A. Phenol Chloroform extractions

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• Phenol/chloroform extraction is a handy way to remove proteins from your nucleic acid samples.

• When done carefully, the method can be used quantitatively.

• Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.

• The method is highly versatile in terms of sample and volumes but can be a bit tiring if dealing with too many samples at a time. So, keep this in mind before planning extractions.

• Other reagents like trizol can b used on similar lines, but I recommend reading the manual before usage.

• For RNA and DNA, pH of phenol is crucial. Acid saturated for RNA while basic for DNA. I suggest buy commercial as saturating yourself can be hazardous and at time inefficient.

1. Dilute your nucleic acid sample to 100–700 µL or divide your samples into tubes such that you have no more than 700 µL per tube. It is difficult to do the extraction with volumes smaller than 100 µL. The sample can be concentrated again after precipitation.

2. Add an equal volume of phenol to the tube, vortex vigorously to mix the phases. Be carful to properly close the tube, you really don’t want to be spilling around phenol.

3. Spin in a microfuge at top speed for 2–5 min to separate the phases, for RNA I prefer to do this at a cooled centrifuge but is not essential. For whole cell or tissue samples, this might take longer as a result of higher protein in the samples.

4. Carefully aspirate to extract the aqueous phase to a new tube.  Avoid picking up any of the protein at the phase interface.

5. Repeat the phenol extraction two more times.

6. Extract the sample two times with an equal volume of chloroform:isoamyl alcohol or just chloroform (for RNA) to remove any trace phenol.

7. Precipitate the nucleic acid. (nucleic acid precipitation)

Reagents

• Phenol equilibrated to pH 7.5 (pH is important, see Notes below)

• Chloroform:isoamyl alcohol in a 24:1 ratio for DNA

Notes

• Equilibrated phenol can typically be purchased from commercial sources. Alternatively, you can equilibrate it yourself. Be advised that this is NOT a fun procedure to carry out. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (arround pH 5). This fact can be used for RNA extraction but it is not a good idea to use acid phenol to clean your chromosomal DNA from proteins.

• Phenol can undergo oxidation. Oxidized phenol will appear yellow or red in color (instead of clear) when saturated with water or buffer.

• Phenol and chloroform should be used in a hood if possible.

• Phenol is a dangerous substance that will burn you if it gets on your skin. WEAR GLOVES and BE CAREFUL. Check out the MSDS to verify the precautions you should take. A solution of PEG 400 is recommended for first aid. Phenol is both a systemic and local toxic agent.

• The protocol can be shortened from two phenol extractions and one chloroform extraction. The number of repetitions also depends on what kind of sample you have and what you want to do with it. If you have whole cell extracts you want to use more phenol steps as if you have only one restriction enzyme to get rid of. In the same way you might not care about residual phenol if you just want so run your DNA or RNA on a gel so one chlorophorm step is sufficient. Some other reactions you can do with nucleic acids are more sensitive to phenol so you should use chlorophorm two times.

• There are also commercial sources of phenol and chloroform mixed together and equilibrated. These are also sufficient for extraction, and I would recommend doing at least two extractions if you decide to go this route. Phenol:Chloroform:Isoamyl alcohol usually has an upper layer of buffer saturated water in the bottle. Do not use this buffer layer -- you want the phenol/chloroform layer underneath.

• Be careful to determine which layer is the phenol. The density of pure phenol (unlike phenol:chloroform:isoamyl alcohol) is almost 1.0. Small changes in the density of your water layer (excess salt, e.g.) can lead to layer inversion.

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