ISOLATE
RNA Kits
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efficiency of RNA isolation.
2. Resuspend the cell pellet in 100µl Te buffer (not supplied). add 2-6µl
lysozyme solution (not supplied). Pipette up and down for a few times until
the solution becomes slightly viscous.
Note: For Gram –ve bacteria: Add 2µl of 20mg/ml lysozyme solution.
For Gram +ve bacteria: Add 6µl of 50mg/ml lysozyme.
3. add 450µl lysis buffer R and vortex vigorously. Incubate for 3 minutes at
room temperature.
Note: To maximize total RNA yield, ensure that no cell clumps are visible.
4. Transfer the sample to spin column R1 placed in a 2ml collection Tube.
centrifuge at 10,000 x g (12,000rpm) for 2 minutes. Discard spin column 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.
5. add 1 volume of 70% ethanol to the filtrate and mix well by pipetting.
6. Transfer 650µl of the sample to spin column R2 placed in a 2ml collection
Tube. centrifuge at 10,000 x g (12,000rpm) for 1 minute. Discard the filtrate
and place spin column R2 in a new collection Tube. Transfer the remaining
sample from step 5 to the same spin column R2 and centrifuge again at
10,000 x g (12,000rpm) for 1 minute. Discard the filtrate and place spin
column R2 into 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.
7. add 500µl Wash buffer aR and centrifuge at 10,000 x g (12,000rpm) for 1 minute.
Discard the filtrate and place spin column R2 into a new collection Tube.
8. add 700µl Wash buffer bR and centrifuge at 10,000 x g (12,000rpm) for 1 minute.
Discard the filtrate and place spin column R2 into a new collection Tube.
9. 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.
10. 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.
11. The isolated Rna is ready for use in downstream applications or for storage
at -20ºc. for long term storage, freeze at -70ºc.
