Peccoud Lab Protocol: purifying and/or concentrating DNA/RNA from various sources by phenol-chloroform extraction and ethanol precipitation.
Sometimes it becomes necessary to concentrate your DNA/RNA or obtain purer DNA/RNA samples. If your DNA is resuspended in nucease-free water, you can concentrate it just by drying it down in the Vacuum Centrifuge . However, if the DNA is in a buffer (e.g. elution buffer or TE) you need to ethanol precipitate the DNA to prevent concentration of the buffer salts. Also, phenol-chloroform extraction of the DNA/RNA sample can remove contaminating proteins left over from the DNA prep, especially when using kits in which the DNA/RNA is concentrated on a silica column and washed on column. Removal of all protein is not usually important for RE digests, cloning or PCR, but is important when sequencing the DNA/RNA.
- DNA sample in screw cap microfuge tube
- phenol:chloroform:isoamyl alcohol mixture, pH6.7/8.0 (25:24:1)
- 96% Ethanol or isopropanol
- 3M Sodium Acetate , or 2M sodium chloride 5M solution , or 8M lithium chloride, or Ammonium acetate solution, 5M
- In the fume hood, add an equal volume of phenol:chloroform:isoamyl alcohol to DNA sample
- Vortex for 2 min
- Centrifuge max speed (14,000 rpm) for 2 min
- Collect the top aqueous layer and place in new screw cap tube, ensuring that you do not aspirate any of the white boundary layer
- Repeat steps 1-4 with fresh tubes.
- Add 1/10th the total volume of 3M sodium acetate (DNA), or 2M sodium chloride (DNA + SDS), or 8M lithium chloride (RNA: but beware – chloride ions will inhibit in vitro translation or reverse transcription), or equal volume of 5M ammonium acetate (for the removal of dNTPs, except for preparation of DNA for T4 polynucleotide kinase reactions). To increase the yield in precipitations of low concentration or small nucleic acid pieces (less than 100 nucleotides), also add MgCl2 to a final concentration of 0.01M.
- For DNA, add 2x the volume of 95% ethanol (or 1x volume of isopropanol) and freeze for 1 hour. Isopropanol is good for low concentrations of DNA but also tends to precipitate more salt, so one needs to wash several times in 70% ethanol to remove salts.
- Centrifuge at max speed (14,000 rpm) for 5 minutes at 4 °C
- Aspirate off ethanol (or isopropanol)
- Add 500 ul of 70% ethanol, let sit for 1 minute, spin down, and aspirate
- Dry pellet for 20 minutes at 45 °C
- Re-suspend in nuclease free water or TE to desired concentration (assume you have lost half of total DNA). Use TE only if you need to store the DNA for an extended time. TE will inhibit some enzymatic reactions.