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DNA prep protocols often include a final precipitation step with alcohol, often isopropanol, where the DNA must be kept in the alcohol, at a low temperature such as -20C or -70C, often overnight.

What is the point of keeping it at low temperature? Is lower better? At what point do you see diminishing returns?

What about the time? Is overnight really necessary, or would, say, an hour suffice? Do lower temperatures result in quicker precipitation?

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1 Answer 1

For the precipitation you use different reagents: First you add salt under slightly acidic conditions to make sure that the DNA precipitates in a less polar environment. This is achieved by adding alcoholes like ethanol or isopropanol.

It is commonly thought that a incubation at low temperatures enhances the results, but this is interestingly not true. There is a paper from the Bethesda Research Laboratories which is called "Ethanol Precipitation of DNA." which analyzes these relationships (temperature, time and alcohol concentration) in great detail, its definitely worth reading.

Figure 1-3 from this paper shows the influence for Ethanol (but I am pretty sure that this is not much different for isopropanol):

enter image description here

I would prefer ethanol precipitation to ispropanol whenever possible. Isopropanol co-precipates salts, so you have to wash a few times with 70% ethanol and it is less volatile than ethanol, so the drying step takes longer. Its also less effective cleaning organic compounds like phenol away and sometimes the pellets are harder to see.

On the other it can be useful when you have to deal with large volumes, since you only need 0,7 volumes of isopropanol compared with 2,5 volumes of ethanol.

Precipitations with Isopropanol are usually not incubated but centrifuged directly after mixing. Here is a protocol from my lab (which originally came from Qiagen, if I remember correct):

  • Adjust the salt concentration, for example, with sodium acetate (0.3 M, pH 5.2, final concentration) or ammonium acetate (2.0–2.5 M, final concentration).
  • Add 0.6–0.7 volumes of room-temperature isopropanol to the DNA solution and mix well.
  • Centrifuge the sample immediately at 10,000–15,000 x g for 15–30 min at 4°C
  • Carefully decant the supernatant without disturbing the pellet.
  • Wash the DNA pellet by adding 1–10 ml (depending on the size of the preparation) of room-temperature 70% ethanol. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, making the DNA easier to redissolve.
  • Centrifuge at 10,000–15,000 x g for 5–15 min at 4°C
  • Repeat the washing and centrifugation steps once more.
  • Carefully decant the supernatant without disturbing the pellet.
  • Air-dry the pellet for 5–20 min (depending on the size of the pellet).
  • Redissolve the DNA in a suitable buffer.
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It seems that overnight incubation is absolutely superfluous based on Fig.2 - or am I mistaken? –  Superbest Feb 21 at 20:56
Thats how I read it and how we do it. I have never had any problems with short incubation times. –  Chris Feb 21 at 21:00
Why can you conclude that overnight incubations are superfluous when they only examined conditions up to 10 minutes? The 5-minute difference between incubating for 5 or 10 minutes seems pretty minor compared to the possibility of incubation for 15+ hours. In general, I'm not so sure I can rule out that temperature might also have a better effect if the samples were left for 15 hours as opposed to 10 minutes. –  A. Kennard Feb 22 at 1:37
Because you should be able to see a difference between 0 (simply mixing) and 10 minutes. And its also my own experience with fits with this data. –  Chris Feb 22 at 8:50
@Chris I find your experience more credible than Fig. 2. What if the amount of precipitate over time looks like a Michaelis-Menten curve (dwb4.unl.edu/Chem/CHEM869K/CHEM869KLinks/www.curvefit.com/… change x-axis to time and y-axis to amount of precipitate)? Then a lot would rapidly precipitate out, but a significant fraction might take a long time to come out in a short time; you wouldn't necessarily see the difference between 5 min and 10 min, but you might notice a difference after 15 hr. –  A. Kennard Feb 22 at 11:39
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