Haven't posted in a while, so this will be quick.
People ask me about their protocols on a pretty regular basis. Even if they don't ask specifically, I often ask to see their workflow so I can tell if I am giving reasonable advice in the context of their whole experiment. One of the most common things I encounter is the insistence on putting samples in the freezer to "help" with precipitating their precious DNA. However, this idea should have been laid to rest almost 30 years ago now with the publication of "Ethanol Precipitation of DNA" in Focus (Fall 1985, Vol 7, No 4) by Zeugin and Hartley.
The key findings of this paper are that precipitation is less efficient at low temperatures than worm temperatures, length of incubation time has minimal effect except for very dilute samples, and centrifugation time is the most important factor in planning your precipitation. The precipitations in this paper were NaOAc/Ethanol precipitations with the final NaOAc concentration being 0.3M (1/10 vol 3M NaOAc) and the final EtOH concentration being 75% (~2.5 vols EtOH).
The authors concluded that cold incubation is not beneficial to the precipitation of DNA and can even be counterproductive if your sample contains only a small quantity of DNA. They speculate that decreased temperature will increase the viscosity of the solution and inhibit the motion of DNA through the ethanol solution during centrifugation. I agree with this idea, but I would also speculate myself that by removing energy from the system, you simply slow down the kinetics of precipitation where free cations associate with the ribo-phosphate backbone while ethanol drives water molecules out of the double helix structure resulting in precipitation of DNA in a salt form. Certainly during centrifugation such interactions will increase, so lately my thinking has been that centrifugation is the only thing that matters much during your precipitation. This is also supported by their data where they looked at length of centrifugation time on precipitation. Again, high concentration samples precipitated readily while low concentration samples needed more time, but most concentrations seemed to taper toward maximum recovery at 30 min, which unfortunately, was the longest centrifugation time they tested.
For me, this has resulted in the following being my standard precipitation conditions. I prefer NaCl over NaOAc because I have some loose data indicating better recovery (not shown). I continue to use NaOAc during EtOH precips of cycle sequencing reactions.
1) Add NaCl to 0.15M. No need to be exact here. Use a 5M NaCl stock and you won't need to add much so the volume compensation is unnecessary. For instance, if you have a 300uL sample, calculate as follows: (300uL*0.15M)/5M = 9uL. So just add 9uL 5M NaCL and save yourself the headache. It's not worth it!!
2) Add 2.5 vols EtOH. Many protocols say add 2-2.5 vols EtOH. I think this is problematic since if you use 2 vols, your final EtOH concentration will be only 66% whereas at 2.5 vols you achieve about 71%. This may seem subtle, but my experience (admittedly anecdotal here) has taught me 2.5 is always better.
3) Mix sample well, and place directly into the centrifuge. For samples with decent concentration, 30 min at maximum speed. For low concentrations, longer is better, so maybe 30 min to 60 min (your choice).
4) Immediately after the centrifuge stops, remove the tubes and decant into the sink (plates can be GENTLY centrifuged inverted on a paper towel at low RPM, low acceleration/deceleration for ~10 sec to remove supernatant as per ABI sequencing protocol). If you happen to be out of the room when your centrifuge stops, start it again for 5 min or so to ensure your DNA is well-adhered to the wall of your tube.
5) Add 70% EtOH. For 1.5mL tubes I usually do 500uL, for 96 well plates, I usually add 50uL. Spin again at max speed, but only 10-15 min is necessary now.
7) Decant again as in step 4. Dry samples down in a vacuum centrifuge, or place on a heat block (~55C) for 10 min to evaporate any residual ethanol. If you have left over EtOH and you try to run a gel, your sample will maddeningly just float away. It can also inhibit downstream enzymatic steps.
8) Resuspend in your favorite solution. Some people like water, but since the pH of "pure" water is often a bit acidic, depurination is a real threat to your sample. Some people like TE, but even small amounts of EDTA can inhibit a PCR reaction. For these reasons my go to solution is Tris-Cl pH 8.5, 10mM.
So, I hope someone finds this useful. Happy precipitating!!
