DNA isolation & EDTA? 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.

Why use ethanol in DNA isolation

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). This is how ethanol (working with cations) works to precipitate DNA.

Function of isopropanol and ethanol in plasmid isolation? Cold ethanol or isopropanol is used to precipitate the plasmid DNA, DNA is insoluble in alcohol and clumps or clings together. Centrifuging will cause the precipitate to form a pellet which can be decanted from the unwanted supernatant.

Where as if compared with RNA isolation isopropanol is less efficient in precipitating RNA, where in presence of Lithium chloride or ammonium ions can give a good yield

Ethanol precipitation

Ethanol precipitation is a method used to purify and/or concentrate RNA, DNA and polysaccharides such as pectin and xyloglucan from aqueous solutions.

DNA Precipitation

Theory

The first hydration shell of a sodium ion dissolved in water

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):

At an atomic level this diminishing of force acting on charges results from water molecules forming hydration shells around them. It makes water a very good solvent for charged compounds like salts. Electric force which normally holds salt crystals together by way of ionic bonds is weakened in the presence of water allowing ions to separate from the crystal and spread through solution.

The same mechanism operates in the case of negatively charged phosphate groups on DNA backbone, even if positive ions are present in solution, the relatively weak electric force prevents them from forming stable ionic bonds with phosphates and precipitating out of solution.

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

DNA is precipitated by first ensuring that the correct concentration of positive ions is present in solution (too much will result in a lot of salt co-precipitating with DNA, too little will result in incomplete DNA recovery) and then adding two to three volumes of at least 95% ethanol. Many protocols advise storing DNA at low temperature at this point but this has been shown to lower precipitation efficiency [2][3]. The best efficiency is achieved at room temperature but when possible degradation is taken into account it is probably best to incubate DNA on wet ice. Optimal incubation time depends on the length and concentration of DNA. Smaller fragments and lower concentrations will require longer times to achieve the same recovery. For very small lengths and low concentrations over-night incubation is recommended. In such cases use of carriers like tRNA, glycogen or linear polyacrylamide can greatly improve recovery.

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.

Isopropanol can also be used instead of ethanol; the precipitation efficiency of the isopropanol is higher making one volume enough for precipitation. However, isopropanol is less volatile than ethanol and needs more time to air-dry in the final step. The pellet might also adhere less tightly to the tube when using isopropanol[1].

Protocol

1. Add 1/10 volume of Sodium Acetate (3 M, pH 5.2). 2. Add 2.5-3.0 X volume (calculated after addition of sodium acetate) of

at least 95% ethanol. 3. Incubate on ice for 15 minutes. In case of small DNA fragments or

high dilutions overnight incubation gives best results, incubation below 0 °C does not significantly improve efficiency [2][3].

4. Centrifuge at > 14,000 x g for 30 minutes at room temperature or 4 °C.

5. Discard supernatant being careful not to throw out DNA pellet which may or may not be visible.

6. Rinse with 70% Ethanol 7. Centrifuge again for 15 minutes.

8. Discard supernatant and dissolve pellet in desired buffer. Make sure the buffer comes into contact with the whole surface of the tube since a significant portion of DNA may be deposited on the walls instead of in the pellet.[1]

DNA Precipitation

Phenol (removes protein)

1. add equal volume of Phenol (= tris-saturated Phenol-Chloroform-Isoamyethanol)

2. vortex 3. spin 2 minutes at 12000 rpm 4°C 4. transfer supernatant to a fresh tube (avoid aspiration of the

interlayer or organic phase)

Chloroform (removes phenol)

1. add equal volume of Chloroform 2. vortex 3. spin 2 minutes at 12000 rpm 4°C 4. transfer supernatant to a fresh tube (avoid aspiration of the

interlayer or organic phase)

100% Ethanol (precipitates DNA)

1. add 0.1 volume 3 M sodium acetate 2. add 2.5 volumes 100 % Ethanol 3. vortex 4. precipitate at:

o -20°C overnight (+++) o -80°C 1 h (++) o dry ice 15min (+)

5. spin 20 minutes at 12000 rpm 4°C 6. carefully pour out / aspirate supernatant (do not lose DNA-pellet)

70% Ethanol (washes out salt)

1. carefully add 1 mL cold 70% Ethanol (do not vortex)

2. spin 10 minutes at 12000 rpm 4°C 3. carefully pour out / aspirate supernatant (do not lose DNA-pellet) 4. air dry 10 minutes at room temperature (do not overdry, because

DNA becomes hard to dissolve) 5. dissolve in:

o 10 mM Tris pH 7.5 (+++) o TE-Buffer (++) - EDTA may inhibit downstream enzymatic

reactions o dH2O (+) - freeze at -20°C because unbuffered DNA undergoes

degradation

The Basics: How Ethanol Precipitation of DNA and RNA Works

Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acid (DNA or RNA) preparations in aqueous solution. The basic procedure is that salt and ethanol are added to the aqueous solution, which forces the nucleic acid to precipitate out of solution. The precipitated nucleic acid can then be separated from the rest of the solution by centrifugation. The pellet is washed in cold 70% ethanol then after a further centrifugation step the ethanol is removed, and the nucleic acid pellet is allowed to dry before being resuspended in clean aqueous buffer. So how does this work?

A bit about solubility…

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.

The role of the salt…

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 role of the ethanol…

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.

The role of temperature…

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. However, according to Maniatis et al (Molecular Cloning, A Laboratory Manual 2nd Edition… 2nd edition?? – I need to get a newer version!), this is not required, as nucleic acids at concentrations as low as 20ng/mL will precipitate at 0-4C so incubation for 15-30 minutes on ice is sufficient.

The wash step with 70% ethanol…

This step is to wash any residual salt away from the pelleted DNA.

A few tips on nucleic acid precipitation…

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.

To increase the yield in precipitations of low concentration or small nucleic acid pieces (less than 100 nucleotides)

o Add MgCl2 to a final concentration of 0.01M o Increase the time of incubation ice before centrifugation to 1

hour.