Product Manual
www.bioline.com/isolate
8
2. centrifuge the lysate at maximum speed for 1 minute. 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 Spin
Column R1 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 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 Spin
Column R2 again until all liquid has passed through the membrane.
4. add 500µl Wash buffer aR to spin column R2. centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place spin column R2 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 700µl Wash buffer bR to the spin column 2. centrifuge at 10,000 x g
(12,000rpm) for 1 minute. Discard the filtrate and place spin column R2 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 R2 in an elution Tube.
7. add 30-80µl Rnase-free water directly to 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.
