Reagents:

Provided by Midi Kit (Qiagen, #75142, 10 samples, $90.00):

  • 45 ml Buffer RLT (contains GITC, add 10ml β-Mercaptoethanol per ml RLT before use)

  • 45 ml Buffer RW1 (contains GITC and ethanol)

  • 11 ml Buffer RPE (concentrate, add 44 ml 100% ethanol before use)

  • 10 ml RNase-free water

  • 10 RNeasy Midi columns (RNA binding capacity up to 1mg each)

 

To be supplied by user:

  • 20 cc Syringe (UW Stores, 40/box, 0056-590, $11.12)

  • 14.3 M β-Mercaptoethanol (Sigma, FW=78.13, 100 ml, M-7522, $15.40)

  • Ethanol (70% and 100%)

 

Protocol:

  1. Wash cells once with PBS.

  2. Lyse cells in buffer RLT (2 ml for up to 50 x 106 cells).

  3. Homogenize cell lysate immediately by passing 10 times through 18 gauge needle with syringe.

  4. Add 1 volume (2 ml) of 70% Ethanol, mix thoroughly.

  5. Transfer sample to RNeasy Midi column in 15 ml tube

  6. Centrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

  7. Add 4ml of buffer RW1.

  8. entrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

  9. Add 2.5ml of buffer RPE.

  10. Centrifuge at 3000-5000 x g for 2 minutes; discard flow-through.

  11. Add 2.5ml of buffer RPE.

  12. Centrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

  13. Transfer RNeasy Midi column to clean 15 ml tube.

  14. Add max. 250 µl RNase-free H2O, incubate for 1 minute.

  15. Centrifuge at 3000-5000 x g for 3 minutes

  16. Add max. 250 µl RNase-free H2O, incubate for 1 minute.

  17. Centrifuge at 3000-5000 x g for 3 minutes; discard column.

  18. Read O.D. 260/280.

  19. Give sample to CEA for Bioanalyzer run.

Department of Microbiology
School of Medicine
University of Washington
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