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extinction coefficient - (Mar/17/2006 )

how to determine extinction coefficient of supercoiled, linear and open form of DNA?


The equation for calculating extinction coefficients is stated by Beer's Law:

A = epsilon.c.l

Where A is the absorbance measurement at a given wavelength

epsilon is the extinction coefficient in M^^-1

c is the concentration of the material in^-3 (molar)

l is the path length in centimetres


i want to calculate the concentraton only after determining the extinction coeff. then hw do i determine the extinction coeff of supercoiled DNA, for example??


if you've your three forms in the sample i don't think it's possible to distinguish them. Hence, you've a general extinction coeff.


As far as I'm aware it's simply not possible. I don't think there is another equation that relates the variables in the first one. If there was you could solve them simultaneously.

What makes you so sure that the different forms of DNA have different extintion coefficients? For calculating the concentration of DNA you simply use the well established fact that for linear, plasmid or any other type of DNA an A260 reading of 1.0 equates to 50 µg/mL of DNA. You can then work out the concentration in Molar if you know the length of the fragment and just use the estimate of Mw 660 per base pair.