[Jefke]

Dear all,

when doing a DNA precipitation you use 100% ethanol and 70%.

Now I am wondering: why the 100% first and than the 70%?
Whats the benefit of using 100% first and than the 70?

[Babcock_Hall]

Without a protocol, all that I can do is to make a guess, and it has been some time since I last precipitated DNA.  When one adds 100% ethanol to an aqueous solution, the ethanol is now less than 100%.  Whether it is 70% or not depends on the two volumes.

[Jefke]

Without a protocol, all that I can do is to make a guess, and it has been some time since I last precipitated DNA.  When one adds 100% ethanol to an aqueous solution, the ethanol is now less than 100%.  Whether it is 70% or not depends on the two volumes.

What I mean is: you first add 100% to the sample (when doing an ethanol precipitation) to precipitate the DNA (2 or 3 times the volume of the "liquid")
After this you remove the ethanol/liquid and you add 70% to wash the DNA.

Now why first 100% and not just right away 70%?

(and yes: I know the 100% is less, because often you start with 95% from a bottle and some of the ethanol is evaporated anyway)

Aha, I see what you mean: if you use 70% you would need to add more to have an overal concentration of "70%" , I guess this is the reason?
That the concentration has to be high enough still to precipitate the DNA?

At what concentration does ethanol still precipitate DNA? is this known?

[Babcock_Hall]

Let's assume for the sake of simplicity that we are starting with 100% ethanol.  Let's also ignore the minor issue of volume changes of mixing*, again for the sake of simplicity.  If I take 1 part aqueous DNA and mix it with three parts ethanol by volume, then final concentration of ethanol is 75%.  If one instead mixes one part aqueous DNA to two parts ethanol, then this gives a final concentration of 67% ethanol.  I am not sure that I made this point clearly in my previous message.  If I do not use 100% ethanol but instead use a mixture close to 95% ethanol/5% water, then the numbers change slightly, but the principle stays the same.  I am not sure at what percentage ethanol stops being effective as a precipitant.
*in other words that volumes of liquids that are mixed are not strictly additive.

[Yggdrasil]

In an ethanol precipitation, you have an aqueous solution of DNA and added salt (for example, sodium acetate) then you add 100% ethanol to make a solution that has a final ethanol concentration of ~ 70%.  If you add 70% ethanol to your aqueous solution of DNA, will that solution ever reach a final concentration of 70% ethanol?

You can use 70% ethanol to wash the pellet after centrifugation because there is not water present that will dilute the ethanol.

[Jefke]

In an ethanol precipitation, you have an aqueous solution of DNA and added salt (for example, sodium acetate) then you add 100% ethanol to make a solution that has a final ethanol concentration of ~ 70%.  If you add 70% ethanol to your aqueous solution of DNA, will that solution ever reach a final concentration of 70% ethanol?

You can use 70% ethanol to wash the pellet after centrifugation because there is not water present that will dilute the ethanol.

Indeed

Let's assume for the sake of simplicity that we are starting with 100% ethanol.  Let's also ignore the minor issue of volume changes of mixing*, again for the sake of simplicity.  If I take 1 part aqueous DNA and mix it with three parts ethanol by volume, then final concentration of ethanol is 75%.  If one instead mixes one part aqueous DNA to two parts ethanol, then this gives a final concentration of 67% ethanol.  I am not sure that I made this point clearly in my previous message.  If I do not use 100% ethanol but instead use a mixture close to 95% ethanol/5% water, then the numbers change slightly, but the principle stays the same.  I am not sure at what percentage ethanol stops being effective as a precipitant.
*in other words that volumes of liquids that are mixed are not strictly additive.

yes yes

I can see your point.


I am just wondering: is 70% (or perhaps a bit less) really the limit for precipitation or would less be ok too.
Just curious of this is known.

[Johte]

http://molbiol.ru/wiki/Ethanol_precipitation

The biggest thing to take away from this page is a lot of the details of precipitation are preferential. That being said, you are typically using your counter ion in excess. The 70% comes after to wash your pellet of residual salt. Why 70%? Well, because, that's why. AFAIK there is no solid answer and my PI gets frustrated with my desire of the "why."   
I've read technical papers that incubation temperature below 0 has no significant effect. For short-mers precipitating overnight will give you the highest yield. However, I'm still told to do -80, 1 hour. c'est la vie.

Could you use less water? Probably. Although I can't find it I'm sure someone at some point had to have figured out that 70% EtOH is ideal.

[Jefke]

http://molbiol.ru/wiki/Ethanol_precipitation

The biggest thing to take away from this page is a lot of the details of precipitation are preferential. That being said, you are typically using your counter ion in excess. The 70% comes after to wash your pellet of residual salt. Why 70%? Well, because, that's why. AFAIK there is no solid answer and my PI gets frustrated with my desire of the "why."   
I've read technical papers that incubation temperature below 0 has no significant effect. For short-mers precipitating overnight will give you the highest yield. However, I'm still told to do -80, 1 hour. c'est la vie.

Could you use less water? Probably. Although I can't find it I'm sure someone at some point had to have figured out that 70% EtOH is ideal.

This is something I have been wondering about too lately.
Everyone keeps telling me to incubate at -20°C or -80°C but the newer researchpapers do indeed mention that room temperature is ok.