Eppendorf BioPhotometer plus - Manual (Page 23)

23
5 Operation
BioPhotometer plus — Operating manual
5.8.3 Frequency of incorporation "FOI"
The FOI is a measure for the concentration of the dye relative to the concentration of the nucleic
acid. It is not provided for protein methods. For calculating the FOI you can select between 2 units
in the method parameters:
• "MOLECULE DYE / kb": dye molecules per 1000 nucleotides.
• "pmole/μg DNA": pmole dye per μg nucleic acid.
The formulae for calculating the FOI can be found separately (see Calculation of the FOI on
page 45).
5.8.4 Correction factors
In addition to the options for turbidity correction described (parameter "CORRECTION A
340
")
(see Nucleic acids on page 19) the affect of the dye on the measurement of the nucleic acid or
the protein can also be corrected for the "DYE methods". If the degree to which the dye absorbs
light is known also for the measuring wavelengths of the biomolecule (260 and 280 nm), then a
correction can be made in the parameter "CORRECTION
550 (or 650)
". If you enable this parameter
you can enter a correction factor for 260 and 280 nm. E.g. for Cy 3 the preprogrammed values
are:
• 0.04 for "CORR.A
550
: F
260
"
• 0.05 for "CORR.A
550
: F
260
"
The use of these factors for the calculation of concentration and ratio of the biomolecule is
described elsewhere (see Correction A
550/650
on page 46).
5.8.5 Measuring procedure and result display
After calling the method you will see the following display (example: "DYE 550 - ssDNA"):
Top: method name and cuvette selected in the parameters.
Center: programmed values for the calculation of the DNA
and dye concentrations: factor for ssDNA and absorbance
coefficient for the dye. In addition the factor calculated from
the absorbance coefficient related to a cuvette optical path
length of 10 mm will be displayed.
Bottom: measuring key for the next measurement.
 Carry out a blank measurement.
 Carry out a sample measurement.
Top: method name and sample number
Center: result for DNA and dye.
Bottom: result for the frequency of incorporation.
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