DNA QUANTITATION

BIOCHEMISTRY agoodrid at vaxa.weeg.uiowa.edu
Wed Oct 2 12:07:00 EST 1991


muriana at aclcb.purdue.edu (murianap)  writes:

>I presume that the presence of individual RNA bases would still
>give absorbance and mask the true levels of DNA quantitation by
>absorbance at 260nm.  Am I correct? and would this be alleviated by
>ethanol  precipitation, whereby the digested bases would not be
>precipitated by ethanol, so the DNA can be revovered again for quantitation.

Peter,

In short the answer to both questions is yes.

The bases do absorb at A260.  However, I would like to say something
about RNase digestions.  In my experience with plasmid preps only extensive
digestions with RNase along with precipitation effectively remove RNA.

Test a sample of your DNA by running a high percentage gel (eg Nuseive 5%)
and look for RNA bases and oligos.  In my experience RNases A and T1
(a common mix for effective removal of RNA) do not digest RNA to completion.
The resulting oligos can also precipitate.  So when I don't use a column
I perform two cycles of digestion and precipitation.  The purity of the
DNA from RNA is good after this procedure but then I suppose that depends
on what you are going to use the DNA for.

I would stay away from isopropanol precipitations since it is effective
in precipitating oligos.

Hopefully, these answers are better than the ones I gave you previously.
:-)

-= Steve =- 	AGOODRID at VAXA.WEEG.UIOWA.EDU



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