Yu:RNA extract

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Contents

RNA Extraction

Day 1

  1. Grow 50 mL cultures to OD 0.2-0.4
  2. Spin down cultures - 3000 RPM for 2 min at RT (prevents cold-shocking the cells)
  3. Pour of supernatant and immediately plunge into Liquid Nitrogen
  4. Store at -80°C until ready for RNA extraction
Freeze cells in liquid nitrogen even if extracting RNA the same day

Day 2

Handle all organic solvents in the fume hood and dispose of organics in the proper disposal container
  1. Thaw cell pellets on ice
    1. Turn on water bath and set to 65°C or turn on thermomixer
  2. Resuspend cells in residual liquid and transfer to eppendorf tube
  3. Spin down 1 min, top-speed at 4°C
    1. Remove supernatant
  4. Resuspend the cell pellet in 500 μL TES (RNase-free)
  5. Add 500 μL acid-phenol and vorext 10"
  6. Incubate at 65°C for 1 hr, vortexing every 10 min
    1. Be sure to use cap-locks on all tubes
  7. Ice tubes for 5 min
    1. Centrifuge 5 min, top-speed at 4°C
  8. Transfer aqueous phase (top layer) to a clean eppendorf tube Do not suck up any of the organic layer!!!
    1. Add 500 μL acid-phenol and vortex 10"
  9. Ice tubes for 5 min
    1. Centrifuge 5 min, top-speed at 4°C
  10. Transfer aqueous phase (top layer) to a clean eppendorf tube Do not suck up any of the organic layer!!!
    1. Add 500 μL chloroform and vortex 10"
    2. Centrifuge 5 min, top-speed at 4°C
  11. Transfer aqueous phase (top layer) to a clean eppendorf tube (~400 μL) to a clean eppendorf tube
    1. Add 50 μL of 3M NaOAc pH 5.3 and mix by pipetting
    2. Add 1 mL ice-cold 100% EtOH (RNase-free)
    3. Precipitate O/N at -20 °C

Day 3

  1. Centrifuge samples for 10 min, top-speed at 4°C
  2. Remove supernatant, either by decanting or pipetting
  3. Add 1 mL ice-cold EtOH (RNase-free)
    1. Vortex briefly (pellet may or may not dislodge)
  4. Centrifuge samples for 10 min, top-speed at 4°C
  5. Remove supernatant, either by decanting or pipetting
    1. Flash-spin samples and remove excess liquid with P-20
  6. Air-dry pellet ≥30min
  7. Dissolve samples in 100 μL H2O (RNase-free)
  8. Dilute sample 1:1000 and measure the OD260 and OD280
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