a new collection Tube.
Note: Ensure that ethanol has been added to Wash Buffer AR according to the instructions
on the bottle.
5. add 650µl Wash buffer bPR to the spin column PR2. centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place spin column PR2 in
a new collection Tube.
Note: Ensure that ethanol has been added to Wash Buffer BR according to the instructions
on the bottle.
6. centrifuge at 10,000 x g (12,000rpm) for 2 minutes to remove all traces of
ethanol. Discard the filtrate and place spin column PR2 in an elution Tube.
7. add 30-80µl Rnase-free water directly to the spin column membrane.
Incubate at room temperature for 1 minute. centrifuge at 6000 x g (8000rpm)
for 1 minute to elute the Rna.
Note: Use a lower volume of RNase-free water if a high concentration of RNA is required.
Increasing the volume of water will increase the yield but decrease the concentration of RNA.
Optionally, perform a second elution step to increase the yield.
8. The isolated Rna is ready for use in downstream applications or for storage
at -20ºc. for long term storage, freeze at -70ºc.
