NanoPhotometer
®
P-Class
User Manual
Version 2.1
Page 67 / 70
12.APPENDIX
12.1
Nucleic acid quantification
For determination nucleic acid concentration in solution the absorbance at wavelength 260 nm is used. The function
describing the concentration to absorbance relation is a modification of the Lambert-Beer equation.
c
nuc
= Abs. 260 * factor
nuc
* lid factor * dilution factor
With background correction:
c
nuc
= (Abs. 260 – Abs. 320) * factor
nuc
* lid factor * dilution factor
c
nuc
nucleic acid concentration (ng/µl)
Abs. 260
absorbance (AU) of nucleic acids
factor
nuc
substance specific factor for nucleic acids (ng * cm/µl) (ds DNA 50, ssDNA 37, RNA 40, Oligo 33)
lid factor
virtual dilution factor 5, 10, 50, 100 and 250; dependent on the used dilution
lid.
12.2
Nucleic acid fluorescent dye incorporation
To determine the nucleic acid concentration and the dye concentration after probe labelling a modification of the
Lambert-Beer equation is used. Background correction is always calculated; possibility to switch the dye correction on
and off.
Calculation of the fluorescence nucleic acid concentration
c
nuc
= (Abs. 260 – Abs. 320) * factor
nuc
* lid factor
*
dilution factor
With dye correction:
c
nuc
= [(Abs. 260 – Abs. 320) – (CF
dye
* (Abs.
max, dye
– Abs. 320)] * factor
nuc
* lid factor
*
dilution factor
c
nuc
nucleic acid concentration (ng/µl)
Abs. 260
absorbance (AU) of nucleic acids
CF
dye
dye-dependent correction factor at 260 nm
Abs.
max, dye
absorbance at absorption maximum of the dye (AU)
factor
nuc
substance specific factor for nucleic acids (ng * cm/µl) (ds DNA 50, ssDNA 37, RNA 40, Oligo 33)
lid factor
virtual dilution factor 5, 10, 50, 100 and 250; dependent on the used dilution lid.
Calculation of the dye concentration
c
dye
= ((Abs.
max, dye
– Abs. 320) * lid factor * dilution factor) / (ε
dye
* 10
-6
)
c
dye
dye concentration (pmol/µl)
Abs.
max, dye
absorbance at absorption maximum of the dye (AU)
lid factor
virtual dilution factor 5, 10, 50, 100 and 250; dependent on the used dilution lid.
ε
dye
dye-dependent molar extinction coefficient (M
-1
* cm
-1
)
Calculation of the frequency of incorporation (FOI) of dye per 1,000 bases:
Formula for dsDNA:
FOI = (6.49 * (Abs.
max, dye
– Abs. 320) / [ε
dye
* 10
-6
* (Abs. 260 – Abs. 320)]
With dye correction:
FOI = (6.49 * (Abs.
max, dye
– Abs. 320) / [ε
dye
* 10
-6
* (Abs. 260 – Abs. 320 – CF
dye
* (Abs.
max, dye
– Abs. 320)]