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
>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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