Adding "volumes" of ethanol to precipitate nucleic acids - (Feb/08/2006 )
When a protocol instructs you to add 0.1 volumes of sodium acetate and then 2.5 volumes of ethanol, are the 2.5 volumes of ethanol supposed to take into account the increased volume due to teh sodium acetate? I doubt if it matters a whole lot but I have always wondered. Usually I do a compromise - somewhere between 2.5 volumes of the original volume and 2.5 volumes of the original plus sodium acetate volume.
After 5 years of randomly asking this question to colleagues and never getting a definitive answer, I thought I'd' try this forum.
I always take it to mean the increased volume, but as you say it really doesn't matter as the difference is so small
I've tried both ways and gotten good yields of the DNA. We all agree that the volume difference due to the addition of acetate is not going to impact the yield much. But when it comes to semantically analyzing the protocol instruction, I'd rather go with thinking 2.5 volumes of the initial volume (before addition of acetate) than otherwise, my reasoning being thus: 0.1 vol refers to the initial volume and since the phrase "2.5 vols" doesn't qualify further, it is reasonable to think this also refers to the initial volume. Right?
You allso have to remember that the sodium acetate (or eny other acetate salt for that mater) it an organic slat. Thier for it will be fond in the EtOH and not in the sample (or aquarius part)
I have always only considered the origional volume of plamid to be precipitated.
So if I have 400ul of plasmid to precipitate, I add 40ul Na Acetate and 800ul Etoh. (I precipitate with 2x vol of Etoh).
If you don't take the added NaOAc into account, the final % Ethanol would be 69.4% (2.5vol/1vol + 0.1vol + 2.5vol)
If you do take it into account the final % Ethanol would be 71.4% (2.5vol*1.1/1vol + 0.1vol + (2.5vol*1.1vol))
Which is 70.4% +/- 1%. So I really don't think it matters!
john buckels rocks ....
Finally, you need to remind that nucleic acids pellet at ethanol concentrations upper than 60%.
I dont care much about amount of alcohol. If it is ethanol I take double the volume and if it is isopropanol equal amount. If Hol has a complication I have one too...
If sodium Acetate should be 1/10 of the solution then after you add X amount of Na Acetate it will be 1/10+Y th amount if Y is amount of Na Acetate added!!! Wondering ??? I too wondered!