Phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation

We use this protocol to generate clean sterile DNA for electroporation into E. coli or Trypanosoma brucei.

The starting volume should be at least 100 μl. If working with a standard ligation volume of 10 or 20 μl, make the volume up to 100 μl with distilled water in a 1.5 ml microcentrifuge tube.

1. Add an equal volume of water saturated phenol:chloroform:isoamyl alcohol, vortex for 5 seconds and centrifuge at >16,000 g in a microcentrifuge for 1 minute; this separates the organic and aqueous phases.
Safety consideration – gloves should be worn and contact with skin should be avoided when handling phenol:chloroform. Phenol is a systemic poison whose absorption by the skin is enhanced by the presence of chlorofrm. It can cause severe burns.

2. Transfer the aqueous phase (containing your DNA) to a fresh 1.5 ml microcentrifuge tube, add 0.2 μl glycogen (a ‘carrier’) and four volumes of ice cold 100% ethanol. Place at -20°C for at least 10 minutes.
Safety consideration – the organic phase should be transferred to your laboratory’s phenol waste and disposed of according to your institutional guidance.

3. Centrifuge at >16,000 g in a microcentrifuge for 5 minutes to pellet your DNA. Pour off the supernatant.

4. Wash the pellet in 1 ml ice cold 70% ethanol, inverting several times, and centrifuge at >16,000 g in a microcentrifuge for 5 minutes. Pour off the supernatant, then repeat the centrifugation for 30 seconds and remove the residual supernatant with a pipette.

5. Open the tube and leave the pellet to dry for 5-10 minutes in a flow hood and resuspend in 10 μl distilled water.
If you require your DNA to be sterile (e.g. for T. brucei electroporation), supernatant removal after the 70% ethanol wash, and pellet drying should be carried out in a suitable flow cabinet.