Dissolving plasmid DNA after precipitation

Curt Ashendel ashendel at aclcb.purdue.edu
Fri Jan 24 11:17:00 EST 1997


>Kevin Mulcahy wrote: 
> I often have problems dissolving plasmid DNA (either uncut or
> restriction fragments) following precipitation with sodium acetate and
> ethanol. 
> I normally do the following:
> - precipitate with 1/10 vol of 3M Na-acetate (pH 5) and 2 vols of 100%
> ethanol 

On Thu, 23 Jan 1997 21:19:02 -0600, 
Rick Bright   <rbright at mnw.net> wrote:

>I have this EXACT same problem and have tried the same things you have,
>even Refrig overnight (which seems to help slightly).  

Kevin: I hope you are adding ethanol to the supernatant from the NaOAc 
step, and not adding NaOAc to the same solution as the ethanol. 

Both of you: Since you are using NaOAc, not KOAc, you will get a large 
amount of protein in your ethanol/isopropanol pellet. If we are going 
to precipitate the DNA without deproteinating Phenol extraction first, 
we always use KOAc on ice since K-SDS is not very soluble in the cold 
solution and when it precipitates it takes a lot of the protein with 
it. (This assumes an alkaline-SDS lysis  method). If you use alkaline 
SDS lysis (vs, say, the boiling method), and you precipitate the NaOAc 
sup, almost all the protein in the bacteria will precipitate with the 
alcohol, but the protein will fail to go back into solution later 
(once a cooked egg, always a cooked egg). The aggregated 
denatured protein will usually hang onto the DNA as well, making it 
difficult to redissolve.

I hope this helps.


Curt Ashendel
Purdue University, West Lafayette, IN
ashendel at purdue.edu



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