DNA precipitation using salt and ethanol is a commonly used protocol in the lab for concentrating nucleic acids such as DNA and RNA. The basic method is salt and ethanol are added to the aqueous solution, which forces the nucleic acid to precipitate out of solution. Ccentrifugation of the precipitated nucleic acids and washing isolates the nucleic acid from the rest of the solution. The nucleic acid containing pellet is then washed with cold 70% ethanol to remove the salt.
Then a second centrifugation step is used to isolate the nucleic acid away from the ethanol allowing the ethanol to be removed. The nucleic acid pellet is then allowed to dry, and is then resuspended in fresh aqueous T (Tris) buffer or water.
How Does DNA Precipitation Work?
One must first understand why nucleic acids are soluble in water.
Water (H2O) is a polar molecule that has a partial negative charge near the oxygen atom due the unshared pairs of electrons, and partial positive charges near the hydrogen atoms.
Due to this polar nature of water, other polar molecules such as DNA or RNA can interact electrostatically with the water molecules, allowing them to easily dissolve in water.
Precipitation breaks this interaction and creates new ones, allowing the DNA or RNA to come out of solution and be isolated easily.
Salt in DNA Precipitation
Salt's role in the protocol is to neutralize the charges on the sugar phosphate backbone of the nucleic acid whether DNA or RNA. Commonly used salts in DNA and RNA precipitation include sodium acetate.
In aqeous solution, sodium acetate breaks up into Na+ and [CH3COO]-. The positively charged sodium ions neutralize negatively charged backbone on the DNA, specifically the PO3- groups on the nucleic acids, making the nucleic acid molecule far less hydrophilic, and therefore much less soluble in water.
Ethanol in DNA Precipitation
Electrostatic attractions between the Na+ ions in solution and the PO3- ions are dictated by Coulomb's Law, which is affected by the dielectric constant of the solution. Water has a high dielectric constant, which makes it fairly difficult for the Na+ and PO3- to come together. Ethanol on the other hand has a much lower dielectric constant, making it much easier for Na+ to interact with the PO3-, shield it’s charge and make the nucleic acid less hydrophilic, causing it to drop out of solution.
Temperature in Precipitation
Incubation of the nucleic acid/salt/ethanol mixture at low temperatures (e.g. -20 or -80C) is commonly cited in protocols as necessary in protocols. Nucleic acids however at concentrations as low as 20ng/mL will precipitate at 0-4C so incubation for 15-30 minutes on ice is sufficient.
DNA Precipitation Protocol
The goal of this protocol is to precipitate DNA, as the name says. This is usually coupled with phenol chloroform extraction and is used as a way of purifying nucleic acids. This can also be used as a method for changing what solution or buffer your nucleic acid is in.
This protocol also works for RNA precipitation (take care to us RNAse free materials in this case).
- 3M NaOAc pH 5.2
- EtOH 95%
- Glycogen (optional)
- 1/10 volume NaOAc.
- 1ul Glycogen.
- 2 volumes EtOH.
- -20°C overnight or 30min -80°C.
- Centrifuge >12k G for at least 15 mins.
- Remove supernatant
- Resuspend in desired volume of water/buffer
-20°C for an hour is fine for using larger (1mL of bacterial culture, plasmid) amounts of DNA
The DNA pellet will not always be visible depending on how much DNA you are precipitating. So always take care in loading your samples in the centrifuge to remember the direction they are facing. The DNA pellet will be on the part of the tube facing the outside of the centrifuge.
This protocol will precipitate all nucleic acids, not just DNA. If you do not want RNA in your sample, one of the many ways to deal with it is to simply resuspend in TE + RNAse at the last step and leave it at room temperature for 15mins-1hr.
If salt does precipitate, it can easily be removed by washing with 70% EtOH. The DNA will not dissolve at room temperature,
but the salt will. (This can also be used to remove CsCl. I find a few washes with 70% EtOH is easier than dialysis when I have to remove CsCl from a plasmid prep. NOTE: do NOT chill when removing CsCl, which has a high temperature coefficient of solution.)
DNA Precipitation Tips
- Choice of salt
- Use Sodium acetate (0.3M final conc, pH 5.2) for routine DNA precipitations
- Use Sodium chloride (0,2M final conc) for DNA samples containing SDS since NaCl keeps SDS soluble in 70% ethanol so it won’t precipitate with the DNA.
- Use Lithium Chloride (0.8M final conc) for RNA. This is because 2.5-3 volumes of ethanol should be used for RNA precipitation and LiCl is more soluble in ethanol than NaAc so will not precipitate, but beware - chloride ions will inhibit protein synthesis and DNA polymerase so LiCl is no good for RNA preps for in vitro translation or reverse transcription. In these cases, use NaAc.
- Use Ammonium acetate (2M final conc) for the removal of dNTPs, but do not use for preparation of DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the enzyme.
- To increase the yield in precipitations of low concentration or small nucleic acid pieces (less than 100 nucleotides)
- Add MgCl2 to a final concentration of 0.01M
- Increase the time of incubation ice before centrifugation to 1 hour.