Biology Reference
In-Depth Information
Table 5.2: Sample Protocol for Precipitating DNA with Ethanol.
For recovery of DNA from a typical reaction (1
μ
g of DNA in 20
μ
l):
1. To 20
μ
l of aqueous DNA sample in a microcentrifuge tube, add 2
μ
l of 3 M sodium acetate (pH 5.5) and
40
μ
l of EtOH.
2. Mix well by vortexing and immerse the tube in a
−
70°C bath composed of methanol plus dry ice for
15 minutes. The mixture should freeze or form a slurry.
3. Centrifuge the DNA precipitate in a bench top microcentrifuge at maximum speed for 10 minutes in a
cold room. A whitish pellet of DNA should appear at the bottom of the tube. In general, pellets of 10
μ
g
are visible, whereas pellets of 2
μ
g are invisible.
4. Remove the EtOH supernatant by using a micropipet, taking care not to disturb the pellet or the area of
the tube where the pellet should be located.
5. Add 100
μ
l 70% EtOH (chilled to
−
20°C) to the sample and vortex. This step removes any solute trapped
in the precipitate.
6. Reprecipitate the DNA by centrifugation for 2 minutes and remove the supernatant as described in step 4.
7. Dry the pelleted DNA for 1-2 minutes in a vacuum desiccator, taking care to release the vacuum gently so
as not to dislodge the dried sample.
8. Resuspend the DNA in TE buffer (pH 8) (TE buffer contains 10 mM Tris-HCl at pH 8.0; 1 mM Na
2
EDTA)
or in sterile water.
(Modified from Berger and Kimmel 1987.)
by ethanol (EtOH) precipitation, isopropanol precipitation, or several other
methods. The most versatile is probably EtOH precipitation, because it can con-
centrate both DNA and RNA and purify DNA after phenol extractions.
DNA can be precipitated by combining the DNA sample with a salt and EtOH
at
−
20°C or lower (
Table 5.2
). The precipitated salt of the nucleic acid is then
pelleted by centrifugation, the EtOH supernatant is removed, and the nucleic-
acid pellet is resuspended in a buffer. Which salt or which buffer is used is
determined by the nature of the sample and by the planned use for the nucleic
acid. Once the DNA is purified, it can be stored at 4°C in TE buffer (pH 8.0) or
sterile water. EDTA in the TE buffer helps to prevent degradation of the DNA
during storage because it chelates heavy metal ions that are commonly required
for DNase activity. For very long-term storage (
≥
5 years), the DNA can be frozen
at
−
80°C but should not be subjected to freeze-thaw cycles or it will be dam-
aged (sheared).
The now-purified DNA can be cut, either by shearing or, preferably, with a
restriction endonuclease. Shearing produces random fragments, but restriction
endonucleases can generate fragments of a desired size and with termini, or
ends, appropriate to the annealing and ligation steps of the experiment out-
lined in
Figure 5.1
.
