Yu:RNA extract

Day 1
Freeze cells in liquid nitrogen even if extracting RNA the same day
 * 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

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
 * 2) Turn on water bath and set to 65°C or turn on thermomixer
 * 3) Resuspend cells in residual liquid and transfer to eppendorf tube
 * 4) Spin down 1 min, top-speed at 4°C
 * 5) Remove supernatant
 * 6) Resuspend the cell pellet in 500 μL TES (RNase-free)
 * 7) Add 500 μL acid-phenol and vorext 10"
 * 8) Incubate at 65°C for 1 hr, vortexing every 10 min
 * 9) Be sure to use cap-locks on all tubes
 * 10) Ice tubes for 5 min
 * 11) Centrifuge 5 min, top-speed at 4°C
 * 12) Transfer aqueous phase (top layer) to a clean eppendorf tube Do not suck up any of the organic layer!!!
 * 13) Add 500 μL acid-phenol and vortex 10"
 * 14) Ice tubes for 5 min
 * 15) Centrifuge 5 min, top-speed at 4°C
 * 16) Transfer aqueous phase (top layer) to a clean eppendorf tube Do not suck up any of the organic layer!!!
 * 17) Add 500 μL chloroform and vortex 10"
 * 18) Centrifuge 5 min, top-speed at 4°C
 * 19) Transfer aqueous phase (top layer) to a clean eppendorf tube (~400 μL) to a clean eppendorf tube
 * 20) Add 50 μL of 3M NaOAc pH 5.3 and mix by pipetting
 * 21) Add 1 mL ice-cold 100% EtOH (RNase-free)
 * 22) 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)
 * 4) Vortex briefly (pellet may or may not dislodge)
 * 5) Centrifuge samples for 10 min, top-speed at 4°C
 * 6) Remove supernatant, either by decanting or pipetting
 * 7) Flash-spin samples and remove excess liquid with P-20
 * 8) Air-dry pellet ≥30min
 * 9) Dissolve samples in 100 μL H2O (RNase-free)
 * 10) Dilute sample 1:1000 and measure the OD260 and OD280