Precipitation
Monovalent salt neutralises the phosphate backbone and alcohol removes the water that solvates it, so the nucleic acid aggregates and can be pelleted. Ethanol at 2–2.5 volumes is the default; isopropanol at 0.6–0.8 volumes suits large or dilute samples but brings more salt down with the pellet.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Ethanol / isopropanol
Monovalent salt neutralises the phosphate backbone and alcohol removes the water that solvates it, so the nucleic acid aggregates and can be pelleted. Ethanol at 2–2.5 volumes is the default; isopropanol at 0.6–0.8 volumes suits large or dilute samples but brings more salt down with the pellet.
How it differs from the standard method
- Sodium acetate at pH 5.2 to 0.3 M is the usual salt; ammonium acetate is preferred before a ligation because it leaves less inhibitory residue.
- A sample below a few ng/µL needs a carrier or most of it stays in the supernatant whatever you do.
- The 70% ethanol wash removes co-precipitated salt; absolute ethanol does not, because salt only dissolves in the water part.
- Dry until the alcohol has gone and no further: an over-dried pellet, particularly of high-molecular-weight DNA, may never fully redissolve.
1Making something leave solution on purpose
Precipitation concentrates a sample by making the thing you want stop being soluble, so it can be spun into a pellet and redissolved in whatever volume and buffer you choose next. It is the oldest trick in the preparative repertoire and it is still used daily, because it concentrates and changes buffer in one step, needs no special equipment, and scales from a microlitre to a litre.
2Nucleic acids: salt first, then alcohol
DNA and RNA are soluble because the negatively charged phosphate backbone is surrounded by water. Adding monovalent salt supplies cations that sit against the backbone and neutralise its charge; adding alcohol lowers the dielectric constant of the solvent so water no longer solvates it well.
2–2.5 volumes
The default. More volume to handle, but salt stays in solution better, so the pellet is cleaner.
0.6–0.8 volumes
For large volumes or dilute samples. Co-precipitates more salt, so the 70% wash matters more.
Below a few ng/µL
Glycogen gives the precipitate something to nucleate on. Carrier tRNA works but is itself nucleic acid, so it ruins A260 quantification.
3The wash is where the method succeeds or fails
Salt co-precipitates with the nucleic acid and sits in the pellet, and salt inhibits most of the enzymes you are about to use. The 70% ethanol rinse works because salt is only soluble in the water part of the wash: absolute ethanol rinses the outside of the pellet and leaves the salt behind.
4Proteins: denature, or compete for water
Trichloroacetic acid and acetone precipitate protein by denaturing it and stripping its hydration shell, which is thorough: recovery is high and detergents and salts are left behind, which is why it is the standard clean-up before a gel or mass spectrometry. The cost is that the pellet is denatured and often stubborn to redissolve, so it is useless when you need activity.
Key terms
Practise these as flashcards →An inert substance, usually glycogen, added to a dilute sample so the precipitate has something to nucleate on and form a visible pellet.
Salt or other solutes coming out of solution along with the target and ending up in the pellet.
Precipitating protein by adding a high concentration of a kosmotropic salt, which competes for the water that keeps the protein soluble. It does not denature.
A pair of ammonium sulfate concentrations defining a fraction: what stays soluble below the first and precipitates by the second.
Ammonium sulfate concentration expressed as a percentage of saturation at that temperature, which is how recipes and nomograms are written.
Drying a nucleic acid pellet past the point where the alcohol has gone, which makes it hard or impossible to redissolve.
How well a solvent screens charge. Alcohol lowers it, which is why water stops solvating the charged phosphate backbone.
When to use it
- Concentrating a dilute nucleic acid sample, or moving it into a smaller volume or a different buffer.
- Cleaning protein away from detergent, salt or chaotrope before a gel or mass spectrometry.
- Taking a first, cheap fractionation step on a crude protein extract before any chromatography.
- Removing an enzyme and its buffer between steps when a column would lose too much material.
Compared with alternatives
The gentler route when the protein must stay folded and the volume does not need to fall.
What actually collects the precipitate; the spin conditions decide how much of it you keep.
Column and bead methods that replace precipitation for routine nucleic acid clean-up.
Where a protein prep usually goes next, often after an ammonium sulfate cut has removed the bulk.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Monovalent salt neutralises the phosphate backbone and alcohol removes the water that solvates it, so the nucleic acid aggregates and can be pelleted. Ethanol at 2–2.5 volumes is the default; isopropanol at 0.6–0.8 volumes suits large or dilute samples but brings more salt down with the pellet.
- Sodium acetate at pH 5.2 to 0.3 M is the usual salt; ammonium acetate is preferred before a ligation because it leaves less inhibitory residue.
- A sample below a few ng/µL needs a carrier or most of it stays in the supernatant whatever you do.
- The 70% ethanol wash removes co-precipitated salt; absolute ethanol does not, because salt only dissolves in the water part.
- Dry until the alcohol has gone and no further: an over-dried pellet, particularly of high-molecular-weight DNA, may never fully redissolve.