Sean's RNA Purification protocol - short
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 
- 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.