Spec vs Flourimeter for [DNA] ?

Tue Nov 30 21:25:53 EST 1993

In article <19931130120206.bloksber at thomashaw-at.css.msu.edu>
bloksber at pilot.msu.edu  (Leonard N. Bloksberg) writes:
>Question about the Hoefer TK100 DNA flourimeter.
>I have 3 samples of plasmid DNA, all are subclones of the same clone in
>pBlueScriptKS- or empty vector, all double CsCl banded, look great on gels,
>etc.  When I measure my DNA concentration on an LKB Biochrom Ultrospec II
>spectrophotometer as A260, I get 1 set of readings and when I measure on the
>Hoefer TK100 DNA flourimeter I get another.  The 260/280 ratios are all 1.80.
>The spec give a very consistent reading of 3x the flourimeter reading.  Which
>do I believe and why?  Can anyone offer some insight or warnings?  The
>exact concentration is important to me for titrating DNA binding proteins.
>Thanks in advance.  I will post a summary of important points if warrented.
>	Leonard N. Bloksberg
>	Bloksber at pilot.msu.edu
>	Dept. of Crop and Soil Science
>	Michigan State University
I am having similar problems with a CTAB DNA extraction procedure I use.
When I do the spec readings I get a 10 fold higher reading than I get
using fluorimetry.  This does not occur when I use CsCl purified plasmid
DNA.  It seems to have something to do with the CTAB extraction procedure
I think.  I am trying to isolate DNA from Physcomitrella patens (a moss),
blot it and then look for single copy sequences.
Any help or suggestions would be appreciated!
Tracey Zacharias, Frustrated Grad Student
University of Regina
Zacharit at max.cc.uregina.ca

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