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Protocol

Precipitation of DNA with Isopropanol

Michael R. Green and Joseph Sambrook

DNA is less soluble in solutions containing isopropanol than in solutions containing ethanol. In
contrast to precipitation with ethanol, which requires 2–3 volumes of alcohol, precipitation with
isopropanol is performed with 0.6–0.7 volume of alcohol. Isopropanol is often the better choice
when precipitating DNA from large volumes of solution. Precipitation with isopropanol, described
here, is performed at room temperature to lessen the risk that solutes like sucrose or sodium chloride
will be coprecipitated with the DNA.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.

Reagents
DNA sample
Ethanol
Isopropanol
Sodium acetate (3 M, pH 5.2)
TE buffer, 10× <R> (pH 8.0)
Dilute stock solution before use.

Equipment
Vacuum aspirator equipped with traps

METHOD

1. Add sodium acetate (3.0 M, pH 5.2) to the DNA solution to a final concentration of 0.3 M.
2. Add 0.6–0.7 volume of isopropanol at room temperature, and mix well.
3. Mark the outside of the tube to allow the pellet to be located. Centrifuge the sample immediately
for 20–30 min in a microcentrifuge tube at 4˚C.
Centrifugation at 4˚C prevents the sample from overheating.

From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.
© 2017 Cold Spring Harbor Laboratory Press
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot093385

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M.R. Green and J. Sambrook

4. Carefully decant the supernatant fluid into a fresh labeled tube. As insurance against loss of the
DNA pellet during decanting, store the supernatant until the recovery of the precipitated DNA
is confirmed.
5. Wash the DNA pellet with ethanol to remove residual isopropanol.
Residual isopropanol hinders the redissolution of the DNA.

6. Centrifuge the sample for 20–30 min in a microcentrifuge tube at 4˚C.

7. Remove the supernatant carefully as aspiration, and allow the DNA pellet to dry.
Do not allow the DNA to dry completely; otherwise, the pellet will be very difficult to dissolve. The pellet
should still be damp when the buffer is added in Step 8.
8. Dissolve the pellet in an appropriate volume of TE (pH 8.0).

DISCUSSION

Isopropanol is often preferable to ethanol for precipitating DNA from large volumes of solution.
However, precipitation of DNA with isopropanol has some disadvantages.
• Salts are less soluble in solutions consisting of 35% isopropanol than in solutions containing
65% ethanol.
• The pellets of DNA are translucent and are difficult to see. Isopropanol is less volatile than ethanol
and is therefore more difficult to remove.
• Finally, DNA precipitated by isopropanol does not stick tightly to the wall of the microcentrifuge
tube and is easily lost when the isopropanol solution is removed or when the pellet is washed with
ethanol (Step 5).
In general, precipitation of DNA with ethanol is preferred, unless it is necessary to keep the volume
of fluid to a minimum. For ethanol precipitation, see Protocol: Precipitation of DNA with Ethanol
(Green and Sambrook 2016).

RECIPE

TE Buffer, 10×

100 mM Tris-Cl (desired pH)

10 mM EDTA (pH 8.0)
Sterilize solutions by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Store the buffer
at room temperature.

REFERENCES

Green MR, Sambrook J. 2016. Precipitation of DNA with ethanol. Cold

Spring Harb Protoc doi: 10.1101/pdb.prot093377.

674 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot093385

 Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on August 2, 2017 - Published by
Cold Spring Harbor Laboratory Press

Precipitation of DNA with Isopropanol

Michael R. Green and Joseph Sambrook

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot093385

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