Discrepancy between OD-value on spec and DNA on agarose gel
hroychow at NMSU.EDU
Mon Aug 12 15:41:05 EST 1996
On Sat, 10 Aug 1996, Botma Visser wrote:
> 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
> Thank you.
> Botma Visser
> pbv at rs.uovs.ac.za
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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