Sean's RNA Purification protocol - short

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based on [Sean Moore's RNA purification protocol]

Materials and solutions

  • Wash buffer: 10mM Tris-Cl pH 8.0, 150mM NaCl
  • Native Lysis buffer: B-Per supplemented with 1mM MgOAc, 0.5mM CaCl, 0.1mM EDTA, 0.01-0.1mg/mL lysozyme, RNase-free DNaseI (10 units/ml final)
  • Salty Extraction buffer: 50mM bis-Tris pH 6.5, 400mM NaCl, 5mM EDTA, 1µL linear polacrylamide at 10mg/ml for every 400µL soln
  • acidic phenol/chloroform: buy from Ambion [1]
  • Isopropanol
  • 75% EtOH
  • 95% EtOH
  • Non-denaturing resuspension soln: 10mM bis-Tris, pH 6.5, 0.1 mM EDTA
  • Denaturing resuspension soln: 95% stabilized formamide, 10mM bis-Tris pH 6.5, 0.1mM EDTA, "some" bromophenol blue)

Procedure for Native RNA purification

  • 1) Grow up cells to desired phase/OD.
  • 2) Spin down cells and resuspend (or dilute original culture?) into ice-cold salty washing buffer. Unless noted, keep cold.
  • 3) Spin down cells again and resuspend in 100µl Native lysis buffer; vortex at room temp and let sit; lysis should occur in <2min.
  • 4) Add 400µl of salty extraction buffer and immediately add add 300-500µl of acidic phenol/chloroform; vortex samples and let sit on bench for 5 min.
  • 6) Spin at 6000rpm for 2 min and remove supernatant to new tube. Repeat extraction by adding phenol/chloroform to aqueous super. Repeat vortex and spin.
  • 7) Add 1.5x volume of isopropanol (so about 600-700µl if added 400µl salty extraction buffer) to supernatant and vortex; leave tubes on ice for 30 min or overnight at -20.
  • 8) Spin for 35min in the cold; the long time is critical!
  • 9) Remove supernatant but don't loose the pellet.
  • 10) Wash w/ 700µl 75% EtOH, re-spin (30s), and remove supernatant.
  • 11) Wash w/ 700µl 95% EtOH, re-spin, remove supernatant, and let the pellet DRY COMPLETELY. --> pellet should be bright white.
  • 12) Resuspend pellet in 50µl/(ml culture harvested) of non-denaturing soln. Drag the droplet over the stuff on the sides to recover all the RNA.
  • 13) Before running Northern, suspend needed amount of RNA in denaturing soln; make denaturing soln no less than 1/2 of the volume of the sample.
  • Expect 50-100µg total RNA/10^9 cells.