Hot Phenol RNA Isolation
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Pipet 5ml of culture from the side-arm flask into a 15ml plastic conical Falcon
tube.
Add 1:10 vol/vol of phenol to ice cold ethanol to 5ml of culture. (4.5ml 100%
ethanol and 500ul water saturated phenol) and centrifuge 10 mins @ 4C to
pellet cells.
Pour off supernatant leaving as little medium behind as possible.
Cell pellets maybe snap frozen using dry ice or liquid nitrogen and stored
@ - 80C for up to a week.
Lyse the cells by adding 700ul buffer TE pH 8.0 and transfer to a 1.5ml
microfuge tube
Add 7ul of 10% SDS and put in a 65C water bath for 1-2 mins.
Add 77ul of 1M NaOac (sodium acetate) and 850ul of hot (65C) water saturated
phenol and invert 10 times.
Place in a 65C water bath for 6 mins. Invert microfuge tube every 60 secs
while in the waterbath.
Centrifuge 10 mins @ 4C (21000 (xG).
Pipet off the top layer and put into a 1.5ml centrifuge tube containing 850ul
chloroform and invert several times.
Centrifuge 10 mins @ 4C (21000 xG).
Pipet off the first 250ul of the top layer and put into a 1.5ml microfuge tube.
Pipet off the remaining 250ul or what is left of the top layer into another 1.5ml
microfuge tube.
To each tube add 800ul cold ethanol (100%), 35ul 3 M NaOac.
Put the tubes in the - 80C freezer for at least two hours to overnight.
Centrifuge for 25 mins @ 4C (21000 (xG).
Carefully decant supernatant.
Wash the RNA pellet by adding 1 ml of cold 80% ethanol.
Centrifuge for 15 mins @ 4C (21000 (xG).
Repeat ethanol wash steps 17 and 18.
Resuspend RNA pellet in 30ul of DEPC water.
Spec RNA samples and store @ -80C. DNAse treat (on column) while cleaning
up RNA using Qiagen DNAse kit and RNeasy mini kit.