User:Neil J. Parikh/Ethanol Precipitation Explained

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Ethanol Precipitation

Ethanol Precipitation (for > 200bp)

®     Ethanol Precipitation is used to concentrate DNA or RNA from dilute, aqueous solution. Such is needed for nearly all protocols involving purification of nucleic acids from cells.

®     Salt and ethanol are added to the aqueous solution, which forces the nucleic acid to precipitate out of solution. This precipitated nucleic acid (DNA) can now be separated from the solution by centrifuging it.

®     Use: 3 volumes of COLD 100% Ethanol (EtOH), 1/10 volume 3M Sodium Acetate, 1µl GlycoBlue

Procedure:

 

General Procedure

For 50µl

For 100µl

1.

Add 3 volumes COLD 100% EtOH

Add 150µl EtOH

Add 300µl EtOH

2.

Add 1/10 volume sodium acetate (NaAc)

Add 20 µl NaAc

Add 40µl NaAc

3.

Add 1µl GlycoBlue

4.

Incubate for 1hour at -80°C

5.

Centrifuge for 30 minutes at 0°C

6.

Remove the supernatant carefully, ensuring not to remove any solid

7.

Air Dry to allow EtOH to evaporate

8.

Resuspend the DNA in the desired buffer (usually H2O)

How Does it Work:

®     DNA is a polar molecule and is hydrophilic because of the negatively charged phosphate (PO3-) groups along the sugar phosphate backbone

®     Water is polar and hydrophilic as well and as such DNA dissolves easily in H2O

®     The salt, Sodium Acetate in this case, is used to neutralize the charges on the sugar phosphate backbone. In the solution, Sodium Acetate breaks up into Na+ and [CH3COO]- and so the positive sodium atoms neutralize the negative charges on the phosphate groups. The DNA becomes more hydrophobic now, and therefore less soluble in H2O.

®     Ethanol, which is added first, is used to change the dielectric constant of the solution. In H2O, which has a high dielectric constant, it is much more difficult for the Na+ and the PO3- to come together. EtOH on the other hand, has a lower dielectric constant, and therefore the sodium can neutralize the phosphate’s charges much easier, thereby making the molecule less hydrophilic and more insoluble.

®     Incubation at -80°C is used to allow the nucleic acids to precipitate

®     Centrifuging the solution will allow the DNA to form a clump at the bottom, and allows for the supernatant to be removed, leaving the DNA behind

®     Air drying allows the EtOH to evaporate from the precipitate, thus leaving behind only DNA

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