Hot Acid Phenol RNA Isolation (revised 6-26-06)

 Use 10-15 ml of cells.  One should get about 300 mg of RNA from 2X108 cells.  This comes from Currents Protocols in Molecular Biology and from HahnÕs web site.

 

Important:  Make sure and use acid phenol not buffered phenol.  (There is a stock of this already made up.) This allows for less DNA contamination.  All reagents and supplies must be RNAse-free.

 

  1. Transfer 10 - 15 ml of cell culture into 50 ml conical. Centrifuge 3 min at 15000 x g at 4oC.  This is assuming the cells are at ~0.5 to 1 X 107 cells/ml.
  2. Resuspend pellet in 1 ml ice-cold (RNase free) water, move to 1.5 microfuge tube.
  3. Pellet cells and decant.
  4. Proceed to step 5 or immediately freeze pellet on dry ice and store at -70 until further use.
  5. Resuspend pellet in 400 ml of TES, add 400ml acid phenol. Vortex 10 sec, incubate for ~60min at 65o with occasional vortexing.
  6. Incubate on ice for 5min. Microfuge 5 min for 4o.
  7. Transfer aqueous (upper layer) to new 1.7 ml tube add 400ml acid phenol. Vortex 10 sec.  Repeat step 6.
  8. Repeat step 7. In other words do 2 acid phenol extractions in total
  9. Do two CHCl3 extractions: add 400 ml chloroform, vortex vigorously, spin 5Õ, 4o.
  10. Add 10% volume of 3M NaOAc, pH5.2 and 2-2.5 volume of 100% EtOH to final aqueous layer.  Incubate on ice for at least 1 hour. 
  11. Microfuge 15min, remove supernatant, wash with 500ml 80%EtOH.  (Can leave overnight at -20C.) Dry pellet at room temperature or in speed vac.  But do not over dry in speed vac or else the pellet will be hard to resuspend.
  12. Resuspend pellet in 50ml of RNase-free water. Store at -70.
  13. Check OD at 260nm in water for quantitation.  To check 260/280/230 dilute in TE.  260/280 > 1.7; Organic contaminants like phenol, ethanol and aromatic compounds absorb at 230 therefore 260/230 should be at least 1.8,  above 2.2 is better.

 

REAGENTS

Acid phenol- To solid phenol add RNase-free water until there is a layer of water on top of the phenol: Heat new bottle (500g) to 65 oC, crack lid.  Add 100 ml of RNase-free water.  Mix and let cool.  Add about 100 mls more of water until a little water remains on top of phenol so that is it completely water saturated. Aliquot into 50 ml tubes and freeze at -20.

 

TES Ð made from RNAse-free stock solns

                                                           For 50 mls

10 mM Tris pH 7.5                       0.5ml  1M Tris pH 7.5

10 mM EDTA                                1.0ml 500 mM EDTA

0.5% SDS                                        2.5 mls of 10% SDS

Water                                                 up to 50 mls