Spec vs Flourimeter for [DNA] ?

Jim Owens jow at helix.nih.gov
Thu Dec 2 09:31:01 EST 1993

In article <CHDoH7.Dv7 at fiu.edu> Derek M. Harkins, harkinsd at solix.fiu.edu
>But then I re-read the initial question and the plasmid nucleic acids
>have been spun through CsCL--therefore no contaminating RNA should be
>to skew the readings of the spectrophotometer.

I would not assume that there is no RNA to contaminate the plasmid unless
there is an agorose gel to prove it.

Years ago, when I routinely used CsCl gradients for plasmid preps, fixed
angle rotors were used for the gradient centrifugations.  RNA pellets
under the conditions used and could be seen smeared along the side of the
tubes away from the center of rotation.  The ccc plasmid band would
always be contaminated by RNA that came off the tube's wall.  A second
CsCl gradient was needed to eliminate the RNA.

An RNaseA treatment before the centrifugation resulted in oligomers of
RNA spread all over the gradient.  The RNA oligomers in the plasmid band
would not go away with a second centrifugation since their density
matched that of the plasmid.

Since so many people have trouble with fluorescent dyes in measuring DNA,
and since you should have so much DNA from a CsCl gradient plasmid prep,
uv absorbance is the method of choice in my humble opinion.

Good luck,

Jim Owens

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