9. Application Note for DNA Quantification
9.3.4
DNA Purity at 260/280 nm
Table 3
Determination of the purity (DNA/protein ratio) of plasmid DNA samples.
The high impurity of the samples may be due to the extraction method used
9.4 Conclusion
Depending on origin and further use of the DNA, the concentration and volume of
samples may not be sufficient for quantitation using absorbance. In order to
obtain comparable accuracy and reduce the amount of DNA required, the use of
fluorescence is recommended. From the plasmid extractions, a stock solution
volume of 50
µ
l was obtained with a DNA concentration high enough for
absorbance measurements. The DNA extractions from human blood yielded
50
µ
l of stock solution with concentrations between 1 and 5 µg/ml, so the amount
of extracted DNA was not sufficient for quantitation with absorbance.
This application example clearly demonstrates the advantage of having 2
detection techniques in one instrument. Both techniques offer a variety of
advantages depending on the method of extraction and amount of available DNA.
DNA quantitation by UV absorbance measurement is easy to perform,
inexpensive and allows a rapid check of DNA purity (260/280nm ratio) although
the extraction method, interference with RNA, ssDNA, or single nucleotides and
contaminants (such as phenol or EDTA) may influence the quality of the results.
In contrast, fluorescence assays provide the perfect combination of sensitivity
and selectivity for dsDNA and extend the detection range significantly.
Acknowledgements
We would like to thank Robert Liebhart for carrying out the practical experiments
at ETH Zürich, Inst. for Plant Sciences (Phytopathology), Universitätsstrasse 2,
CH-8092 Zürich (Laboratory Dr. Cesare Gessler).
June 2002
Operating Manual for GENios, GENios FL, and GENios
Plus
No: I 112 904 Rev No: 1.1
9-5
