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

[H. ROYCHOWDHURY]

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.
> 
> Thank you.
> 
> Botma Visser
> 
> pbv at rs.uovs.ac.za
> 
> 
Hi,
	You can not determine NA concentrations by looking at the 260nm
reading only, since the nucleotides have absorption maxima at 260nm. A
solution containing very small oligo-ntds will also absorb strongly at
260nm, leading one to believe that it has high DNA/RNA conc.

			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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