(II) Total RNA Purification
Perform all steps at room temperature and centrifugation at 10,000-16,000 x g for 30
seconds, unless specified.
1. Pellet 1-5 x 107cells (1-1.5 ml culture) by centrifugation at 500 x g for
2 minutes. Then carefully remove the supernatant.
2. Add 80 µl of the YR Digestion Buffer and 5 µl of the Zymolyase1to
the cell pellet and resuspend the pellet completely by pipetting up and
down.
3. Incubate the suspension at 30-37°C for 40-60 minutes2.
If the volume of the cell pellet is ≤ 25 µl, incubate for 40 minutes. For cell pellet volume
≥ 25 µl, incubate for 60 minutes.
4. Add 160 µl of the YR Lysis Buffer and mix thoroughly by vortexing.
5. Add an equal volume ethanol (95-100%) (1:1) and mix well.
6. Transfer the mixture to the Zymo-Spin™IIICG Column3in a
Collection Tube and centrifuge. Discard the flow-through.
7. Add 200 µl RNA Wash Buffer to the column and centrifuge. Discard
the flow-through.
8. Add 200 µl RNA Wash Buffer to the column and centrifuge. Then
carefully, transfer the column into a nuclease-free tube (not provided).
9. Add 60 µl of DNase/RNase-Free Water directly to the column matrix
and centrifuge
Alternatively, for highly concentrated RNA use ≥ 35 µl elution.
The eluted RNA can be used immediately or stored frozen.
1 Prior to use, resuspend the lyophilized Zymolyase according to Buffer Preparation, page 4.
2. Incubation time will vary based on cell number. Optimization may be required.
3 To process samples > 700 µl, columns may be reloaded.
