RNeasy RNA isolation

  1. Bacterial culture is grown (in LB at 37°C) to wanted OD600 (0.5).
  2. T0 sample is removed and processed as below, remainder is transferred to specialised conditions and incubation is continued and then processed as below.
  3. <1*109 bacterial cells (approximately 3 ml 0.5 OD600 for Salmonella typhimurium in LB) are transferred to 15 ml Greigner tubes.
  4. Spin for 10 minutes at 5000g (Full speed in tabletop centrifuges in lab and cold room).
  5. The supernatant is carefully and completely removed from tubes by decanting and gentle tapping to remove any remaining liquid.
  6. Pellet is resuspended in 100µl of lysozyme (1mg/ml in TE (not treated with DEPC)) by vortexing.
  7. Transfer to eppendorf tube and incubate at room temperature for 5 minutes with vortexing for 10 seconds after 2 and 4 minutes.
  8. To the wanted amount add 1µl of β-Mercaptoethanol per 100µl to buffer RLT from Qiagen RNeasy kit (350µl per preparation).
  9. Add 350µl of the RLT buffer with β-Mercaptoethanol to the lysed cells and vortex thoroughly for complete mixing.
  10. Centrifuge at maximum speed for 2 minutes.
  11. Transfer supernatant to fresh eppendorf tube for use in subsequent steps.
  12. Add 250µl of ethanol (96%) and vortex.
  13. Add everything (approximately 700µl) including any precipitate to an RNeasy column in the provided 2ml collection tube.
  14. Centrifuge for 15 seconds at 8000g (10,000rpm) and discard flow through but keep the collection tube.
  15. Add 350µl buffer RW1 from RNeasy kit to the column.
  16. Centrifuge for 15 seconds at 8000g (10,000rpm) and discard flow through but keep the collection tube.

    DNase Step

  17. Add 10µl DNase solution from Qiagen RNase-Free DNase Set to 70µl buffer RDD from same and mix gently without vortexing.
  18. Add the 80µl DNase mix to the membrane taking care to avoid any of the mix sticking to the sides.
  19. Incubate at room temperature for 15 minutes.

    RNeasy Mini RNA prep protocol continued

  20. Add 350µl buffer RW1 from RNeasy kit to the column.
  21. Centrifuge for 15 seconds at 8000g (10,000rpm).
  22. Transfer spin column into new provided 2ml collection tube.
  23. Add 500µl buffer RPE from RNeasy kit to the column.
  24. Centrifuge for 15 seconds at 8000g (10,000rpm) and discard flow through but keep the collection tube.
  25. Add 500µl buffer RPE from RNeasy kit to the column.
  26. Centrifuge for 15 seconds at 8000g (10,000rpm).
  27. Transfer spin column into 1.5ml eppendorf tube with the lid removed.
  28. Centrifuge for 2 minutes at full speed.
  29. Transfer spin column to 1.5ml collection tube.
  30. Add 30µl of RNase-free water from RNeasy kit to the membrane again taking care to avoid any sticking to the sides.
  31. Centrifuge for 1 minute at 8000g (10,000rpm).
  32. Add another 30µl of RNase-free water from RNeasy kit to the membrane again taking care to avoid any sticking to the sides.
  33. Centrifuge for 1 minute at 8000g (10,000rpm).
  34. Transfer 50µl to a fresh eppendorf and label both tubes, the remaining 10µl can be used for quality control.

The yield from this should be between 15µg and 60µg of RNA, giving a concentration of between 0.25µg/µl and 1.00µg/µl.

The RNA can be stored in a -70°C freezer

Solutions

DNase solution
Dissolve solid DNase I from DNase kit with 550µl of the provided RNase-free water in provided glass vial, take to avoid any DNase escaping when opening vial. Mix by inverting vial and aliquot into tubes of 10µl or 20µl each and store in freezer.

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