Concentration Calculation (Beer's Law) - Thermo Scientific NanoDrop 1000 V3.7 User Manual

Concentration Calculation (Beer's Law)

General
The Beer-Lambert equation is used to correlate the calculated
absorbance with concentration:
A = E * b * c
Where A is the absorbance represented in absorbance units (A), E is
the wavelength-dependent molar absorptivity coefficient (or extinction
coefficient) with units of liter/mol-cm, b is the path length in cm, and c
is the analyte concentration in moles/liter or molarity (M).
Fluorescent Dyes (Microarray Measurement)
The software uses the general form of the Beer-Lambert equation to
calculate fluorescent dye concentrations in the Microarray module.
The table of extinction coefficients for each dye is below:
Dye Type
Cy3
Cy5
Alexa Fluor 488
Alexa Fluor 546
Alexa Fluor 555
Alexa Fluor 594
Alexa Fluor 647
Alexa Fluor 660
Cy3.5
Cy5.5
Nucleic Acids
For nucleic acid quantification, the Beer-Lambert equation is modified
to use an extinction coefficient with units of ng-cm/ml. Using this
extinction coefficient gives a manipulated equation:
c = (A * e)/b
Where c is the nucleic acid concentration in ng/microliter, A is the
absorbance in AU, e is the wavelength-dependent extinction
coefficient in ng-cm/microliter and b is the path length in cm.
The generally accepted extinction coefficients for nucleic acids are:
• Double-stranded DNA: 50 ng-cm/ul
• Single-stranded DNA: 33 ng-cm/ul
Extinction
Coefficient
(liter/mol-cm)
150000
250000
71000
104000
150000
73000
239000
132000
150000
250000
19-2
Measurement
Wavelength
(nm)
550
650
495
556
555
590
650
663
581
675
Section 19- Appendices