Apply 500 µl Buffer BR4 to the PAXgene column, and centrifuge for 1 min at ³8000 x g (³10,000 rpm). Place the PAXgene column in a new 2 ml processing tube, and discard the old processing tube containing flow-through.
Note: Buffer BR4 is supplied as a concentrate. Ensure that ethanol is added to Buffer BR4 before use (see “Important notes before starting”).
12. Add another 500 µl Buffer BR4 to the PAXgene column. Centrifuge for 3 min at maximum speed to dry the PAXgene column membrane.
After this washing step, the silica-gel membrane may be light or dark brown in color. This does not influence the quality of the RNA isolated.
13. Discard the tube containing the flow-through, and place the PAXgene column in a new 2 ml processing tube (not supplied). Centrifuge for 1 min at full speed. DO this, NOT optional – need to be sure to remove all BR4 from sample!
14. To elute, discard the tube containing the flow-through, transfer the PAXgene column to a 1.5 ml elution tube, and pipet 40 µl Buffer BR5 directly onto the PAXgene column membrane. Centrifuge for 1 min at ³8000 x g (³10,000 rpm).
It is important to wet the whole membrane with Buffer BR5 in order to achieve maximum elution efficiency. Be careful to add BR5 to middle of membrane, and not to touch membrane with pipet tip.
15. Repeat the elution step (step 14) as described, using 40 µl Buffer BR5.
16. Store samples at –70°C or –80°C. We store the purified total RNA immediately and denature prior to further manipulation. However, the official protocol provides the option of denaturing RNA at 65°C for 10 minutes before storing at -80°C.
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