DNA Precipitation

Ben Carter, 9/24/2014

''Mechanism: Nucleic acids are negatively charged. In order to precipitate them, ethanol is added to reduce the polarity of the solvent and therefore reduce the solubility of the nucleic acids. Sodium acetate is added to provide positively charged ions that will bind to the nucleic acid and cause it to precipitate out of the less-polar solution.''

Note: You can expect a DNA yield of 50-80% of the theoretical yield.

Precipitation

 * 1) Add 1/10 volume of 3M sodium acetate pH 5.2 to the samples. Optional: Add nuclease-free glycogen to the solution to a concentration of 0.1mg/mL. This makes the resulting pellet more visible. Always add glycogen if your sample contains less than 1µg of DNA.
 * 2) Add 2.5 volumes of ethanol. Invert five times to mix.
 * 3) Incubate samples on ice for 15min. For fragments less than 200bp or for samples less than about 20ng/µL, incubate overnight. Incubation below 0°C does not significantly improve efficiency.

Washing and Collection

 * 1) Centrifuge samples at max speed for 20min in a room temperature microcentrifuge.
 * 2) Carefully pipette away supernatant. The pellet may not be visible if you did not add glycogen.
 * 3) Carefully add 500µL of 70% ethanol to the samples. Invert five times to mix.
 * 4) Repeat centrifugation at max speed for 10min.
 * 5) Carefully pipette away supernatant. Perform a 10s centrifugation to collect residual solution at the bottom of the tube. Use a P200 to carefully pipette away any residual supernatant. Residual ethanol will poison reactions such as PCR.
 * 6) Incubate the open sample tubes at room temperature for 10min in a laminar flow hood to allow residual ethanol to evaporate. Some pellets can over-dry: do not exceed 15min.
 * 7) Suspend the pellet in Milli-Q water or TE Buffer. Vortex briefly to mix. Ensure that the buffer comes into contact with the tube’s entire interior surface as much of the nucleic acid may be deposited on the walls instead of in the pellet.

TE Buffer

 * 5mL of 1M Tris pH 8.0
 * 1mL of 0.5M EDTA pH 8.0
 * Dilute to 500mL using Milli-Q water
 * Filter sterilize