260/280 ratio

Rimantas.Plaipa at lt.VU.GF Rimantas.Plaipa at lt.VU.GF
Mon May 22 11:39:23 EST 1995

>On 18 May 1995, Barry Moore wrote:
>> I have been using a Beckman DU-64 spectrophotometer to quantify
>> concentrations of DNA suspended in H2O or TE.  I expect and sometimes
>> get a 260/280 ratio of 1.8 for pure DNA, and when the ration is 
>> around 2.0 I suspect the presence of RNA.  However, I have not been
>> able to find an explination for the occasional 260/280 readings of
>> 2.5-3.0.  What type of contamination or situation would elevate the
>> 260/280 ratio above 2.0?
>> Thanks for any comments that you might have.
>> Barry Moore
>Are you using Phenol in your extraction of the DNA? If so, there may be 
>residual phenol left in your sample. Since it has one lambda max at 
>270nm, it can cause you to get a higher ratio, and an overestimation of 
>your DNA concentration. This info is from a lab manual for recombinant 
>DNA by Zyskind and Bernstein.
>Hope this info helps
>Debbie Sellers  

>This posting have intrigued me, because I always believed that phenol has UV
>absorbtion spectrum similar to that of protein, since Tyr is derivative of
>phenol. I checked one handbook on UV spectroscopy of organic compounds, and
>also found 270 nm as the lambda max for phenol in H2O. I've recorded spectrum
>of phenol disolved in water. Indeed it has a maximum at 270 nm, but a 260/280
>ratio is 1.35, so, the admixture of phenol may only decrease the 260/280 ratio
>of nucleic acids. Adsorbtion at 270 nm is about 2-fold higher then at either
>260 and 280 nm, therefore such an admixture should be easily recognizable
>from UV absorbtion spectrum (maximum shifted to 270 nm).
>There are many of (in)organic compounds, which have short-wavelength
>UV absorbtion bands, whose tails extends up to 260 nm, i.e, compounds with
>carboxylic groups (acetates, EDTA). For example, 10 mM EDTA has A260=0.17 and
>A280=0.01. The admixtures of such coumpounds may really increase a 260/280
>ratio of NA.

>Rimantas Plaipa
>Depart. of Biochemistry and Biophysics, Vilnius University, Lithuania
>Rimantas.Plaipa at GF.VU.LT

I would have to concur with Rimantas. This makes intuitive sense unless there is 
some chemical interaction between the phenol and DNA/RNA bases. This explanation 
is supported by Maniatis, Molecular Cloning. V3, p E.5 which states " If there is 
contamination with protein or phenol, the OD260/OD280 will be significantly less 
than the values given above [DNA RNA, 1.8 2.0 respectively], and accurate 
quantitation of the amount of nucleic acid will not be possible."


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