Phenol-Chloroform Extraction

9/30/04

The purpose of phenol-chloroform extraction is to remove protein from a nucleic acid sample (DNA or RNA).

Note: Phenol can cause burns and smells bad. You should not breathe chloroform. Use these in the hood and always wear gloves. You must dispose of liquid waste as hazardous.

Take DNA solution. You may need to add water to make the volume a little higher. Ideal is >50uL.

Add an equal volume phenol/chloroform/IAA (25:24:1). For DNA this solution should have a pH of 8.0 (for RNA it will be 4.5 and have different ratios). The phenol is heavier than water so will be underneath a layer of Tris that will lie on the very top. Therefore pipette from the bottom.

Vortex the mixture until well mixed (30sec).

Spin 14K 5’.

You will see two phases in the tube, with the protein in a white band between them. Pipetting from the top, remove as much of the top phase (this is where the DNA/RNA is) as possible without bringing along any of the white interface. Leave behind some of the top phase if you must to avoid the protein. Place top phase in a fresh tube.

Dispose of bottom phase (phenol) as hazardous waste in the hood.

Add an equal volume of chloroform to the new tube with the top phase. Extracting with chloroform helps to remove any residual phenol. Vortex, spin, and take the top phase again.

If the volume is too large for the next step, consider precipitating DNA.