- All centrifugation should be done at room temperature with a swing-bucket centrifuge.
- Add 10µl ß-mercaptoethanol (ß-ME) per 1 ml RX Buffer or PRX Buffer.
- Add 100 ml of ethanol (96 ~ 100 %) to the WS Buffer bottle when first open the bottle.
- Grind 1 g (or less) plant sample under liquid nitrogen to a fine powder and transfer to a new tube.
- Add 4.5 ml of RX Buffer or PRX Buffer (ß-ME added) to the tissue powder and vortex vigorously. In most cases RX Buffer is the buffer of choice to lyse plant tissue. However, plant tissues contain sticky secondary metabolites (for example, maize with milky endosperm or mycelia of filamentous fungi), PRX Buffer is used instead.
- Apply lysate to the Shearing Tube sitting in a Collection Tube and centrifuge at full speed (about 3,000 rpm or 2,500 x g) for 2 minutes. Transfer flow-through sample from the Collection Tube to a new tube. Avoid pipetting any debris and pellet in the Collection Tube.
- Add 2.3 ml (about half of the sample volume) 96-100% ethanol to the clear lysate and mix by pipetting. If sample lysate is lost during the preparation, reduce ethanol volume proportionally.
- Apply 6.8 ml of the ethanol added sample (including any precipitate) from step 4 to a Plant RNA Maxi Column sitting in a Collection Tube, close the cap, centrifuge at full speed for 3 minutes, and discard the filtrate. If the solution remains above the membrane, centrifuge again for another 5 minutes.
- Repeat step 5 for rest of the sample.
- Grind 1 g (or less) plant sample under liquid nitrogen to a fine powder and transfer to a new tube.
- Add 4.5 ml of RX Buffer or PRX Buffer (ß-ME added) to the tissue powder and vortex vigorously. In most cases RX Buffer is the buffer of choice to lyse plant tissue. However, plant tissues contain sticky secondary metabolites (for example, maize with milky endosperm or mycelia of filamentous fungi), PRX Buffer is used instead.
- Apply lysate to the Shearing Tube sitting in a Collection Tube and centrifuge at full speed (about 3,000 rpm or 2,500 x g) for 2 minutes. Transfer flow-through sample from the Collection Tube to a new tube. Avoid pipetting any debris and pellet in the Collection Tube.
- Add 2.3 ml (about half of the sample volume) 96-100% ethanol to the clear lysate and mix by pipetting. If sample lysate is lost during the preparation, reduce ethanol volume proportionally.
- Apply 6.8 ml of the ethanol added sample (including any precipitate) from step 4 to a Plant RNA Maxi Column sitting in a Collection Tube, close the cap, centrifuge at full speed for 3 minutes, and discard the filtrate. If the solution remains above the membrane, centrifuge again for another 5 minutes.
- Repeat step 5 for rest of the sample.
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