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.