RNA preparation from plates with Trizol (PJ Keller)

/0 Comments/in , , /by

RNA Prep Trizol (Click to Download)

  1. Aspirate media, wash plates with PBS 1X and aspirate off excess liquid
  2. Add 0.5 ml Trizol to each well in the hood, pipet up and down a few times to cover the wells, pipet the mixture into autoclaved microfuge tubes, incubate for 5’ at RT (combine the replicates into one tube)
  3. Add 0.2 ml Chloroform to each tube, cap tightly and shake vigorously for 15 seconds, incubate at RT for 2-3 min.
  4. Spin at 12,000 rpm for 15 minutes at 4C, RNA will be in the aqueous top phase.
  5. Transfer the aqueous phase to a fresh microfuge tube
  6. Add 500 ml of RNA only isopropanol to each tube, incubate at RT 10’
  7. Spin at 12,000 rpm for 10 minutes at 4C, look for RNA pellet
  8. Wash the pellet once with 1ml 75% ethanol prepared with Rnase-free water, vortex the sample to mix and spin at 7500 rpm for 5 min
  9. Remove the ethanol, invert tube and allow to air dry for 10-15 min
  10. Resuspend the pellet in 50 ml of Rnase-free water, incubate for 10’ at 50C to dissolve, store at –80