The old but good way to isolate/clean-up DNA
DNA can be purified for DNA sequencing or for restriction digestion by precipitation in an alcohol/water mixture in the presence of a high concentration of inorganic salt. DNA is recovered from the aqueous solution by addition of salt to final concentrations of 0.8M LiCl, 0.3-0.5M NaCl, NaOAc, or 2.5M NH4Ac and an appropriate volume of alcohol (30%-50% final percentage isopropanol; 60%-80% final percentage ethanol), storage for a brief period of time at -20°C or -70°C, followed by centrifugation. Subsequent desalting of the DNA pellet involves rinsing in 70% alcohol, recentrifugation and re-suspension in appropriate buffer.
About 1/2 as much isopropanol as ethanol is required to precipitate DNA, although higher concentrations may be helpful when the DNA is at low concentration. Since isopropanol is less volatile than ethanol, and since many salts are less soluble in isopropanol than in ethanol, a second 70% alcohol rinse of the pellet is recommended to more efficiently desalt the DNA pellet. NaCl is not as soluble as NH4OAc, NaOAc or LiCl in ethanol/water or isopropanol/water; it can be replaced by the latter salts when feasible.
Precipitations are commonly performed at -70°C, -20°C, 0°C, or room temperature for periods of 5 min to overnight. For low DNA concentrations: higher final concentrations of alcohol, longer precipitations (1 hr to overnight), lower temperatures (-20°C to -70°C) and longer centrifugation times (up to 30 min) may be required for efficient recovery.
At the lower temperatures, the viscosity of the alcohol is greatly increased and centrifugation for longer times may be required to effectively pellet the precipitated DNA. The efficiency of precipitation for small concentrations or amounts of DNA may be increased by incubation at -70°C, but these reactions should be brought to 0°C before centrifugation.
When SDS is present, one must take care not to precipitate it along with the DNA. By adding NaCl to a final concentration of only 0.2M or using LiCl (0.5M) before adding the alcohol, the SDS will remain in the supernatant. Finally, one should take care to remove all NH4+ ions if subsequent enzymatic steps are to be utilized, as it inhibits many enzymes (especially T4 polynucleotide kinase).
Example: For 100 ug/ml DNA solution:
1. Add 1/10th volume 5M NaCl, (or 1/2 volume 7.5M NH4Ac, 1/10 volume 8M LiCl, etc).
2. Add 1 volume isopropanol or 2 volumes ethanol.
3. Mix well and place at -20°C for 30 min.
4. Let warm to 0°C.
5. Microcentrifuge 15 min and remove supernatant (Save the supernatant until recovery is assured).
6. Gently rinse the pellet with 70% alcohol and recentrifuge as above.
NOTE: The pellet may not stick to the side of the microcentrifuge tube after this step and care must be taken not to lose it.
7. Aspirate the liquid and air dry or place in a vacuum centrifuge until the pellet is dry.
8. Resuspend the pellet to the required concentration in 0.01M Tris, 0.001M EDTA, pH 7.5.
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