Dissolving plasmid DNA after precipitation
Kevin A. Mulcahy
K.Mulcahy at sheffield.ac.uk
Sat Jan 25 06:38:55 EST 1997
thanks for your reply. Could I ask a couple of things about your
In your reply you wrote:
> 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.
I don't follow what you are trying to say here - could you please
You also wrote:
>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.
The strange thing is that I never have any problems re-dissolving
mini-prep DNA following the initial precipitation (in addition, I
always double-extract with phenol-choloroform before I precipitate the
DNA so no significant amounts of protein should be present). The
re-dissolving problem occurs when I re-precipitate either restriction
fragments (in order to change buffer or following phenol-chloroform
extraction and ethanol precipitation) or highly purified maxi-prep
plasmid DNA (Qiagen Tip500 method).
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