Improved Alcohol Precipitation of DNA
This method was suggested in a BRL "Focus" article several years ago (Zeugin & Hartley, 1985). It is a useful way to avoid coprecipitation of proteins and accumulation of salt in the final desiccated preparation.
0. For small amounts of DNA, add 1 µL glycogen, "Molecular Biology Grade", 20 µg/µL, as a coprecipitant; this amount will always produce a small but visible pellet.
1. Add the following amount of 7.5 M NH4OAc, a volatile salt:
2. Add 2.5 new volumes 95% ethanol.
- Crude DNA: 0.5 volumes
- Pure DNA: 0.1 volumes
3. After >10' on ice, spin 10' full-speed in a microcentrifuge at RT.
4. Aspirate supernatant, wash pellet once with an excess of 70% ethanol.
5. A final desiccation under a vacuum or in a SpeedVac will remove all traces of salt.
We have used undiluted DNA prepared in this fashion for electroporation without any arcing.
A subsequent BRL article (-, 1982) pointed out that precipitation in the cold is unnecessary. The process is an irreversible one, and takes place at room temperature, albeit somewhat more slowly. The same article showed that incubation at -70° can actually inhibit complete precipitation.
-, "DNA Precipitation in the Presence of Ammonium Acetate", Focus 4(3), 12, 1982.
J.A. Zeugin & J.L. Hartley, "Ethanol Precipitation of DNA", Focus 7(4), 1-2, 1985.
This page has been viewed times.
eckofoid at ucdavis.edu