Purity of DNA (Ratio of OD = 1.8-2.0) ?

wmelchior at ntet.nctr.fda.gov wmelchior at ntet.nctr.fda.gov
Wed Jan 13 13:14:20 EST 1993


Andre Hamel has added some useful information to this discussion:
> May I also add that the absorbance measured at
> 230 nm is also VERY useful in addition to the 260 and 280 values because,
> in addition to "other stuff" being detected,  ALL PROTEINS should absorb
> "strongly" at 230 nm due to the presence of peptide bonds (amino ester
> linkage if my memory offirst year chemistry still holds true). Is this not
> why, for instance, many HPLC protein purification systems employ 190- 210 nm
> wavelength light to more sensitively detect the outcoming protein? Those
> proteins with aromatic prosthetic groups will also exhibit additional
> wavelengths of absorbance, around 280 nm. 
> 
> 	Budget permitting, ideally a scanning spectrophotometer would be used as
> opposed to simply taking only 230, 260 and 280 nm measurements. One then
> would use the highest absorbance value (as long as it's around 260 nm,
> plus/minus about 10 nm). This maximum absorbance (peak) should ideally be
> about twice as high in value than the values near 230 and 280 nm. 

At the risk of providing more information than people want, let me note
that the absorbance peak (eg, ca. 254 nm for calf thymus DNA) is generally
NOT 260 nm.  When I'm using the ratio method to check a DNA preparation, I
use the 260 nm value rather than the peak.  The values for different DNAs
given in the Felsenfeld and Hirschman JMB paper are at 10 nm intervals, not
the peak.  As several others have noted, this method is only a crude check, 
any way, so simplicity and reproducibility are the prime requisites.

I strongly endorse Andre's suggestion of scanning a complete spectrum.  
With a little experience, the overall spectrum shows problems other those 
described above, including high backgrounds due to contaminated cuvets or 
solutions, and problems with the spectrophotometer.  Simple ratios can be 
misleading.  (For instance, even low concentrations of EDTA can lead to high 
absorbances at short wavelengths, in a concentration- and pH-dependent 
manner.  The appropriate solvent must be used for a baseline, at least until 
the parameters for your particular situation are well established.)
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||     OMNISCIENCE
National Center for Toxicological Research   ||    Knowing what
Jefferson, AR  72079                         ||    thou knowest not
(501) 543-7206                               ||    is in a sense
                                             ||    omniscience.
WMELCHIOR at NTET.NCTR.FDA.GOV                  ||       from Grooks, Piet Hein



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