DNA Quantification-Spec. Absorbancy

J C Ford jford at grove.iup.edu
Mon Jan 6 17:19:39 EST 1997


In article <32CC1F90.4B71 at hawaii.edu>, Nicholas <tachino at hawaii.edu> wrote:
>When performing 260nm absorbancy for quantification of DNA after
>extraction, what is the accuracy absorbancy range?
>I have read that it is between .03 and .5 for the most accurate
>absorbancy value due to the reduced %transmittance as the absorbancy
>values increase.
>Any thoughts?
>
>Happy New Year :)

In the old days, UV spectrophotometers were limited by thermal noise and the 
result was a minimum in the relative concentration error vs. absorbance curve 
at 0.434.  The minimum was also rather sharply defined.  That means that the 
most accurate analysis involved absorbance readings near that value.  Good 
modern instruments tend to be shot noise limited, and the most accurate value 
is then 0.868 [Willard, Merritt, Dean, & Settle, Instrumental Methods of 
Analysis, 7th Ed., Wadsworth, 1988, p 166-7]. In this case, the minimum 
also becomes broader, so the recommended range becomes 0.3-2.0. 

However, most inexpensive (<$10K US) spectrophotometers start to show some 
curvature in absorbance vs conc plots due to stray light above 1.0 in my 
experience.  We measure this as part of an instrumental analysis laboratory 
exercise.  Only our ($12K?) Perkin-Elmer 552 remains linear above 1.50.  We 
have three LDC/Milton Roy 601s which all show noticable curvature about 1.50; 
one starts to curve at about 1.00.  I suspect someone "munged" the lamp shield 
while changing lamps at some point.

I personally tell my students to adjust the DNA concentrations to keep the 
absorbance between 0.1 and 0.75.

Hope this helps

Dr. John Ford



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