Introduction

This protocol was developed for the MultiPROBEΠII HT Liquid Handling System with a Gripper Integration Platform. Time required: 1 hr 20 min.

For more information on the protocol, see the RNAqueous-96 Instruction Manual (pdf).

Protocol

  1. Add 150 µl Lysis Solution to each well.

  2. Add 150 µl 64% ethanol to each well.

  3. Mix and transfer to the 96-well filter plate.

  4. Apply vacuum suction (20 mm Hg) for 3 min (RNA remains bound to the filter).

  5. Add 400 µl Wash Solution A to each well and apply vacuum suction (20 mm Hg) for 2 min.

  6. Add 40 µl (100 units) DNase I.

  7. Incubate 20 min at room temperature.

  8. Add 400 µl Wash Solution B to each well and apply vacuum suction (20 mm Hg) for 2 min.

  9. Add 400 µl Wash Solution C/D to each well and apply vacuum suction (20 mm Hg) for 2 min.

  10. Add 400 µl 100% ethanol to each well and apply vacuum suction (20 mm Hg) for 2 min.

  11. Move 96-well filter plate to dry heat block covered with fresh paper towels and heat at 42­C for 3 min (this removes any residual wash solution).

  12. Move Spacer and Collection plates to the vacuum manifold. Place 96-well filter plate on top.

  13. Add 80 µl Elution Buffer to each well.

  14. Apply vacuum suction at 10 mm Hg for 30 sec then at 20 mm Hg for 2 min (RNA is transferred to collection plate).

  15. (optional) Manually check for fluid remaining on filter plate. If fluid is present, centrifuge the filter plate/collection assembly to recover residual sample.

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