Alcohol Precipitations

of Nucleic Acids
Unraveling DNA, page 300
Adding 2 volumes of cold
EtOH
Isopropanol can also be used, but it results in
more protein contamination
When using isopropanol, you use less of it
This reduces overall product
This is an advantage if your tubes have limited
capacity
Factors affecting nucleic acid
precipitations
Cations for charge shielding
Temperature during the precipitation step
Concentration of the nucleic acids
Time and speed of centrifugation
Charge shielding
There must be at least 0.2 M of a monovalent
cation present to shield the high negative charge
of the phosphate in nucleic acids

Without shielding, intrastrand repulsion is too
great, and there will be no aggregation.

Salts that are used
Sodium acetate is most common for routine DNA
isolations

Sodium chloride (from our TES buffer!!) may be
used if the sample contains/will contain SDS
This allows the detergent to remain soluble in an
ethanol solution
Temperature During
Precipitation
It was commonly thought that precipitation needed
to be at lower temperatures (-20 or -70 C)
The recovery is actually better at 0 C (on ice) or at
room temperature
Concentration of nucleic
acids
Precipitations occur effectively within 10 minutes
when the nucleic acid concentration is greater
than 0.5 ug/mL.

If the concentration is less than 0.5 ug/mL,
incubation time must be increased (from 30
minutes to overnight)
Centrifugation
In a microcentrifuge, nucleic acids are usually
collected at 12,000-14,000 x g. between 4 C and
room temperature
Ten minutes is long enough if the concentration is
high (>0.5 ug/mL)
If the concentration is low, centrifuge for 30-60
minutes OR increase the g-force (using adaptors
in an ultracentrifuge)