Role of sodium acetate in DNA extraction/precipitation
Posted 22 May 2013 - 06:07 AM
I got a bit confused about the role of sodium acetate in DNA extraction protocols.
I was told it helps to neutralise the DNA charge so that DNA could be precipitated easier with the ethanol added.
However I also saw on some webpages that it also helps precipitng the DNA by binding to proteins (bound to the DNA).
I can understand it helps precipitating the DNA, but if it precipitates with the DNA + proteins , than how do you get rid of those protains after the precipitation?
Because you simple "clean" it with ethanol to remove salts, but you dont remove the proteins , or do you?
Posted 22 May 2013 - 01:02 PM
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Posted 22 May 2013 - 11:15 PM
The role is to increase the number of ions in solution to a point where the DNA can be precipitated by the addition of an alcohol primarily. In some extractions such as plasmid preps, it is used to neutralize the alkaline component of the lysis (step 2 NaOH and SDS) and precipitate the proteins and genomic DNA from this step, again through ionic strength.
Yeah, I understand this.
But in the case of genomic DNA: if you precipitate the DNA with the proteins, does this cause problems? Or is it not a problem to have a lot of proteins too for (for example) a DNA sequencing test?
Posted 23 May 2013 - 12:50 AM
Posted 23 May 2013 - 01:21 AM
Ok I see what you mean.
Most DNA extractions have a chaotropic step which denatures proteins which are then removed before the DNA precipitation step.
When you do a genomic DNA extraction/precipitation you have an extra step to remove the proteins before you precipitate the DNA.
Posted 23 May 2013 - 12:41 PM
Posted 24 May 2013 - 08:13 AM
So the salt has two reasons: helping the DNA bind the colum and helping in salting out the denatured proteins?
If you want reasonably clean DNA, yes. Even relatively impure DNA extractions such as those used for tail-tip screening of mice will have a process where the proteins are digested and salted out before the DNA is precipitated.
Posted 24 May 2013 - 05:41 PM