NanoPhotometer
®
P-Class
User Manual
Version 2.1
Page 13 / 70
4.
NANOVOLUME APPLICATIONS AND CUVETTE APPLICATIONS
The NanoPhotometer
®
P-Class offers a complete solution for NanoVolume and standard volume applications. With the
NanoPhotometer
®
P-Class
Submicroliter Cell the required sample volume ranges from 0.3 µl to a maximal sample
volume of 5 µl. Standard volume applications can be performed with 10 mm pathlength quartz, glass or plastic
cuvettes.
Note:
Within the Utilities folder the user has the possibility to select various options that define data output (please see also
7.3 Output Options / Printer).
The
NanoVolume Applications
folder and the
Cuvette Applications
folder contain different sub folders:
Nucleic Acids
,
Protein
and
OD 600
(Cell Density). Contents of these sub folders are detailed below.
4.1
Characterization of DNA, RNA and Oligonucleotides
4.1.1
General Information
Nucleic Acid Quantification (NAQ)
Nucleic acids can be quantified at 260 nm because it is well established that a solution of dsDNA in a 10 mm
pathlength cell with an optical density of 1.0 has a concentration of 50 µg/ml, ssDNA of 37 µg/ml or 40 µg/ml in
the case of RNA. Oligonucleotides have a corresponding factor of 33 µg/ml, although this does vary with base
composition; this can be calculated if the base sequence is known. Please refer to 12.1 Nucleic acid quantification
for further details.
The instrument uses factors 50, 37, 40 and 33 as default settings for dsDNA, ssDNA, RNA and Oligonucleotides,
respectively, and compensation factors for dilution and use of cells which do not have 10 mm pathlength. Dilution
factor and cell pathlength can be entered.
Nucleic Acid Purity Checks
Nucleic acids extracted from cells are accompanied by protein, and extensive purification is required to separate
the protein impurity. The 260/280 ratio gives an indication of purity; it is only an indication, however, and not a
definitive assessment. Pure DNA and RNA preparations have expected ratios of
1.8 and
2.0, respectively;
deviations from this indicate the presence of impurity in the sample, but care must be taken in interpretation of
results.
The 260 nm reading is taken near the top of a broad peak in the absorbance spectrum for nucleic acids, whereas
the 280 nm reading is taken on a steep slope (i.e. small changes in wavelength cause large changes in
absorbance). Consequently, small variations in wavelength at 280 nm will have a greater effect on the 260/280
ratio than variations will at 260 nm. Thus different instruments of the same and different types may give slightly
Folder
Application
Recommended Measurement Cell
Nucleic Acids
DNA
Concentration, purity check and dye incorporation for
DNA samples
Submicroliter Cell / Cuvette
RNA
Concentration, purity check and dye incorporation for
RNA samples
Submicroliter Cell / Cuvette
Oligo
Concentration, purity check and dye incorporation for
Oligo samples
Submicroliter Cell / Cuvette
Protein
Protein UV
(Christian-Warburg)
Protein determination at 280 nm
Submicroliter Cell / Cuvette
Protein Dye
Protein determination at 280 nm and dye
incorporation
Submicroliter Cell / Cuvette
BCA
Protein determination at 562 nm
Cuvette
Bradford
Protein determination at 595 nm
Cuvette
Lowry
Protein determination at 750 nm
Cuvette
Biuret
Protein determination at 546 nm
Cuvette
Cell Count
OD600
Cell density at 600 nm
Cuvette