9. Application Note for DNA Quantification
9.3.2
Sensitivities in comparison:
260 nm absorbance:
approximately 50 – 250 ng DNA/ml
PicoGreen
fluorescence:
approximately 300 pg DNA /ml
The comparison between absorbance and fluorescence measurements show that
fluorescence quantitation is 100 to 1000 times more sensitive. The calibration
curves show the linear range of the different methods. It should be emphasized,
that the sensitivities are dependent on filling volumes (especially for absorbance),
flash numbers, type of diluent and even the type of DNA can influence the final
result.
9.3.3
Results from Plasmid and Blood DNA Sample
(a) DNA
from
Blood
In some cases the amount of blood and DNA is not sufficient for UV quantitation.
DNA from human blood was extracted with different treatments. The volume and
concentration of the extracted samples did not allow to quantify with absorbance.
1
10 µl blood fresh/EDTA
5
10 µl blood old/EDTA
2
20 µl blood fresh/EDTA
6
20 µl blood old/EDTA
3
10 µl blood fresh/Citrate
7
10 µl blood old/Citrate
4
20 µl blood fresh/Critrate
8
20 µl blood old/Critrate
Treatment
1
2
3
4
5
6
7
8
485/535
1.02 1.06 2.99 4.15 3.88 3.92 1.23 2.27
Table 1
Calculated DNA concentrations in µg/ml stock solution of the same
samples with different treatment.
(b) Plasmid
DNA
The same samples from a plasmid extraction have been measured with both
methods. Since the fluorescence method is a lot more sensitive, the samples
were diluted 1:100 before the measurement – both of the detection techniques
show comparable results.
485/535 nm
46 44 46 49 59 50 39 48 48 51 43 48 36
Abs 260 nm
44 45 45 65 53 46 36 49 38 46 55 54 34
Table 2
Comparison of the stock concentrations (referring to the calibration
curves above) in µg/ml of the samples measured with fluorescence (485/535) and
absorbance (260).
9-4
Operating Manual for GENios, GENios FL, and GENios
Plus
No: I 112 904 Rev No: 1.1
June 2002
