Discrepancy between OD-value on spec and DNA on agarose gel

Quentin E. Low qlow at orion.it.luc.edu
Fri Aug 16 17:35:01 EST 1996

On Sat, 10 Aug 1996, Botma Visser wrote:

> Hi
> I have a problem in determining the concentration of plasmid 
> DNA on a spectrophotometer.  I extracted plasmied DNA using 
> the Qiagen midiprep columns.  When I determined the 
> concentration of the plasmid DNA at 260 nm, I got a very 
> high reading, with the concentration going up to 1.1 mg/mL 
> from a 100 mL prep.  I then ran 500 ng plasmid DNA 
> (calculated using the determined concentration) on an 
> agarose gel, and saw nothing.  The prep contained no RNA.  
> Have you encountered similar problems, and if yes, what can 
> I do about it?  It is critical for me to have accurate 
> concentrations.

I have the same problem with the OD method. What I have previously done 
was to include reading with OD 280 with my readings so I can ascertain 
the purity of the DNA. Although knowing the concentration of the DNA is 
important to me, I also need to know if there is any DNA 
(false readings can be obtained with spectroscopy) So what I do is 
to run a small sample out on a gel along with three amounts of lambda ladder:
0.5 ug, 1.0ug and 1.5ug. Not only do I know that there is DNA, but I can 
guesstimate the concentration. When the DNA is pure, the two methods give 
very similar results. 

Hope that this helps

Quentin Low

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