Problem with minimal isopropanol precipitation - (Oct/30/2006 )

Using isopropanol to precipitate plasmid DNA is supposed to help avoid contaminants that might interfere with enzymatic manipulations (cleavage, sequencing, etc.) The standard method (I think) is to have your DNA in 0.3M sodium acetate and add 0.6 volumes isopropanol (final 37.5%).

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I put my DNA in 0.3M sodium acetate (pH 7.something) prior to phenol extraction. After phenol extraction and chloroform:isoamyl extraction, I added 0.6 volumes isopropanol.

I lost all my DNA during the precipitation. Fortunately, I was able to recover enough DNA from some of the leftover aqueous phase in the choloroform extraction, and an ethanol precipitation on these dregs (a little more sodium acetate plus 2 volumes ethanol) seemed to work fine. Still, I am wondering what I did wrong during the isopropanol extraction.

Q: Is it possible that the sodium acetate was diluted (into the organic phase) during the phenol extraction/chloroform:isoamyl extraction? In this case, the sodium acetate would not have been 0.3M when I added isopropanol. From a hydrogen-bonding standpoint, I don't think the salt would go into the organic phase. ??

Q: Do I have the final concentration of isopropanol correctly calculated (or should it be 60% final?)?

What else might cause a simple isopropanol precipitation to fail?

Thanks!

I found this to be full of tips... i, on the other hand, am extraordinarily bad with ispropranol precipitation (i never have a good yield with it), and have resigned myself to sticking with ethanol precipitation.

V