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now: Thu Jun 24 07:30:59 2021 ... mod: Sun Apr 4 18:35:39 2021
Ethanol Precipitation of Nucleic Acids
DNA and RNA can be efficiently precipitated from aqueous solution by the addition of ethanol in the presence of salt.
- measure the volume of aqueous nucleic acid solution to be precipitated
- add 1/10th volume of 8M LiCl
- use care when making the 8M LiCl stock. LiCl gives off a lot of heat when it dissolves and can clump up. add LiCl powder gradually to stirring water
- add 2.5 volumes of room temperature 100% ethanol and mix thourougly
- based on the aqueous volume before the addition of LiCl
- 2.5 volumes of ethanol addition is a good all-purpose amount. 2.0 volumes can be used if there is a lot of nucleic acid to precipitate and/or the solution is already quite salty (e.g. after addition of solution III from a PlasmidMiniprep), or 3.0 volumes can be helpful sometimes for very small amounts of nucleic acid
- for precipitating RNA efficiently, 3.0 volumes of ethanol is generally recommended
- incubate the ethanol / nucleic acid mixture
- for larger amounts of nucleic acids incubation at room temperature for 3 - 5 minutes is generally sufficient or
- for small amounts of nucleic acids (less than 0.5 μg) incubate at -20C for 10 minutes
- it is helpful to give tubes a brief and gentle vortex after this incubation step; this helps the nucleic acid pellet to the bottom of the tube rather than smearing up the side as it can do sometimes
- centrifuge at 12000g (minimum)
- the time of centrifugation is more important for recovery than the time of incubation with ethanol
- 3 minutes for large amounts of nucleic acid (more than 1μg) or
- 10 minutes for small amounts of nucleic acid (less than 1μg)
- decant the supernatant
- add 500 μl room temperature 75% ethanol. rinse around the sides of the tube by gentle inversion
- the 75% ethanol rinse is not particularly effective at dissolving any salt trapped in the DNA pellet; the purpose of the rinse is to wash salt from the sides of the tube
- respin at 12000g for 2 minutes
- this step is optional for large amounts of DNA
- decant the 75% ethanol
- spin down briefly
- pipette aspirate the remaining 75% ethanol, allow the pellet to air dry and resuspend in an appropriate volume of water, TE, etc.
- never use an evaporator such as a SpeedVac to dry the pellet
- do NOT store ethanol solutions in the freezer or refrigerator. it is not necessary for the ethanol to be cold for efficient precipitation. ethanol solutions that are stored cold are subject to progressive dilution by condensation of water from the air every time the solution is accessed
- other salts can be used instead of LiCl such as ammonium acetate, sodium acetate and sodium chloride (each used in different amounts), however LiCl has the advantage of being essentially infinitely soluble in ethanol so that there is no danger of the salt precipitating with the nucleic acids.
- nucleic acids can also be precipitated with 0.7 volumes of isopropanol instead of 2.5 volumes of ethanol. this is useful when precipitating relatively large aqueous volumes. there is more danger of salt precipitation with isopropanol however so never chill an isopropanol precipitation reaction.
- nucleic acids in amounts of 1 μg or larger will show a visible pellet after precipitation. smaller amounts of nucleic acid will still precipitate in essentially quantitative amounts even if the pellet is not visible. if desired, 1 μg of either tRNA or glycogen can be added as a carrier so that a visible pellet will form. tRNA has the advantage of being removable by RNAse digestion. glycogen has the advantage of not absorbing at 260nm. store tRNA in frozen 1 μg/ml solution, store 1 μg/ml glycogen solution at 4C