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complete disruption and lysis of cells walls and plasma membranes of cells
and organelles is an absolute requirement for all genomic DNA isolation
procedures. Incomplete disruption results in significantly reduced yields.
Disruption generally involves use of a lysis buffer that contains a detergent
for breaking down cellular membrane. Buffers containing SDS and EDTA are
recommended for DNA isolation. EDTA chelates divalent metal ions to
inhibit DNases and to destabilize cell membrane for lysis.
Ethanol has a lower dielectric constant than water (it is less electronegative
by a fair margin), so when mixed with a pure water/ DNA sample, it
effectively lowers the dielectric constant of the solution. When monovalent
cations are also added to the solution, the hydration shell that is keeping
DNA dissolved (water molecules bonded to and surrounding the dissolved
DNA) is replaced by ionic (salt bonds) and the DNA molecule is knocked out
of solution (precipitates).
Ethanol precipitation
DNA Precipitation
Theory
DNA is polar due to its highly charged phosphate backbone. This polarity,
based on the principle of "like dissolves like", makes it soluble in water,
which is also highly polar. The high polarity of water, reflected by high value
of its dielectric constant 80.1 (at 20 °C), means that electrical force
between any two charges in aqueous solutions is highly diminished
compared to force in vacuum or air.
This relation is reflected in Coulomb's law, which can be used to calculate
force acting on two charges q1 and q2 separated by a distance r, the
dielectric constant (also called relative static permittivity) of the medium
is present in the denominator of the equation ( is an electric constant):
Ethanol is much less polar than water, its dielectric constant is 24.3 (at 25
°C). This means that adding ethanol to solution disrupts screening of
charges by water. If enough ethanol is added electrical attraction between
phosphate groups and any positive ions present in solution becomes strong
enough to form stable ionic bonds and precipitate DNA. This usually
happens when ethanol makes around 64% of the solution. As the
mechanism suggests solution has to contain positive ions for precipitation
to occur, usually Na+, NH4+ or Li+ play this role [1].
Practice
During incubation DNA and some salts will precipitate from solution, in the
next step this precipitate is collected by centrifugation in a microcentrifuge
tube at high speeds (~12,000g). Time and speed of centrifugation has the
biggest effect on DNA recovery rates. Again smaller fragments and higher
dilutions require longer and faster centrifugation. Centrifugation can be
done either at room temperature or in 4 °C or 0 °C. During centrifugation
precipitated DNA has to move through ethanol solution to the bottom of
the tube, lower temperatures increase viscosity of the solution and larger
volumes make the distance longer, so both those factors lower efficiency of
this process requiring longer centrifugation for the same effect. [2][3] After
centrifugation the supernatant solution is removed, leaving a pellet of
crude DNA. Whether the pellet is visible depends on the amount of DNA
and on its purity (dirtier pellets are easier to see) or the use of co-
precipitants.
In the next step, 70% ethanol is added to the pellet, and it is gently mixed
to break the pellet loose and wash it. This removes some of the salts
present in the leftover supernatant and bound to DNA pellet making the
final DNA cleaner. This suspension is centrifuged again to once again pellet
DNA and the supernatant solution is removed. This step is repeated once.
Finally, the pellet is air-dried and the DNA is resuspended in water or other
desired buffer. It is important not to over-dry the pellet as it may lead to
denaturation of DNA and make it harder to resuspend.
Protocol
DNA Precipitation
The Basics: How Ethanol Precipitation of DNA and RNA Works
First we need to know why nucleic acids are soluble in water. Water is a
polar molecule – it has a partial negative charge near the oxygen atom due
the unshared pairs of electrons, and partial positive charges near the
hydrogen atoms (see the diagram on the right).
Because of these charges, polar molecules, like DNA or RNA, can interact
electrostatically with the water molecules, allowing them to easily dissolve
in water. Polar molecules can therefore be described as hydrophilic and
non-polar molecules, which can’t easily interact with water molecules, are
hydrophobic. Nucleic acids are hydrophilic due to the negatively charged
phosphate (PO3-) groups along the sugar phosphate backbone.
Ok, so back to the protocol. The role of the salt in the protocol is to
neutralize the charges on the sugar phosphate backbone. A commonly used
salt is sodium acetate. In solution, sodium acetate breaks up into Na+ and
[CH3COO]-. The positively charged sodium ions neutralize the negative
charge on the PO3- groups on the nucleic acids, making the molecule far
less hydrophilic, and therefore much less soluble in water.
The electrostatic attraction 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.
This step is to wash any residual salt away from the pelleted DNA.
Choice of salt
o Use Sodium acetate (0.3M final conc, pH 5.2) for routine DNA
precipitations
o 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.
o 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.
o 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.