Phenol-Chloroform Extraction
PROTOCOL DUMP PLEASE FORMAT
For removal of proteins and most of the free nucleotides after T7_in vitro_transcription (or any other_in vitro_transcription method), phenol:chloroform extraction and ethanol precipitation of RNA transcripts is the preferred method.
To prepare beforehand:
Reagent | Amount per sample |
3 M sodium acetate, titrated to pH 5.2, and DEPC treated | 20 uL |
1:1 phenol:chloroform (store in glass container, and wrap in foil to keep away from light) | 200 uL |
70% ethanol, prepared with nuclease-free water and ethanol | 500 uL |
Nuclease-free microcentrifuge tubes, (a) through (f) | 6 tubes |
Clean the hood thoroughly and put foil down. Spray with RNase-Away.
PHENOL AND CHLOROFORM ARE TOXIC AND MUTAGENIC, AND CAN BE ABSORBED THROUGH YOUR SKIN!
IF YOU GET ANY ON YOUR GLOVES: remove the glove and put in the phenol:chloroform waste jar in the hood
IF YOU GET ANY ON YOUR SKIN: rinse thoroughly withglycerol, as rinsing with water will increase phenol penetration into your skin. Wash with soap and water for 15 minutes afterwards. Call EHS.
Phenol:chloroform extraction
Synthesize RNA in a T7in vitrotranscription reaction.
To 20 uL transcribed RNA, add 160 uL nuclease-free water. Transfer to microcentrifuge tube (a).
Add 20 μl of 3 M sodium acetate, pH 5.2, to each sample. Vortex 10 s and centrifuge.
Move to the hood. Phenol and chloroform are both toxic to inhale and upon skin contact.
Add 200 uL of 1:1 phenol:chloroform to each sample. Vortex 20 s to mix.
Spin at max RPM in a tabletop centrifuge, 1 min, to separate the layers.
Take aqueous supernatant (TOP layer), being sure to not collect any of the bottom (organic) layer. Place in new microcentrifuge tube (b). Discard previous tube.
To tubeb, add 200 uL chloroform. Vortex 20 s to mix.
Spin at max RPM in centrifuge, 1 min.
Take aqueous supernatant and place in new microcentrifuge tube (c). Discard previous tube.
Repeat chloroform extraction, and place supernatant in tube(d). Record the volume of supernatant.
Ethanol precipitation
Precipitate the RNA by adding 2 x (volume supernatant, should be ~150 uL) of ethanol to tube(d). Invert to mix. Incubate at -20°C for 1 hr.
Put the microcentrifuge in the 4C deli fridge immediately after placing RNA in the -20C.
Collect RNA pellet by centrifugation for 30 min at 4C. Remove supernatant, making sure to avoid disturbing the pellet.
Wash the pellet with 500 μl of cold 70% ethanol. Centrifuge 5 min at 4C. Remove supernatant.
Open tubes and cover in clean aluminum foil. Dry at 37C for 15 min.
Resuspend the RNA in 50 μl of DEPC-treated water. Store the RNA at -80°C.
Run native agarose gel and measure concentration
While your samples (in tube (d)) are in the -20C and in the centrifuge at 4C, prepare an agarose-TAE gel.
Put 9 uL DEPC-H2O in tubee. Transfer 1 uL of RNA sample from tube(d)into this tube, and vortex to mix.
Place tube (d)into the -80C.
Transfer 2 uL from tube (e)to tube (f).
Measure the concentration from(f)on the NanoDrop
Add 8 uL loading dye to tube (e)and mix well.
Load tube (e)into the gel, and run at 60-80V.
Tips and tricks
Before you start the extraction, check the color of the phenol:chloroform solution. If it’s yellow, your phenol has oxidized. Do not use!
If you get any solvent in your pipet tip during the extraction, slowly eject onto the side of the tube. It is heavier than the aqueous layer, and will be at the bottom of the tip.
Do not transfer any organic solvent with the supernatant between extractions! You'll see a low A260/A280, indicating phenol contamination
During the ethanol precipitation, over-drying the pellet will make it difficult to re-suspend. If pellet is over-dried, add 50 uL DEPC-H2O and place on ice 15 min. Vortex 20 s to resuspend, and centrifuge.
How do I know if my pellet is over-dried? If it looks glassy and dry it may be difficult to resuspend it.
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