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.
- 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.
- Resuspend pellet in 1 ml ice-cold (RNase free) water,
move to 1.5 microfuge tube.
- Pellet cells and decant.
- Proceed to step 5 or immediately freeze pellet on dry
ice and store at -70 until further use.
- Resuspend pellet in 400 ml of TES, add 400ml
acid phenol. Vortex 10 sec, incubate for ~60min at 65o
with occasional vortexing.
- Incubate on ice for 5min. Microfuge 5 min for 4o.
- Transfer aqueous (upper layer) to new 1.7 ml tube add
400ml acid phenol. Vortex
10 sec. Repeat step 6.
- Repeat step 7. In other words do 2 acid phenol
extractions in total
- Do two CHCl3 extractions: add 400 ml chloroform, vortex vigorously, spin
5Õ, 4o.
- 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.
- 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.
- Resuspend pellet in 50ml
of RNase-free water. Store at -70.
- 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