Yu:RNA extract
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RNA Extraction
Day 1
- Grow 50 mL cultures to OD 0.2-0.4
- Spin down cultures - 3000 RPM for 2 min at RT (prevents cold-shocking the cells)
- Pour of supernatant and immediately plunge into Liquid Nitrogen
- 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
- Thaw cell pellets on ice
- Turn on water bath and set to 65°C or turn on thermomixer
- Resuspend cells in residual liquid and transfer to eppendorf tube
- Spin down 1 min, top-speed at 4°C
- Remove supernatant
- Resuspend the cell pellet in 500 μL TES (RNase-free)
- Add 500 μL acid-phenol and vorext 10"
- Incubate at 65°C for 1 hr, vortexing every 10 min
- Be sure to use cap-locks on all tubes
- Ice tubes for 5 min
- Centrifuge 5 min, top-speed at 4°C
- Transfer aqueous phase (top layer) to a clean eppendorf tube Do not suck up any of the organic layer!!!
- Add 500 μL acid-phenol and vortex 10"
- Ice tubes for 5 min
- Centrifuge 5 min, top-speed at 4°C
- Transfer aqueous phase (top layer) to a clean eppendorf tube Do not suck up any of the organic layer!!!
- Add 500 μL chloroform and vortex 10"
- Centrifuge 5 min, top-speed at 4°C
- Transfer aqueous phase (top layer) to a clean eppendorf tube (~400 μL) to a clean eppendorf tube
- Add 50 μL of 3M NaOAc pH 5.3 and mix by pipetting
- Add 1 mL ice-cold 100% EtOH (RNase-free)
- Precipitate O/N at -20 °C
Day 3
- Centrifuge samples for 10 min, top-speed at 4°C
- Remove supernatant, either by decanting or pipetting
- Add 1 mL ice-cold EtOH (RNase-free)
- Vortex briefly (pellet may or may not dislodge)
- Centrifuge samples for 10 min, top-speed at 4°C
- Remove supernatant, either by decanting or pipetting
- Flash-spin samples and remove excess liquid with P-20
- Air-dry pellet ≥30min
- Dissolve samples in 100 μL H2O (RNase-free)
- Dilute sample 1:1000 and measure the OD260 and OD280