DNAse inactivation, washing and RNA elution 

The following steps are required to inactivate the DNAse and to clean the RNAs by removing all the non-nucleic acid particles from the column.

Material and reagents :

  • 2 X Collection tube 2 ml;
  • 2 X collection tube 1,5 ml;
  • 2 X Eppendorf 1,5 ml;
  • RA2 buffer;
  • RA3 buffer;
  • RNAse-free water.

Procedure :

  1. Add 200 μL Buffer RA2 to the NucleoSpin® RNA Plant Column. Buffer RA2 will inactivate the rDNase.
  2. Centrifuge for 30 s at 11,000 x g.
  3. Place the column into a new Collection Tube (2 mL).
  4. Add 600 μL Buffer RA3 to the NucleoSpin® RNA Plant Column.
  5. Centrifuge for 30 s at 11,000 x g.
  6. Discard the flow through (= jeter le liquide du tube dans la poubelle et conserver la colonne) and place the column back into the Collection tube.
  7. Add 250 μL Buffer RA3 to the NucleoSpin® RNA Plant Column.
  8. Centrifuge for 2 min at 11,000 x g to dry the membrane completely.
  9. Place the column into a nuclease-free Collection Tube (1.5 mL, supplied).
  10. Add 50 μL RNase-free H2O and centrifuge at 11,000 x g for 1 min.
  11. Transfert the solution to a new 1,5 ml Eppendorf tube and place the tubes back on ice.
This last step of the RNA extraction itself will retrieve the RNA bound to the membrane. It can be used using a buffer (e.g. TE) or, as in this case, using sterile water.