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Ethanol Precipitate DNA


To ethanol precipitate nucleic aci ds, add salt, mix, add ethanol, mix, then freeze. Centrifuge to pellet the material.

Choice of salts:
        Sodium acetate - estimate aqueous volume of DNA
                Add 1/10 of this volume from a 3M sodium acetate stock

        Ammonium acetate - estimate aqueous volume of DNA
                Add 1/2 of this volume from a 7.5 M ammonium acetate stock.
                Ammonium ions help keep free nucleotides in solution.

        Use absolute ethanol (i.e. 200 proof = 100%) only.
      &nbs p; Add 2.5 - 3 times the volume of the DNA/salt mixture. Mix well before freezing.

Freezing Conditions:
        Freeze in a dry ice /ethanol bath for 20 minutes, a -70°C freezer for a minimum of 40 minutes, or a -20°C freezer overnight. The latter must be used for precipitating DNA in concentrated CsCl solutions since the CsCl salt will fall out of solution at lower temperatures.

3M Sodium Acetate, pH 5.5
18.46 g sodium acetate
Adjust pH with glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.
7.5 M Ammonium Acetate (mw=77.08)
57.81 g ammonium acetate per 100 ml solution
Dissolve 57.81 g ammonium acetate in about 45ml autoclaved water in a sterile container. QS to 100 ml, filter sterilize with Nalgene 0.45 or 0.2 micron filter. DO NOT HEAT or AUTOCLA VE!

    Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF

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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D. on information above this statement.
Proper citation for the above document is: "Frank, M. B. Ethanol Precipitation of Nucleic Acids. In: Frank, M. B. ed. Molecular Biology Protocols. ( 1997. Oklahoma City. Revision Date: October 2, 1997."
Notes that appear below were taken from a discussion on this topic from one of the bionet newsgroups.

Why it works...

A discussion on this topic was posted to the "bionet.molbio.methds-reagnts" bulletin board in July 1992. A thorough discussion was presented by Scott Keeney, of the Biochemistry and Molecular Biology Department, University of California Berkeley ( I have shortened the opening questions and extracted his answer from the newsgroup and present them below for your convenience.

Q: What is the role of ethanol in precipitating DNA from aqueous solutions?
A: The purpose of adding salts is to neutralize the charge on the sugar-phosphate backbone of the DNA, but ethanol's task is a little more complex than "removing" the water. For a precipitation, you're interested in forming ion pairs between the polyanion (DNA) and the cation (Na+, Mg++, spermidine, protamine, etc). In dilute aqueous solution, DNA and counterions like Na+ and Mg++ are more or less in the free ion form rather than the ion pair form (that is, they are surrounded by one or more layers of water molecules).

Water has a high dielectric constant (e), which from Coulomb's Law tells us that the electrostatic force (F) between two ions of opposite charge is very low in water:

F = (Q1 * Q2)/(e * r2)

where Q is the charge on each ion and r is the distance between them. Adding organic solvent *decreases* the dielectric constant o f the solution. As e goes down, F goes up and *BANG*, anion and cation form an ion pair and promptly swoon out of solution.

Q: Why does NaCl increase the stability of DNA duplexes, although you might expect salts to interfere with hydrogen bonds, rather than strengthen them?
A: The Na+ neutralizes the charge. Each strand of DNA has an enormous charge density (charge per unit volume), so the two strands tend to push each other apart. Cations added to the solution for m a "cloud" of positive charges around the DNA. This cloud of counterions lowers the effective charge density and relieves the repulsion between the strands.

As for the effect of salt on hydrogen bonds, you have to realize that the hydrogen bonds formed between bases in duplex DNA contribute little to the stability of the duplex. For an interaction to stabilize the duplex, the interaction between bases must be stronger than the interaction of the bases with water (if bases are not paired wi th one another in a duplex, then they are surrounded by water). Hydrogen bonding between the amines, carbonyl oxygens, etc. of G-C or A-T is of the same energy (sometimes even less) than the hydrogen bonds these same groups would form with water if the DNA were single-stranded. (The H-bonds do contribute *something*: GC base pairs with three H-bonds are harder to melt than AT pairs with two.)

So, what drives DNA strands together? Entropy and enthalpy, of course. Entropy in the form of "hydrophob ic" interactions between the bases (those big aromatic rings are quite hydrophobic, you know). Enthalpy in the form of favorable, stabilizing interactions between the pi electrons of the aromatic rings of bases as they stack on top of one another.

So, what do hydrogen bonds do, if they don't stabilize the duplex? They enforce the *specificity* of base pairing. Correct base pairing is nice, but doesn't add much. Incorrect base pairing, on the other hand, takes a lot away. Forcing unpaired H-bond donors and acceptors (i.e. hydrophilic groups) into a hydrophobic environment makes everybody unhappy.

    Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF

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