Eppendorf BioPhotometer plus - Manual (Page 45)

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11 Evaluation procedure
BioPhotometer plus — Operating manual
11.4 Special evaluation procedures for the dye methods
11.4.1 Calculating the factor for the dye from the absorbance coefficient
In the dye methods a factor is calculated for the dye from the absorbance coefficient entered in
the parameters and is displayed in the startup screen of the method procedure.
The factor is calculated as follows:
F
d, Dye
= factor for the dye with automatic allowance for the cuvette optical path length
ε
Dye
= absorbance coefficient for the dye programmed in the method parameters; unit: cm -1 M -1
d = cuvette optical path length, unit: cm
11.4.2 Calculation of the FOI
As a value for the ratio of dye molecules to the number of nucleotides in the nucleic acid the
frequency of incorporation (FOI) is calculated and displayed for the dye methods. The calculation
can be selected for two different result units:
Unit DYE MOLECULE DYE / kb
Unit pmole/μg DNA (or RNA)
A
X50
= measured absorbance of the dye at 550 or 650 nm
ε
Dye
= absorbance coefficient for the dye, unit: cm -1 M -1
MM
nt
= average molecular mass of the nucleotides; unit: g/mol; for dsDNA/ssDNA/Oligo DNA:
325; for RNA / Oligo RNA: 337
A
260
= measured absorbance of the nucleic acid
F = factor for calculating the nucleic acid programmed in the method parameters. The factor
relates to the cuvette optical path length of 10 mm and does not need modifying if the parameter
CUVETTE is modified.
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