Hello Appu,
Small amounts of strongly UV absorbing contaminants can throw off
your measurements. If you want to be very accurate, try a fluorescent
dye specific for double stranded DNA such as PicoGreen.
Ho
Ho Leung Ng
University of Hawaii at Manoa
Assistant Professor, Department of Chemistry
h...@hawaii.edu
> Deal CCP4 member's
> Sorry for asking Off topic question.
> Please correct me if i am doing wrong measurements. I am determining the
> double stranded concentration using the absorbance spectrophotometer
> reading at 260A and extinction coefficients for individual NMPs . But
> protein to DNA stoichiometry is coming really off in ITC. I am doubting
> weather i am using right method to determine the DNA concentration
> accurately or i am doing some wrong calculation. I am calculating the DNA
> extinction coefficients by simply multiplying the total number of
> individual nucleotide by it extinction coefficients which are as follow
> A = 15.4 mM-1 cm-1
> C = 7.4 mM-1 cm-1
> G = 11.8 mM-1 cm-1
> T = 9.6 mM-1 cm-1
>
> The formula which i am using to calculate the concentration is
> DNA concentration(M) = ( Abs@260-Abs@330)/Molar extinction
> coefficients of DNA
> I am multiplying this by dilution factor.
